a molecular analysis of opsin integration at the
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1
A Molecular Analysis of Opsin
Integration at the
Endoplasmic Reticulum
A thesis submitted to The University of Manchester for the degree of
Doctor of Philosophy in the Faculty of Life Sciences.
2005
Nurzian Ismail
Faculty of Life Sciences
The University of Manchester
2
Contents
List of Figures 6
List of Tables 10
Abbreviations 11
Abstract 12
Declaration 13
Acknowledgements 14
List of Publications 15
Chapter 1 - Introduction 16
1.1 The endoplasmic reticulum 17
1.2 ER-targeting signals 19
1.3 Targeting of polypeptide chains to the ER 23
1.4 The ER translocon 26
1.5 Translocon-associated components 29
1.5.1 The Sec62/63 complex 29
1.5.2 Signal peptidase complex 30
1.5.3 Oligosaccharyltransferase (OST) complex 31
1.5.4 TRAP 34
1.5.5 Glycoprotein specific ER chaperones 34
1.6 Structure and composition of the ER translocation site 37
1.7 The biosynthesis of polytopic membrane proteins at the ER 41
1.7.1 Targeting and insertion of polytopic membrane proteins at the
translocon 41
1.7.2 Lateral exit of TM domains into the ER membrane 44
1.7.3 Models of polytopic membrane protein integration into the ER
membrane 45
1.8 Membrane chaperones and polytopic proteins 46
3
1.9 This study 47
1.9.1 Opsin as a model polytopic membrane protein 47
1.9.2 Use of site-specific cross-linking in the analysis of opsin integration 50
1.9.3 Overview 52
Chapter 2 - Materials and Methods 53
2.1 Materials 54
2.2 Site-directed mutagenesis 55
2.3 Generation of OPTM1-3PPL[cys115] and OPN/5-7[cys-null] mutants 58
2.4 Synthesis of truncated transcription templates lacking a stop codon 60
2.5 Preparation of semi-permeabilised cells 62
2.6 In vitro transcription and translation 63
2.7 Isolation of ribosome-nascent chain complexes 64
2.8 Cross-linking and modifications of nascent chains with sulphydryl specific
reagents 64
2.9 Solubilisation of ribosome-nascent chain complexes in C12E8 64
2.10 Immunoprecipitation 65
2.11 Endoglycosidase H digestion 65
2.12 SDS PAGE and sample analysis 66
Chapter 3 - The use of site-specific cross-linking approach to examine opsin
integration 67
3.1 Introduction 68
3.2 Optimisation of the experimental system 69
3.3 Cross-linking adducts are formed with glycosylated opsin chains 71
3.4 Cross-linking adduct formation is cysteine-dependent 74
3.5 Summary 78
Chapter 4 - The integration of the N-terminal region of opsin: TM1 to TM3 79
4.1 Introduction 80
4.2 TM1 is adjacent to discrete sets of ER components during its integration 82
4.3 TM1 environment is influenced by subsequent TM domains 84
4
4.4 TM2 has exited the translocon in the OP204 integration intermediate 88
4.5 TM3 is associated with the ER translocon in the OP164 integration
intermediate 90
4.6 TM3 exits the translocon upon chain extension 92
4.7 TM3 relocation is independent of the presence of subsequent TM domains 94
4.8 Nascent opsin chains are associated with a single copy of the Sec61
complex during integration 96
4.9 Summary 98
Chapter 5 - The integration of the C-terminal region of opsin: TM4 to TM7 99
5.1 Introduction 100
5.2 TM4 exits the translocon upon chain extension 100
5.3 Opsin TM5 engages the translocon at 259 residues 104
5.4 Opsin TM5 is adjacent to a PAT-10-like molecule during its integration 106
5.5 Opsin TM1 and TM5 are adjacent to a single copy of PAT-10 110
5.7 Opsin TM7 is associated with the translocon throughout opsin biosynthesis 115
5.8 Summary 118
Chapter 6 - Probing the environment of a translocating nascent opsin chain 119
6.1 Introduction 120
6.2 AMS and QSY can modify cysteine probes in the ER lumen 121
6.3 Opsin nascent chains are modified by AMS in the absence of membranes 124
6.4 The environment of cys124 in the OP150 and OP164 integration
intermediates is altered in the presence of the ER translocon 126
6.5 Cys124 in OP150 and OP164 integration intermediate is in a hydrophobic
environment 129
6.6 The loop region C-terminal to TM3 is in a hydrophilic environment at 164
residues 131
6.7 The OP164 nascent chain represents a true integration intermediate that is
attached to the ribosome 134
6.8 Summary 136
5
Chapter 7 - Discussion 137
7.1 Introduction 138
7.2 The integration of TM1, TM2 and TM3 is a ‘co-ordinated’ process 140
7.3 An alternative analysis of TM3 environment during opsin integration 142
7.4 Opsin TM4 exits the translocon upon nascent chain extension 145
7.5 Opsin TM5 is engaged with the ER translocon throughout opsin
biosynthesis 145
7.6 Opsin TM6 and TM7 are associated with the Sec61 complex throughout
opsin biosynthesis 147
7.7 Nascent opsin chains engage a single copy of the Sec61 complex during
opsin biosynthesis 147
7.8 The possible role of PAT-10 as a TM chaperone 148
7.9 Conclusion 149
Chapter 8 - Bibliography 151
Appendices 171
Publications 176
6
List of Figures
Chapter 1
Page
Figure 1.1 A schematic representation of the secretory pathway.
18
Figure 1.2 A schematic representation of a typical cleavable ER-targeting signal sequence.
19
Figure 1.3 Topologies resulting from cleaved and uncleaved signal sequences.
20
Figure 1.4 SRP-dependent targeting of proteins destined for the ER.
25
Figure 1.5 The topology of components of the ER translocon.
27
Figure 1.6 Schematic diagram of the subunits of signal peptidase, TRAP and the oligosaccharyltransferase complex.
33
Figure 1.7 Regulation of glycoprotein folding in the ER.
36
Figure 1.8 The crystal structure of the Sec61 heterotrimer from the (a)-(b) top view and the (c) side view.
38
Figure 1.9 A schematic diagram of the putative arrangement of Sec61 complexes within the oligomer.
40
Figure 1.10 A classical model of the translocation of polytopic membrane protein into the ER membrane.
42
Figure 1.11 Schematic representation of (a) sequential integration of polytopic membrane proteins, and (b) integration of polytopic membrane proteins upon completion of translation.
45
Figure 1.12 A diagrammatic representation of bovine opsin sequence.
49
Figure 1.13 A schematic diagram of a rod photoreceptor cell.
50
Figure 1.14 Structure and chemical reaction of the homobifunctional cross-linking reagent, bismaleimidohexane (BMH).
51
Chapter 3
Figure 3.1 Rationale for HA tagging of nascent opsin chains.
70
7
Figure 3.2 Immunoprecipitation with α-HA antisera allows selection of authentic opsin chains.
72
Figure 3.3 Cross-linking products are formed with glycosylated opsin chains.
73
Figure 3.4 Formation of BMH cross-linking adducts is cysteine dependent.
76
Figure 3.5 Radiolabelled endogenous Sec61α molecules were generated during translation.
77
Figure 4.1 A diagrammatic representation of artificial opsin integration intermediates generated for site-specific cross-linking analysis of distinct TM domains.
81
Figure 4.2 BMH-mediated cross-linking of OP[cys56] integration intermediates of increasing chain length to ER components.
83
Figure 4.3 A) A schematic representation of the OPTM1PPL[cys56] polypeptide. B) BMH cross-linking of OPTM1PPL[cys56] integration intermediates of to translocon associated components.
86
Figure 4.4 A plot of the relative efficiency of cross-linking to Sec61α versus the chain length of the integration intermediate.
87
Figure 4.5 BMH cross-linking of integration intermediates with cys89.
89
Figure 4.6 BMH cross-linking of OP164 integration intermediates with cysteine probes in three different locations within TM3.
91
Figure 4.7 BMH mediated cross-linking of integration intermediates containing TM3 specific cysteine probes to ER translocon components.
93
Figure 4.8 A) A schematic representation of OPTM1-3PPL[cys115] polypeptide chain. B) BMH cross-linking of OPTM1-3PPL[cys115] integration intermediates to translocon components.
95
Figure 4.9 BMH cross-linking with OP164 and OP204 integration intermediates containing double cysteine probes.
97
Chapter 5
Figure 5.1 BMH cross-linking of OP204 integration intermediates from two different single cysteine probes within TM4.
102
8
Figure 5.2 Cross-linking of OP[cys154] integration intermediates of increasing chain lengths to translocon components.
103
Figure 5.3 BMH cross-linking of OP259 integration intermediates using different TM5 specific probes.
105
Figure 5.4 A) A schematic diagram of the OPN/5-7 polypeptide chain. B) Predicted topology of OPN/5-7 polypeptide chain. C) Cross-linking adducts are formed with glycosylated OPN/5-7 nascent chains.
107
Figure 5.5 BMH cross-linking of cys229 integration intermediates of A) normal-length opsin polypeptide chain, and B) OPN/5-7 polypeptide chain.
109
Figure 5.6 Double probe analysis of the OP304 integration intermediate.
111
Figure 5.7 BMH dependent cross-linking from single cysteine probes in opsin TM6.
113
Figure 5.8 BMH cross-linking of cys275 integration intermediates of A) normal-length opsin polypeptide chains, and B) OPN/5-7 polypeptide chains.
114
Figure 5.9 BMH mediated cross-linking from distinct cysteine probes in opsin TM7.
116
Figure 5.10 BMH cross-linking of cys287 integration intermediates of A) normal-length opsin polypeptide chain, and B) OPN/5-7 polypeptide chain.
117
Chapter 6
Figure 6.1 Structures of (a) 4-acetamido-4′-maleimidylstilbene-2-2′-disulfonic acid (AMS) and (b) QSY 9 C5-maleimide (QSY).
120
Figure 6.2 AMS and QSY modification of OP96[cys14] integration intermediates.
123
Figure 6.3 AMS modification of various OP130 to OP164 chains.
125
Figure 6.4 AMS modification of various OP130 to OP164 integration intermediates.
128
Figure 6.5 QSY modification of various OP130 to OP164 integration intermediates.
130
Figure 6.6 AMS and QSY modification of cys140 in (a) OP164 and (b) OP174 chains.
132
9
Figure 6.7 AMS and QSY modification of (a) OP164[cys140] and (b)
OP174[cys140], in the presence of semi-permeabilised mammalian cells.
133
Figure 6.8 Isolation of ribosomes and associated OP164[cys124] chains.
135
Chapter 7
Figure 7.1 A working model for the integration of opsin into the ER membrane.
139
Figure 7.2 The roles of TM1 and TM3 during opsin integration.
141
Figure 7.3 Possible models of TM3 integration into a hydrophobic environment.
143
10
List of Tables
Chapter 1
Page
Table 1.1 Yeast and mammalian homologues of the OST complex and their putative functions.
31
Chapter 2
Table 2.1 Primers used for the introduction of cysteine residues into opsin.
57
Table 2.2 PCR primers used in the generation of OPTM1-3PPL[cys115] and OPN/5-7 constructs.
59
Table 2.3 Primers used for the removal of cysteine residues from the preprolactin coding sequence of OPTM1-3PPL[cys115] construct.
59
Table 2.4 Primers used to generate truncated opsin transcription templates.
60
Table 2.5 Primers used to generate truncated OPTM1PPL[cys56] and OPTM1-3PPL[cys115] transcription templates.
61
11
Abbreviations
AAPs amino acid permeases
AMS 4-acetamido-4′-maleimidylstilbene-2-2′-disulfonic acid
BiP immunoglobulin heavy chain binding protein
BMH bismaleimidohexane
DMSO dimethylsulphoxide
ER endoplasmic reticulum
ERAD ER-associated degradation
HA haemagglutinin
OST oligosaccharyltransferase
PDI protein disulphide isomerase
PPL preprolactin
SDS sodium dodecyl sulphate
SPC signal peptidase complex
SRP signal recognition particle
TCA trichloroacetic acid
TM transmembrane
QSY QSY® 9 C5-maleimide
12
Abstract
A major step in the biosynthesis of many membrane proteins is their insertion into the
membrane of the endoplasmic reticulum (ER). The insertion of a multi-spanning
membrane protein is a complex process since several transmembrane (TM) domains
have to be correctly integrated in order to enable its correct assembly. At present it is
unclear how the integration of multiple TM domains is co-ordinated by the ER
translocon. The aim of this study was to analyse the molecular environment of the TM
domains of a model seven TM domain protein, opsin, so as to better understand the
mechanism by which integration occurs.
For this purpose, stable ‘integration intermediates’ of defined lengths representing
distinct stages of opsin biosynthesis were generated by in vitro translation of truncated
mRNA in the presence of semi-permeabilised cells. Cysteine-mediated, site-specific
cross-linking and immunoprecipitation were employed to examine the environment of
these integration intermediates. In addition, cysteine-specific modification reagents with
different physical properties were used to investigate the environment of opsin TM3
during its insertion at the ER membrane.
Opsin TM domains exhibit unique patterns of adduct formation with the ER translocon
components, Sec61α and Sec61β. TM1 associates with the Sec61 complex at two
distinct stages during nascent chain extension, and this behaviour is dependent on the
presence of subsequent TM domains. The re-association of TM1 with the translocon
may well facilitate the co-ordinated integration of TMs 1-3 into the lipid bilayer. Opsin
TM4 exits the Sec61 complex as soon as the subsequent TM domain is synthesised,
while TM5, TM6 and TM7 remain associated with the ER translocon throughout
protein synthesis, suggesting their concerted release upon chain termination. Evidence
is provided that opsin is integrated via a single Sec61 heterotrimer, despite the fact that
the ER translocon appears to consist of multiple copies of the Sec61 complex. On the
basis of this work, a model is presented describing the complete integration of opsin at
the ER membrane.
13
Declaration
No portion of the work referred to in the thesis has been submitted in support of an
application for another degree or qualification of this or any other university or other
institute of learning.
1) Copyright in text of this thesis rests with the Author. Copies (by any process)
either in full, or of extracts, may be made only in accordance with instructions
given by the Author and lodged in the John Rylands University Library of
Manchester. Details may be obtained from the Librarian. This page must form
part of any such copies made. Further copies (by any process) of copies made in
accordance with such instructions may not be made without the permission (in
writing) of the Author.
2) The ownership of any intellectual property rights which may be described in this
thesis is vested in The University of Manchester, subject to any prior agreement
to the contrary, and may not be made available for use by third parties without
the written permission of the University, which will prescribe the terms and
conditions of any such agreement.
3) Further information on the conditions under which disclosures and exploitation
may take place is available from the Head of Faculty of Life Sciences.
14
Acknowledgements
I would like to thank Samuel Crawshaw for kindly providing the OPTM1PPL[cys56]
construct and Figure 4.5.
I would like to dedicate this thesis to my parents, Ismail Ahmad and Azizah Mohamed
Amin. I am grateful to my family for the opportunities they have given me throughout
my life and would like them to know that I would not have been able to make it this far
without their enduring love, support and encouragement.
I wish to thank my fantastic supervisor, Professor Steve High, for his excellent guidance
and unwavering encouragement during my postgraduate studies in his laboratory. His
tireless optimism and enthusiasm have been both personally affirming and
professionally inspiring – I could never have wished for a better supervisor!
I would like to thank all the members of the High lab for their exceptional help, advice
and support not only in work but in other aspects of my life too. I am also grateful to the
badminton gang for making Thursdays so enjoyable.
Finally I would like to express my appreciation to all my friends here – in particular
Jifang, Bee Bee, Ray, Ay Lin, Anna, Maggie, Nafisa and Jane – for listening to all my
moans and groans! I wish to thank them for making my life in the UK so rich and
memorable and I hope we can continue our happy friendships together regardless of
future locations.
This work was funded by the Wellcome Trust.
15
List of Publications
Lecomte, F.J.L., Ismail, N. and High, S. (2003). Making membrane proteins at the
mammalian endoplasmic reticulum. Biochemical Society Transactions 31: 1248-1252.
(Joint first author)
Wilson, C.M., Kraft, C., Duggan, C., Ismail, N., Crawshaw, S.G. and High, S. (2005).
Ribophorin I associates with a subset of membrane proteins after their integration at the
Sec61 translocon. J. Biol. Chem. 280: 4195-206.
Chapter 1 - Introduction
16
CHAPTER 1 Introduction
Chapter 1 - Introduction
17
1.1 The endoplasmic reticulum
One important feature of eukaryotic cells is the presence of membrane-bound
subcellular compartments that perform various specialised functions. These
compartments are unique, having characteristic environments with different sets of
proteins. Regulating the flow of components into and out of these compartments is
crucial to the maintenance of their specific environments. Since most proteins are
synthesised in the cytosol, many must be targeted to their respective compartments
during or after their synthesis.
A major compartment in the cell is the endoplasmic reticulum (ER). The ER is
organised into a network of branching tubules and flattened sacs which are all
interconnected via a single internal space known as the ER lumen. The ER may appear
‘rough’ or ‘smooth’, depending on the presence of ribosomes on the cytosolic face of its
membrane. The ribosomes present on the rough ER identify it as a major site for protein
biosynthesis, and most of the membrane proteins found in the organelles of the
secretory pathway and the plasma membrane, together with soluble proteins destined
for secretion, are synthesised at the ER (Palade, 1975) (Fig 1.1).
Given that many proteins are synthesised at the ER, it follows that the ER is an
important site for protein folding and post-translational modification. Nascent chains
may undergo a number of covalent modifications at the ER, including the cleavage of
signal sequences, the addition of a carbohydrate group (N-linked glycosylation), the
replacement of a C-terminal transmembrane domain with a glycosylphosphoinositol
(GPI) anchor and the formation of disulphide bonds (Ellgaard & Ruddock, 2005; Mayor
& Riezman, 2004; Paetzel et al., 2002; Yan & Lennarz, 2005). In the case of N-linked
glycosylation, a carbohydrate group is added to an asparagine residue in the consensus
sequence Asn-X-Ser/Thr within a nascent chain, a process that is catalysed by the
oligosaccharyltransferase (OST) complex present in the ER membrane (Yan & Lennarz,
2005). These oligosaccharides function to facilitate protein folding and protein sorting
within the secretory pathway. The ER lumen also facilitates the formation of disulphide
bonds by providing a relatively oxidising environment and the process is catalysed by
ER-resident enzymes, including protein disulphide isomerase (PDI) and related proteins
(Ellgaard & Ruddock, 2005).
Chapter 1 - Introduction
18
Figure 1. 1 A schematic representation of the secretory pathway. As most protein synthesis occurs in the cytosol, targeting of proteins to their appropriate compartment is crucial. Secretory proteins and membrane proteins which are found in the organelles of the secretory pathway and at the plasma membrane, will normally transit through the endoplasmic reticulum first before reaching their final destination.
Other ER-resident proteins include molecular chaperones and folding factors, such as
calnexin, calreticulin, ERp57 and BiP (immunoglobulin heavy chain binding protein),
act to regulate the maturation and folding of the newly-synthesised proteins (Hebert et
al., 2005). Some of these ER chaperones also function as part of a ‘quality control’
machinery that disposes of misfolded or unassembled proteins. Misfolded or
unassembled proteins are retrotranslocated across the ER membrane back to the cytosol
where they are deglycosylated and polyubiquitinated before degradation (Ahner &
Brodsky, 2004). Once the proteins have attained their correct tertiary or quartenary
structures, they may be directed to other organelles in the secretory pathway, including
the Golgi apparatus, lysosomes, endosomes and secretory vesicles, or to the plasma
membrane via vesicular transport (Fig 1.1).
It is evident that before proteins can be sorted to other organelles in the secretory
pathway, they must first be targeted to the ER. Hence, the presence of targeting signals
in these proteins play a crucial role in ensuring that they are properly directed to the ER.
At the ER membrane, a specialised translocation machinery exists to facilitate the
Chapter 1 - Introduction
19
passage of these proteins into and across the lipid bilayer (Alder & Johnson, 2004;
Johnson & van Waes, 1999; Lecomte et al., 2003).
1.2 ER-targeting signals
Newly-synthesised proteins are sorted to their appropriate compartment in the cell by
having distinctive signals within their sequence. Generally, an ER-targeting signal
comprises of a stretch of hydrophobic amino acids next to a short region of basic
residues (Nothwehr & Gordon, 1990). For example, secretory proteins destined for the
ER have a short, positively-charged N-terminal region (n-region), a hydrophobic core
(h-region) and a polar C-terminal region (c-region) which contains the recognition site
for signal peptidase, within their signal sequence (Fig 1.2) (Emanuelsson & von Heijne,
2001; von Heijne, 1998). The presence of such an ER-targeting signal in a protein
allows components in the cytosol or the ER membrane, such as the signal recognition
particle (SRP) and the ER translocon, to recognise it as being destined for the ER (Belin
et al., 1996; Powers & Walter, 1996).
Figure 1. 2 A schematic representation of a typical cleavable ER-targeting signal sequence. The signal sequence contains a positively-charged N-terminal region (n), a hydrophobic region (h) and a C-terminal region which contains a signal peptidase complex (SPC) cleavage site (indicated with an arrow).
Even though the ER-targeting signals of different precursors share very little sequence
identity, the most consistent feature is the presence of the central hydrophobic core (von
Heijne, 1985). The diverse signal sequences do not seem to affect the targeting
efficiency of the proteins to the ER, but may result in different rates of initiation of
nascent chain translocation (Kim et al., 2002). Despite such sequence variation, ER-
targeting signals can be swapped between substrates without any obvious consequences
Chapter 1 - Introduction
20
for the function of the protein (Gierasch, 1989). Targeting usually occurs during the
translation of polypeptide chains in mammalian cells, while it may be co-translational or
post-translational in yeast cells, depending on the substrate. Once the signal sequence
has been translocated into the ER membrane, it may be cleaved or retained to function
as a transmembrane anchor for the protein (see Fig. 1.3).
Figure 1. 3 Topologies resulting from cleaved and uncleaved signal sequences. Cleavage of the signal sequence (orange) of a secretory protein would release it into the ER lumen (1) while cleavage of the signal sequence of a membrane protein would result in a type I membrane protein that is anchored via a stop-transfer sequence (brown) located after the signal sequence (2). Membrane proteins with an uncleaved signal sequence, i.e. a signal-anchor (blue), may have either a type I (3) or type II (4) orientation, depending on the properties of the hydrophobic core and the regions flanking it (see text for details). Tail-anchored proteins (5) have a type II orientation resulting from the uncleaved signal sequence (blue) near their C-termini. Polytopic membrane proteins have both signal-anchor and stop-transfer sequences, and may have either the N-terminus (6) or C-terminus (7) or both (not shown) translocated across the membrane.
Chapter 1 - Introduction
21
Cleavable ER-targeting signals are normally located at or towards the N-terminus of a
protein and are eventually oriented such that their N-terminus is in the cytosol when the
signal is inserted into the ER membrane. This orientation allows the cleavage of the
signal sequence by the signal peptidase complex (SPC) in the lumen to release the
signal peptide into the lipid bilayer. Secretory proteins tend to have cleavable signal
sequences as they have to be completely translocated across the ER membrane into the
lumen (Fig. 1.3). Some membrane proteins also have a cleavable signal sequence but
this is followed by another stretch of hydrophobic residues which functions as a ‘stop-
transfer’ sequence. This hydrophobic region prevents further translocation and anchors
the protein within the ER membrane. As cleavage of the signal sequence occurs on the
lumenal side of the ER membrane, membrane proteins with a cleavable signal sequence
will always have a so called type I topology with the N-terminus located in the lumen of
the ER (Fig. 1.3).
In the case of non-cleavable ER targeting signals, the signal sequence is referred to as a
signal-anchor sequence, and such signals are normally found in integral membrane
proteins (Meacock, 2000). A signal anchor sequence is responsible for both targeting
the protein to, and anchoring it in, the ER membrane. Single-spanning and multi-
spanning integral membrane proteins with a signal anchor sequence at their N-terminus
may adopt either a type I orientation, in which the N-terminus is in the lumen, or a type
II orientation, in which the N-terminus is in the cytosol (High & Laird, 1997; Meacock,
2000) (see also Fig. 1.3). Signal-anchor sequences may also be found at the C-terminus
of single-spanning integral membrane proteins. These proteins are known as tail-
anchored proteins, since the bulk of their polypeptide chain is located in the cytosol
(Fig. 1.3).
The orientation that a signal anchor sequence adopts during insertion into the ER
membrane is influenced by the distribution of positively charged side chains flanking
the central hydrophobic region (Goder & Spiess, 2001; Hartmann et al., 1989; Levy,
1996; van Geest & Lolkema, 2000). This phenomenon is sometimes referred to as the
‘positive-inside’ rule where the more positive flanking region of a signal sequence
remains in the cytosol while the less positive flanking region is translocated across the
membrane (Hartmann et al., 1989; von Heijne, 1989). Therefore, a net positive charge
towards the N-terminal region of a signal anchor sequence would result in the
Chapter 1 - Introduction
22
translocation of its C-terminal flanking region and vice versa. In many cases, the
topology of a membrane protein may simply be inverted by the addition of positively-
charged residues to the appropriate flanking region (Hermansson et al., 2001; Szczesna-
Skorupa & Kemper, 1988). Whilst a cleavable signal sequence adopts a loop like
topology during translocation (c.f. Fig. 1.3), a recent study suggests that this orientation
may be achieved ‘kinetically’. Hence, the N-terminus of a signal sequence was shown
to be translocated into the ER lumen before its subsequent inversion to the opposite
orientation occurs. A more positive N-terminal region results in a faster rate of
inversion to orientate the N-terminus in the cytosol (Goder & Spiess, 2003). The basis
for the positive-inside rule for both cleavable signal sequences and signal anchors is
most likely due to interactions with the Sec61 complex of the ER translocon since
mutation of conserved residues in the Sec61 complex allowed ‘less stringent’
orientation (Goder et al., 2004).
The length and the hydrophobicity of the hydrophobic region of a signal anchor
sequence can also have a role in determining the orientation of a membrane protein, and
a longer hydrophobic segment seems to promote N-terminal, rather than a C-terminal,
translocation (Abell et al., 2002; Rosch et al., 2000). The ‘more hydrophobic’ end of the
signal sequence is also more efficiently translocated across the ER membrane (Harley et
al., 1998). Amino acid residues with greater hydrophobicity seemed to promote
translocation of the N-terminal region of the signal sequence and experiments have been
performed to rank amino acids in order of their capacity to promote N-terminal
translocation (Liu & Deber, 1998; Rosch et al., 2000). In a recent study, it was shown
that TM domain insertion may also be influenced by the position of residues within the
TM helix (Hessa et al., 2005). For example, tyrosine and tryptophan residues promote
membrane insertion when they are placed away from the centre of the helix, while a
proline residue introduced at the N-terminal end of the TM domain allow more efficient
integration.
Other factors which influence the topology of membrane proteins include, folding of the
N-terminal domain, interaction with adjacent transmembrane domains and glycosylation
(Goder & Spiess, 2001; van Geest & Lolkema, 2000). Since the N-terminal region of a
signal sequence is exposed to the cytosol first, folding of this region may affect its
ability to be translocated, thus retaining it in the cytosol. The topology of some
Chapter 1 - Introduction
23
transmembrane (TM) domains is also dependent on adjacent TM domains. For example,
in the Band 3 protein, the stop-transfer sequence in the second transmembrane domain
must interact with the preceding signal anchor sequence for its integration into the
membrane. This is likely to reflect its ‘weak’ stop-transfer function, and it is capable of
being completely translocated when the loop region connecting the two TM spans is
elongated (Ota et al., 2000). Glycosylation may influence protein topology by simply
sterically trapping segments of the nascent chain in the ER lumen. In one case, when
chimeric polypeptide chains with two conflicting signal sequences were engineered with
a glycosylation site in between the signals, the nascent chains preferentially adopt a
topology in which the glycosylation site was in the ER lumen (Goder et al., 1999).
Additional trans-acting factors may also govern protein topology during translocation as
indicated by studies performed on proteins with topological heterogeneity such as the
prion protein and ductin (Dunlop et al., 1995; Hegde et al., 1998). In the case of the
prion protein, the TRAP complex (see section 1.5.3) seems to play some role in the full
translocation of the protein into the ER lumen (Fons et al., 2003).
1.3 Targeting of polypeptide chains to the ER
The ER-targeting signal of most proteins in mammalian cells is first recognised by a
ribonucleoprotein complex, consisting of one 7S RNA molecule and six polypeptides,
which is known as the signal recognition particle (SRP) (Luirink & Sinning, 2004). The
protein subunits have apparent molecular masses of 9, 14, 19, 54, 68 and 72 kDa. The
54 kDa subunit, SRP54, is the most highly conserved of the subunits (Hann et al., 1989)
and it plays a central role in the recognition of ER-targeting signals by SRP (Keenan et
al., 2001; Powers & Walter, 1996).
Three regions have been identified within the SRP54 subunit, known as the N, G and M
domains (Luirink & Sinning, 2004; Walter & Johnson, 1994). The C-terminal M
domain is rich in methionine and it interacts with both the RNA component (Römisch et
al., 1990) and the hydrophobic ER-targeting signal (High & Dobberstein, 1991). The
crystal structure of the SRP54 homologue of Thermus aquaticus, Ffh, showed that a
hydrophobic groove lined with methionine residues is present in the M domain and it
has been proposed that this could mediate the signal sequence recognition event
(Keenan et al., 1998). This region of SRP54 seems fully adapted to recognise the
Chapter 1 - Introduction
24
variable h-regions that can occur within the signal sequence of different nascent chains
(Keenan et al., 1998). In addition, an arginine-rich α-helix within a helix-turn-helix
motif present in the M domain of SRP54 is implicated in binding to SRP RNA (Keenan
et al., 1998). The central G domain of SRP54 has a role in binding and hydrolysing
GTP which is essential for SRP function (Römisch et al., 1989), while the N-terminal N
domain of SRP54 subunit is thought to increase the efficiency of signal sequence
recognition (Keenan et al., 2001; Newitt & Bernstein, 1997).
Another subunit, SRP19, is essential in the assembly of SRP, and a Saccharomyces
cerevisiae homologue of SRP19, the Sec65 protein, has also been identified (Stirling &
Hewitt, 1992). SRP19 and Sec65p are involved in the recruitment of SRP54 to SRP and
hence have an important role in maintaining SRP integrity (Hann et al., 1992; Regnacq
et al., 1998; Stirling, 1999). SRP9, together with SRP14, is involved in slowing down of
the rate of nascent chain elongation by the ribosome after SRP binding to the signal
sequence. Mutations which prevent this elongation arrest decrease efficiency of nascent
chain translocation, thus implying that it is important for the function of SRP (Mason et
al., 2000). Cryo-electron microscopy structure of mammalian SRP in complex with an
active ribosome indicated that elongation arrest is achieved by direct binding of SRP to
the elongation-factor-binding site of the ribosome (Halic et al., 2004).
SRP is able to target nascent chains to the ER due to the presence of an SRP receptor in
the ER membrane. In mammalian cells, the SRP receptor consists of two components,
SRβ and SRα (Luirink & Sinning, 2004). SRα is peripherally attached to the ER
membrane on the cytosolic face by its strong interaction with SRβ which is a single-
spanning membrane protein. Like SRP54, both subunits of the SRP receptor are
GTPases (Millman & Andrews, 1997). During SRP-dependent targeting to the ER
membrane, SRP first binds to the signal sequence as soon as it emerges from the
ribosome, forming a complex that consists of SRP, the nascent chain and the ribosome.
At this stage, the SRP54 subunit of SRP is positioned very close to the exit site of the
ribosome (Halic et al., 2004; Pool et al., 2002) and translation of the polypeptide chain
is arrested. The ribosome then promotes GTP-binding by the SRP54 subunit of SRP and
this targets the complex to the ER by allowing a high affinity interaction between SRP
and its receptor in the ER membrane (Bacher et al., 1996).
Chapter 1 - Introduction
25
Figure 1. 4 SRP-dependent targeting of proteins destined for the ER. SRP (in yellow) binds to the ER-targeting signal as soon as it emerges from the ribosome (stage 1). Binding of GTP by SRP subsequently allows a high affinity interaction of SRP with its receptor on the ER (stage 2, 3 and 4). This binding event releases the nascent chain from SRP and translocation through the membrane occurs (stage 5). GTP hydrolysis then allows SRP to be released from its receptor (stage 6) and it returns to the cytosol to bind to other nascent chains.
At the ER membrane, SRP54 in the complex binds to SRα, which then binds GTP.
Binding of SRP to its receptor repositions SRP54 away from the exit site of the
ribosome, possibly allowing the ribosome to bind to the ER translocon (Sec61 complex)
(Pool et al., 2002). The presence of the translocon subsequently induces the β subunit of
the SRP receptor to bind GTP, resulting in the release of the signal sequence from SRP
and initiating translocation of the nascent chain through the translocon (Fulga et al.,
2001) (Fig. 1.4). The release of the nascent chain from SRP only occurs in the presence
of a functional ER translocon, implying that an acceptor for the signal sequence is
necessary (Song et al., 2000). As SRP delivers an incomplete polypeptide chain to the
ER insertion site, the process of translocation is coupled to subsequent translation, thus
circumventing the problem of translocating large folded regions of polypeptide. GTP
hydrolysis by SRP54 and SRα subsequently allows the release of SRP from the SR
receptor thereby completing a ‘cycle’ of targeting.
Chapter 1 - Introduction
26
Some proteins with classical ER-targeting signals do not seem to require SRP to be
directed to the ER, and in fact, S. cerevisiae is still viable in the absence of a functional
SRP pathway, although growth is dramatically perturbed (Walter & Johnson, 1994).
SRP-independent proteins usually possess a signal sequence with lower average
hydrophobicity (Ng et al., 1996), or other ill-defined features that confer SRP-
independent targeting (Matoba & Ogrydziak, 1998). To date, such precursors have
largely been characterised in yeast and their translocation tends to be post-translational.
This pathway involves other molecules such as the cytosolic Hsp70s and Ydj1p,
lumenal Hsp70s and an additional ER membrane protein complex, the tetrameric
Sec62/63p complex (Caplan et al., 1992; Rapoport et al., 1999). Many cytosolic
chaperones are upregulated as part of the adaptive response to the lack of an SRP
pathway consistent with their role in promoting an alternative targeting route (Mutka &
Walter, 2001).
1.4 The ER translocon
The ER translocon is composed of a heterotrimeric complex known as the Sec61
complex which is evolutionarily conserved in archea, bacteria and yeast cells (Rapoport
et al., 1996). The first component of the ER translocon, Sec61p, was initially identified
in yeast from a genetic screen for secretion mutants (Deshaies & Schekman, 1987) and
is found to be in complex with two other components, Sbh1p (Panzner et al., 1995) and
Sss1p (Esnault et al., 1993; Hartmann et al., 1994). In S. cerevisiae, two homologues of
the Sec61 complex are present, each performing slightly different functions. The
Sec61p/Sbh1p/Sss1p complex mediates co-translational translocation of nascent chains
into the ER but may additionally associate with the Sec62/63p complex to mediate post-
translational translocation of protein substrates (Panzner et al., 1995). (see also section
1.5.1). The second yeast homologue of the Sec61 complex consists of Ssh1p, a non-
essential Sec61p homologue, Sbh2p, a second Sbh1p homologue, and Sss1p (Finke et
al., 1996). This complex does not associate with the Sec62/63p complex and thus, is
proposed to be exclusively involved in co-translational translocation at the ER (Finke et
al., 1996). In bacteria and archaea, the SecY and SecE proteins are homologous to
Sec61α and Sec61γ respectively. A Sec61β homologue of archaea, Secβ, also exists but
no obvious homology was observed between Sec61β and the corresponding SecG
Chapter 1 - Introduction
27
component in bacteria (Hartmann et al., 1994; Matlack et al., 1998). The bacterial
SecYEG complex and archaeal SecYEβ complex are important components of the
prokaryotic protein export machinery.
Figure 1. 5 The topology of components of the ER translocon. Sec61α and TRAM are polytopic membrane proteins with ten (confirmed) and eight (estimated) transmembrane domains respectively, while Sec61β and Sec61γ are both tail-anchored proteins. The TRAM protein is also glycosylated at an asparagine residue (shown by Y).
The major component of the mammalian Sec61 complex is the Sec61α subunit which
has 50% identity to yeast Sec61p (Görlich et al., 1992b). Sec61α is a multispanning,
non-glycosylated, integral membrane protein with ten transmembrane regions
(Wilkinson et al., 1996) (Fig. 1.5). Initial studies using photocross-linking indicated that
translocating nascent chains are in close contact with Sec61α, suggesting that it is a
component of the ER translocon (High et al., 1991; Krieg et al., 1989; Thrift et al.,
1991; Wiedmann et al., 1989). Even though Sec61α is 53 kDa in molecular mass, it was
initially detected as a 35-kDa protein when analysed by SDS-PAGE in cross-linking
studies, thus it was initially referred to as P37 and imp34 (High et al., 1991; Kellaris et
al., 1991). The Sec61α protein is essential for the translocation of both membrane and
secretory proteins across the ER membrane (Oliver et al., 1995).
Sec61α may be purified as a complex with Sec61β, the mammalian homologue of
Sbh1p, and Sec61γ, the mammalian homologue of Sss1p (Görlich & Rapoport, 1993).
Chapter 1 - Introduction
28
Sec61β is a 14-kDa tail-anchored integral membrane protein whose N-terminus is in the
cytosol (Hartmann et al., 1994) (Fig. 1.5). Although S. cerevisiae Sec61β homologues,
Sbh1p and Sbh2p, are not essential for cell viability (Esnault et al., 1993; Finke et al.,
1996), in vivo studies using Drosophila showed that mutations in Sec61β are lethal and
resulted in severe defects in embryonic development (Valcarcel et al., 1999). The role
of Sec61β is still unclear but in vitro studies show that it enhances the rate of protein
translocation across the ER membrane (Kalies et al., 1998). Sec61β could also be cross-
linked to the 25-kDa subunit of the signal peptidase complex (SPC25), implying it may
play a role in the recruitment of this enzyme complex to the ER translocon (Kalies et
al., 1998). Another putative role for Sec61β is that of ribosome-binding which may
occur alongside ribosomal interactions with other components of the Sec61 complex
(Levy et al., 2001). A functional interaction between Sec61β and the nascent chain may
also be possible since it can be cross-linked to a number of translocating nascent chains
(Knight & High, 1998; Laird & High, 1997; Meacock et al., 2002).
Like Sec61β, the other component of the Sec61 complex, Sec61γ, is also a tail-anchored
integral membrane protein with a molecular mass of around 10 kDa. A single Sec61γ
homologue, Sss1p, is present in yeast and it is clearly essential for cell viability and
protein translocation, although its role is still uncertain (Esnault et al., 1993; Esnault et
al., 1994). The overexpression of Sss1p could restore translocation in a SEC61 mutant
while its depletion resulted in the accumulation of secretory and membrane proteins that
were not post-translationally modified (Esnault et al., 1993). In C. elegans, deletion of
Sec61γ gave a lethal phenotype while its mutation resulted in defects in oogenesis
(Iwasaki et al., 1996). One significant observation is that mammalian Sec61γ can
functionally replace S. cerevisiae Sss1p, indicating functional conservation of the ER
translocation complex (Hartmann et al., 1994).
Functional reconstitution of translocon components identified an additional protein that
is necessary for the translocation of several proteins across the ER (Görlich et al.,
1992b). This component is known as TRAM (translocating chain-associating membrane
protein) and it is a 34 kDa integral membrane protein believed to have eight
transmembrane regions (Fig. 1.5). To date, TRAM appears to be restricted to higher
eukaryotes and no definitive S. cerevisiae equivalent has been discovered. TRAM is
required for the efficient in vitro translocation of most, but not all, secretory and
Chapter 1 - Introduction
29
membrane proteins (Görlich & Rapoport, 1993; Oliver et al., 1995; Voigt et al., 1996).
Cross-linking studies indicate that TRAM interacts primarily with the N-terminal region
preceding the hydrophobic core of the signal sequence and show that this association
occurs at early stages of the membrane translocation process (Mothes et al., 1994). The
requirement for TRAM was later shown to be dependent upon the structure of the
hydrophobic core and the length of the charged, N-terminal, region of the signal peptide
(Voigt et al., 1996).
1.5 Translocon-associated components
1.5.1 The Sec62/63 complex
The Sec62/63p complex of the ER membrane of S. cerevisiae comprises of Sec62p,
Sec63p, Sec71p and Sec72p. Sec62p is a 30-kDa integral membrane protein with two
transmembrane regions while Sec63p is a 73-kDa integral membrane protein with three
transmembrane regions (Deshaies et al., 1990). Both Sec62p and Sec63p are essential
for yeast cell viability, whilst Sec71p and Sec72p are non-essential proteins (Deshaies
& Schekman, 1989; Fang & Green, 1994; Feldheim & Schekman, 1994; Rothblatt et
al., 1989). Sec71p is a 31.5-kDa glycoprotein which spans the ER membrane once
(Fang & Green, 1994). Sec72p is a 23-kDa peripheral ER protein which interacts very
strongly with Sec71p on the cytosolic side of the membrane and it is known to bind the
signal sequence of protein precursors (Feldheim & Schekman, 1994).
The Sec62/63p complex associates with the trimeric Sec61p translocon to form a
complex of seven proteins that can mediate post-translational translocation when
purified and reconstituted into proteoliposomes (Panzner et al., 1995). Sec62p was
shown to bind this Sec complex via its cytosolic N- and C-terminal domains. It was also
shown to bind the last 14 residues of Sec63p via its N-terminal domain (Wittke et al.,
2000). In addition, the region immediately after the second transmembrane domain of
Sec62p may be important for signal sequence recognition (Wittke et al., 2000). Efficient
post-translational translocation does not only require the Sec62/63p complex, both
Kar2p (a lumenal Hsp70) and ATP are also necessary (Panzner et al., 1995). Although
Sec63p was initially characterised to be involved in post-translational translocation,
other studies have shown that it also has a role in the co-translational SRP-dependent
pathway (Willer et al., 2003; Young et al., 2001). Depletion of Sec63p from yeast cells
Chapter 1 - Introduction
30
prevented the translocation of both SRP-independent and SRP-dependent precursors,
indicating the dual role of Sec63p (Young et al., 2001).
Although no such post-translational pathway has been characterised for mammalian
cells, human homologues of Sec62p and Sec63p have been identified, termed as Sec62
and Sec63 respectively (Meyer et al., 2000). The mammalian proteins are ubiquitous
and are also able to associate with the Sec61 complex. Despite the abundance of the
various subunits in the ER membrane, only low concentrations of Sec61-Sec62-Sec63
complexes were found (Meyer et al., 2000). It is not known if this complex also has a
role in post-translational protein translocation, as to date, no efficient post-translational
translocation has been observed in mammals. However, mammalian Sec62p and Sec63p
have both recently been shown to be in close proximity to membrane proteins during
their insertion at the ER membrane (Abell et al., 2003). In humans, mutations in the
SEC63 gene can give rise to autosomal dominant polycystic liver disease, potentially
via a defect in the secretion of particular proteins (Davila et al., 2004).
1.5.2 Signal peptidase complex
An important post-translational modification for a nascent chain with a cleavable signal
sequence (Fig. 1.3) is the removal of its signal peptide from the polypeptide chain after
its translocation at the ER. Cleavage of the signal sequence occurs in the ER lumen and
is mediated by the signal peptidase complex (SPC) which is composed of five
membrane proteins, named after their corresponding molecular masses: 12, 18, 21,
22/23 and 25 kDa (Evans & Blobel, 1986). SPC18, SPC21 and SPC22/23 are type II
single-spanning membrane proteins with most of their C-terminal region in the lumen of
the ER (Shelness et al., 1993), while SPC12 and SPC25 have two TM domains with
both the N- and C-termini in the cytosol (Kalies & Hartmann, 1996) (Fig. 1.6).
SPC22/23 is also singly glycosylated at its C-terminus (Fig. 1.6).
Since cleavage of the signal sequence occurs in the lumen, it is not surprising that
SPC18, SPC21 and SPC22/23 appear to have a more direct role in cleavage activity
consistent with their topologies. The yeast homologue of SPC18 and SPC21, termed
Sec11p, and the homologue of SPC22/23, Spc3p, are both essential for viability and
cleavage activity in yeast cells (Bohni et al., 1988; Meyer & Hartmann, 1997). The
Chapter 1 - Introduction
31
catalytic sites of SPC reside in SPC18 and SPC21, and their cleavage activity relies on a
serine, histidine and two aspartic acid residues (VanValkenburgh et al., 1999). On the
other hand, yeast homologues of SPC12 and SPC25, known as Spc1p and Spc2p
respectively, are non-essential under normal growth conditions and do not have roles in
signal sequence cleavage (Fang et al., 1996; Mullins et al., 1996). These subunits may
have roles in modulating cleavage activity since overexpression of Spc1p can suppress
sec11 temperature-sensitive mutants while the depletion of Spc2p renders yeast cells
defective in signal sequence cleavage at a high temperature (Mullins et al., 1996).
1.5.3 Oligosaccharyltransferase (OST) complex
Yeast Mammalian Putative function(s)
Ost1p Ribophorin I Substrate binding, ER retention of complex
Ost2p DAD1 ?
Swp1p Ribophorin II ER retention of complex
Wbp1p OST48 ER retention of complex
Ost4p OST4 ?
Ost3p N33, DC2 ?
Ost6p IAP, DC2 ?
Stt3p STT3A/B Catalytic site
Ost5p ?
KCP2 ?
Table 1. 1 Yeast and mammalian homologues of the OST complex and their putative functions.
Asparagine-linked (N-linked) glycosylation occurs in the lumen of the ER and is
catalysed by a multisubunit enzyme complex known as the oligosaccharyltransferase
(OST) complex which is closely associated with the ER translocon (Chavan et al.,
2005). The OST complex catalyses the en bloc transfer of a preformed oligosaccharyl
moiety (Glc3Man9GlcNAc2) from the lipid carrier dolichyl phosphate to asparagine
within selected Asn-X-Ser/Thr consensus sequences, where X can be any residue except
for proline (Bause & Hettkamp, 1979; Yan & Lennarz, 2005). In S. cerevisiae, nine
proteins have been identified that make up the OST complex (Table 1.1). Five of these
Chapter 1 - Introduction
32
proteins, Ost1p, Ost2p, Stt3p, Wbp1p and Swp1p, are essential for cell viability, while
one protein, Ost4p, is necessary for growth at 37 °C but not at 25 °C, and the other
subunits, Ost3p, Ost5p and Ost6p, are only required for optimal glycosylation activity
(refer to Knauer & Lehle, 1999 and references therein).
Several mammalian homologues of the yeast OST subunits have been identified (Yan &
Lennarz, 2005) (Table 1.1). A recent study has additionally identified two as yet
uncharacterised proteins, DC2 and KCP2, which were copurified with the mammalian
OST complex (Shibatani et al., 2005). DC2 is a ~17 kDa protein which has a weak
homology to the C-terminal region of Ost3p and Ost6p, while KCP2 is a novel ~14 kDa
protein (Shibatani et al., 2005). Of these proteins, the most conserved subunit is STT3
with more than 50% identity between yeast and mammalian homologues (Knauer &
Lehle, 1999). Photo-cross-linking studies using translocating nascent chains showed
that STT3 is in close proximity to the glycosylation consensus site (Nilsson et al.,
2003). Since cross-linking to this consensus site was abolished in the presence of a
competitive peptide substrate, STT3 became the primary candidate for the enzymatic
activity of the OST complex. Two isoforms of STT3, STT3A and STT3B, have recently
been found in human cells. These STT3 isoforms are found in different OST complexes
and have distinct catalytic activity (Kelleher et al., 2003).
Not much is known about the functions of the other subunits, but some of these proteins
are likely to have a role in peptide binding or the retention of the OST complex at the
site where it functions in the ER. Photolabelling experiments suggest that Ost1p (yeast
homologue of ribophorin I) has a possible role in substrate binding since it can bind a
I125-labelled photoreactive peptide (Yan et al., 1999). A recent study also showed that
ribophorin I can be cross-linked to a subset of newly-synthesised substrates (Wilson et
al., 2005). Ribophorin I, ribophorin II and OST48 have specific ER localisation signals
and may function to retain the complex in the ER (Fu & Kreibich, 2000).
Chapter 1 - Introduction
33
Figure 1. 6 Schematic diagram of the subunits of signal peptidase, TRAP and the oligosaccharyltransferase complex. N-glycosylation sites are indicated with ‘Y’ while black rectangles represent TM domains or hydrophobic regions. (The diagram is not to scale)
Chapter 1 - Introduction
34
1.5.4 TRAP
Another complex that is found associated with the ER translocon is the TRAP
(translocon-associated protein) complex. TRAP consists of four different subunits,
TRAPα, TRAPβ, TRAPγ and TRAPδ (Hartmann et al., 1993) (Fig 1.6). TRAPα was
initially thought to be an ER localised ‘signal sequence receptor’ and was consequently
called SSRα (signal sequence receptor α subunit), but depletion of SSRα did not inhibit
translocation activity at the ER (Migliaccio et al., 1991) and thus, it was renamed
TRAPα (Hartmann et al., 1993). TRAPβ (previously known as SSRβ) was initially
identified in a complex with TRAPα when the detergent Nonidet P-40 was used to
solubilise canine pancreatic microsomes (Gorlich et al., 1990), but the use of digitonin
as a ‘milder’ detergent resulted in the further identification of TRAPγ and TRAPδ
(Hartmann et al., 1993).
Cross-linking studies indicate that TRAPα is adjacent to various translocating nascent
chains (Görlich et al., 1992a), but its function is not entirely clear. A recent study using
the prion protein indicated that its proper translocation into the ER requires the TRAP
complex (Fons et al., 2003). Interestingly, the dependency of substrates on TRAP seems
to be influenced by the nature of their signal sequences. Substrates with signal
sequences which display a low efficiency in initiating translocation at the translocon are
more likely to be TRAP-dependent (Fons et al., 2003). One model suggests that TRAP
may function to stabilise the nascent chain by having an indirect effect on the translocon
structure (section 1.6), thus allowing the nascent chain to have an easier access to the
lumen, while a second model suggests that TRAP stabilises nascent chains with weak
signal sequences by interacting directly with them at the translocon.
1.5.5 Glycoprotein specific ER chaperones
The majority of proteins synthesised at the ER, including opsin, are N-glycosylated.
One of the most important functions of this modification is to promote the correct
folding of polypeptides in the ER. The addition of a large polar carbohydrate group
helps to orient the local region of the polypeptide at the surface of the protein and
decreases the likelihood of protein aggregation by increasing its solubility. In the ER,
folding of glycosylated proteins is regulated by a unique chaperone system known as
Chapter 1 - Introduction
35
the calnexin-calreticulin cycle. Both calnexin and calreticulin are ER resident lectins
which interact with the glycan moiety of newly-synthesised glycoproteins, in
combination with their co-chaperone, ERp57 (Helenius & Aebi, 2004). Calnexin is a
type I single-spanning membrane protein of 65 kDa, while calreticulin is a soluble
homologue of calnexin of 46 kDa. ERp57 is a ~60 kDa thiol oxidoreductase and a
member of the protein disulphide isomerase family (Freedman et al., 1994).
When a nascent chain is first N-glycosylated, the glycan group contains three terminal
glucose residues (Fig. 1.7). Two glucose residues are successively removed from the
glycan moiety by ER glucosidases I and II to give a monoglucosylated form which is
recognised by calnexin or calreticulin (Hammond et al., 1994; Spiro et al., 1996). This
interaction is likely to be co-translational especially when the N-glycosylation site is
located towards the N-terminus of the polypeptide chain (Molinari & Helenius, 2000).
The further removal of the third glucose residue by glucosidase II prevents the
interaction of calnexin/calreticulin with the glycoprotein. If the polypeptide chain fails
to attain a native structure, it is bound by another ER lumenal enzyme, uridine
diphosphate (UDP)-glucose:glycoprotein glucosyltransferase (UGGT), which catalyses
the addition of a glucose residue back to the glycan thereby allowing
calnexin/calreticulin binding to take place again (Parodi, 2000). By recognising the
folding status of polypeptide chains, UGGT acts as a ‘folding sensor’ that forces
misfolded proteins to remain in the calnexin-calreticulin cycle.
Proteins may escape the calnexin/calreticulin cycle after the removal of a mannose
residue by another ER enzyme, α-(1,2)-mannosidase I, but its action is slow, allowing
time for the glycoproteins to undergo several rounds of the calnexin/calreticulin
mediated folding cycle (Wang & Hebert, 2003). At this stage, if the protein is properly
folded, it will be transported to the Golgi apparatus, but if it is incorrectly folded, it will
be reglucosylated by UGGT and recognised by calnexin. The misfolded protein is then
transferred to another lectin, known as EDEM (ER degradation-enhancing α-
mannosidase-like protein) which targets it to the ER-associated degradation pathway
(ERAD), where retrotranslocation of the protein back to the cytosol and degradation by
the proteasome occur (Molinari et al., 2003; Oda et al., 2003).
Chapter 1 - Introduction
36
Figure 1. 7 Regulation of glycoprotein folding in the ER. Glucosidase I and II cleave two glucose residues from the oligosaccharide group, allowing calnexin or calreticulin to bind the monoglucosylated glycan group on the nascent chain. The last glucose residue is subsequently cleaved off by glucosidase II, followed by the removal of a mannose residue by α-(1,2)-mannosidase I. Properly folded glycoproteins are allowed to proceed to the Golgi, while a glucose residue is re-added onto the glycan group of polypeptide chains with non-native conformations by the action of UDP-glucose:glycoprotein glucosyltransferase (UGGT). The re-addition of a glucose residue after the action of mannosidase I results in calnexin/calreticulin binding of the nascent chain and subsequent transfer of the nascent chain to EDEM. The nascent chain is then degraded via ER-associated degradation (ERAD).
Chapter 1 - Introduction
37
1.6 Structure and composition of the ER translocation site
Early structures of the ER translocon were obtained by cryo-electron microscopy and
image reconstruction techniques using purified, detergent solubilised mammalian and
yeast Sec61 complexes. These studies indicated that the Sec61 complex exists as a
cylindrical oligomeric structure consisting of three to four Sec61 heterotrimers (i.e.
Sec61α, Sec61β and Sec61γ) (Hanein et al., 1996). This ring structure has an overall
diameter of ~85 Å, a height of ~50-60 Å and an internal diameter of ~20 Å. The number
of the oligomeric particles increases when components such as the ribosome or the
Sec62/63 complex, are present, suggesting that these factors may induce the
oligomerisation of the Sec61 complexes (Hanein et al., 1996). This type of regulated
assembly is also consistent with the observation that Sec61β can only be cross-linked to
Sec61α in the presence of ribosomes (Kalies et al., 1998). The shape of the ring
structure may also be influenced by the presence of TRAP. Hence, reconstitution of
proteoliposomes containing Sec61 complexes after TRAP depletion resulted in a
structure similar to that obtained using purified Sec61 but distinct from that of Sec61
complex obtained from native membranes (Menetret et al., 2005). This implies that
TRAP may contribute to the ER translocon.
It is well-known that the ribosome has an intrinsic affinity for the translocon and it can
bind directly to the Sec61 complex (Lauring et al., 1995; Prinz et al., 2000b). In fact,
the Sec61 complex displays the properties of a major ribosomal receptor and is
protected from protease digestion in the presence of ribosomes (Kalies et al., 1994).
Limited protease digestion of the Sec61α subunit also suggests that it plays a key role in
ribosome binding (Raden et al., 2000) and such a role is entirely consistent with EM-
derived structural data (Beckmann et al., 2001). The interaction between the ribosome
and the Sec61 complex is mediated by the 28S rRNA of the large ribosomal subunit in
eukaryotes (Prinz et al., 2000a). The interaction between rRNA and the Sec61 complex
seems to be conserved since eukaryotic ribosomes can bind the prokaryotic SecYEG
complex and vice versa (Prinz et al., 2000a). The C-terminal tail or the cytoplasmic
loop 8 of Sec61α seems to be important for ribosomal interaction as proteolytic
digestion of these regions abolishes binding activity (Raden et al., 2000).
Chapter 1 - Introduction
38
It was initially thought that a central cavity in the oligomeric ring structure that was
apparent in the structure deduced by cryo-EM formed the pore of the ER translocon (c.f.
Fig. 1.9). However, a recent high resolution crystal structure of the archaeal homologue
of the Sec61 complex implies a different scenario. This 3.2 Å resolution structure
suggests that one copy of the Sec61 heterotrimer is sufficient to form the active
translocation pore (Van den Berg et al., 2004) (Fig 1.8). In this model, the multi-
spanning Sec61α subunit is arranged in two halves, consisting of TM1-5 and TM6-10,
forming a clamp-shaped structure linked together by the loop between TM5 and TM6
with a central hourglass-shaped pore of ~5-8 Å (Fig. 1.8). The view that the
translocation channel is formed by one Sec61 heterotrimer is supported by the evidence
that translocating nascent chains formed the strongest adducts with residues located in
the putative pore of the comparable SecYEG complex (Cannon et al., 2005). Sec61β
makes limited contact with Sec61α and is found near the TM1-5 half, while Sec61γ
contacts TM1, TM5, TM6 and TM10 of Sec61α and clamps the two halves together. In
addition, a lateral opening between TM2-3 and TM7-8 of the Sec61α subunit is
postulated to allow the lateral exit of a signal sequence or a TM domain of a
translocating nascent chain into the phospholipid bilayer of the ER membrane (Van den
Berg et al., 2004).
Figure 1. 8 The crystal structure of the Sec61 heterotrimer from the (a)-(b) top view and the (c) side view. (a) Sec61α, Sec61β and Sec61γ subunits are represented by multi-coloured, pink and magenta strands respectively. (b) TM2a of Sec61α which acts as a plug in the pore is shown in green while all the other strands of Sec61α, β and γ are represented in white. (c) The plug (TM2a) is indicated in green and its movement during gating of the pore is shown with an arrow. The sidechains of hydrophobic residues composing the pore ring are shown in yellow. (Adapted from van den Berg et al., 2004)
Chapter 1 - Introduction
39
Van den Berg et al. (2004) suggest that Sec61 complexes are arranged into an
oligomeric assembly in a back to back fashion on the basis of a 2D crystal structure of
the bacterial SecYEG translocase (Breyton et al., 2002) (see Fig. 1.9). This arrangement
is supported by several cross-linking studies with bacterial SecYEG complexes. In one
study, the N- and C-termini of two molecules of SecY (bacterial Sec61α homologue) in
a functional complex could be cross-linked together (van der Sluis et al., 2002), while in
a separate study, cross-linking experiments indicated that two SecE subunits (the
presumptive bacterial Sec61γ equivalent) are in close proximity to one another
(Kaufmann et al., 1999). This model implies that the ‘central cavity’ observed in the
ring structure seen in the low resolution EM images is simply an indentation that is
filled with lipids, rather than a water-filled channel for translocation (Dobberstein &
Sinning, 2004).
Although the crystal structure indicated the size of the translocation pore is ~5-8 Å at its
narrowest point (Van den Berg et al., 2004), other studies using fluorescent probes
suggest that the size of the pore may be larger. Fluorescence quenching agents of
different sizes were employed to establish access to fluorescent probes that were
introduced into nascent chains within the pore (Hamman et al., 1997). By determining
the largest reagent that could quench the probes, the diameter of the pore was found to
be ∼40 to 60 Å. The discrepancy in pore size may simply be a reflection of the
functional states of the translocon. The inactive translocon, as represented by the crystal
structure, may have a smaller pore which is capable of expansion in response to the
presence of a translocating nascent chain. The lifetimes of the fluorescent probes
incorporated into the signal sequence of a translocating nascent chain also indicated that
the nascent chain is in an aqueous environment during its transport across the ER
membrane (Crowley et al., 1993; Crowley et al., 1994). If the translocon pore truly has
an aqueous environment, then such an aqueous channel would allow high conductivity
when examined by electrophysiological techniques. This was indeed the case and it was
found that such a conductance was observed when empty ribosomes were bound to the
ER translocon, but the conductance was lost when the ribosomes were removed
allowing the channel to close (Simon & Blobel, 1991).
Chapter 1 - Introduction
40
Figure 1. 9 A schematic diagram of the putative arrangement of Sec61 complexes within the oligomer. Individual Sec61 heterotrimers associate in a back to back manner such that the lateral opening of each Sec61 complex is facing the exterior of the structure (Adapted from Dobberstein and Sinning, 2004).
Evidence for the regulation of the translocon in order to maintain the permeability
barrier of the ER membrane suggests that, rather than being an inert structure, the
translocon is highly dynamic. Gating of the channel occurs on both cytosolic and
lumenal sides of the ER membrane. After targeting, signal sequence recognition by the
translocon occurs, leading to the formation of a seal at the ribosome-membrane junction
(Belin et al., 1996; Jungnickel & Rapoport, 1995). Some evidence suggests that the seal
may not be continuous. A three-dimensional analysis of purified Sec61 complex in
detergent in the presence of non-translating ribosomes indicated that only a single
connection exists between the complex and the ribosome (Beckmann et al., 1997). More
recently however, it was reported that there were seven connections between Sec61
complex and the ribosome with a lateral opening into the cytosol (Menetret et al.,
2005). It was speculated that these openings may allow some regions of the nascent
chain to escape into the cytoplasm during translocation. It was also proposed that the
gap between the Sec61 complex and the ribosome allows the exit of a nascent chain in
the event that the ribosome is translating a cytosolic protein (Menetret et al., 2000). It
could also be that the gap is filled with the flexible regions of the translocon
components that could not be perceived in the low resolution EM structures.
As translation continues, the pore opens to the lumen only after the length of the nascent
chain reaches about 70 residues (Crowley et al., 1994; Liao et al., 1997). During the
translocation of membrane proteins, the lumenal gate closes when the transmembrane
Chapter 1 - Introduction
41
segment begins to be synthesized by the ribosome. The folding of the TM segment
within the ribosome may act as a signal to allow ribosome-induced alterations in the
conformation of the translocon (Woolhead et al., 2004). Earlier studies suggest that BiP,
a lumenal Hsp70, has a role in the closure of the lumenal end of the translocon channel
in an ATP-dependent manner (Haigh & Johnson, 2002). Pore closure requires BiP to be
ADP-bound while opening of the pore occurs only after BiP binds ATP (Alder et al.,
2005). The crystal structure of the Sec61 complex also indicates that a short TM helix
denoted TM2a may act as a ‘plug’ to block the diffusion of ions across the translocon
pore (Van den Berg et al., 2004) (Fig. 1.8(b) and (c)). In addition, a ring of hydrophobic
residues is present at the narrowest point of the translocon channel and these may form
a seal around the translocating nascent chain, thus maintaining ER membrane barrier
during substrate translocation (Van den Berg et al., 2004). Despite the need for further
studies to resolve the relative contributions of BiP and the Sec61 subunits to gating, it is
clear that the alternate opening and closing of the cytosolic and lumenal gates seem to
ensure that the permeability barrier of the ER membrane is maintained during
translocation of nascent chains.
1.7 The biosynthesis of polytopic membrane proteins at the ER
1.7.1 Targeting and insertion of polytopic membrane proteins at the translocon
The fundamental principles that underlie the biosynthesis of polytopic membrane
proteins at the ER are the same as those already described for secretory proteins and
simple membrane proteins. Hence, their targeting is SRP dependent (Friedlander &
Blobel, 1985; Wessels & Spiess, 1988) and their insertion into the ER membrane
requires the Sec61 complex (Görlich & Rapoport, 1993; High et al., 1991; Meacock et
al., 2002; Oliver et al., 1995). The biosynthesis of multi-spanning membrane proteins at
the ER is less well characterised than that of the single-spanning membrane proteins.
An early model proposed that the ribosome cycles between membrane bound and
unbound states during the biosynthesis of proteins with multiple transmembrane
domains (Blobel, 1980). In this model, the ribosome is bound to the translocon of the
ER membrane when it is synthesising domains destined for the lumen, such as regions
just after an appropriate signal sequence. The ribosome then detaches from the ER
membrane when it is translating a cytosolic segment so that this segment is synthesized
directly into the cytosol (see also Hegde & Lingappa, 1996). The next TM domain
Chapter 1 - Introduction
42
would then direct the ribosome back to the translocon acting as an internal signal
sequence (Fig. 1.10).
Figure 1. 10 A classical model of the translocation of polytopic membrane protein into the ER membrane. The nascent chain is first targeted to the ER membrane via an SRP-dependent pathway (1). The ribosome binds to the translocon and translocation of the nascent chain is initiated (2). During the translation of a cytosolic domain, the ribosome detaches from the translocon such that it is synthesized directly into the cytosol (3). Synthesis of the subsequent transmembrane region directs the ribosome back to the translocon where it binds again (4). The cycle continues until the polypeptide chain is fully synthesized (5 and 6).
Experimental evidence indicates that such a model cannot account for the biosynthesis
of all polytopic membrane proteins. Cross-linking studies showed that the cytosolic
region of a nascent chain can still be cross-linked to the translocon components in the
ER membrane. Furthermore, the ribosome was not released from the membrane even
after the cytosolic domain between the transmembrane segment of the nascent chain and
the ribosome was severed by protease treatment. The region of the nascent chain that
was still attached to the ribosome after cleavage could still be cross-linked to the
translocon (Mothes et al., 1997).
One implication of the model shown in Figure 1.10 is that the orientation of the
transmembrane segments of a protein is defined by the first transmembrane sequence.
As an example, the first transmembrane segment might act as the signal sequence to
allow insertion while the second transmembrane segment acts as a ‘stop-transfer’
sequence to prevent further translocation of the nascent chain. The third transmembrane
Chapter 1 - Introduction
43
region again initiates insertion while the fourth region stops translocation. Thus, the
transmembrane segments function as alternating signal and stop-transfer sequences to
eventually direct the full insertion of the polytopic polypeptide chain (Wessels &
Spiess, 1988).
In the context of this model, it is unclear if the reinitiation of subsequent signal
sequences requires SRP. By definition, the presence of a signal sequence in a
transmembrane segment would imply that SRP is needed. However, in practice, it was
shown that a short cytoplasmic segment between two transmembrane regions allowed
‘SRP-independent’ re-insertion while longer cytoplasmic regions resulted in the SRP-
dependent re-insertion of subsequent transmembrane regions (Kuroiwa et al., 1996). In
fact, not all transmembrane domains from a polytopic protein can function as a signal or
stop-transfer sequence when analysed separately (Audigier et al., 1987; Moss et al.,
1998). In some cases, it has also been found that the first transmembrane domain of a
polytopic protein does not necessarily dictate the orientation of subsequent
transmembrane domains (Sato et al., 1998). Manipulating the charge difference between
regions flanking the first TM of Glut1 glucose transporter in an attempt to invert the
topology of the entire protein resulted only in a local inversion; the topology of the
downstream TM domains of Glut1 was not affected.
A more likely scenario is that, instead of ribosomal detachment during synthesis of the
cytosolic domains of membrane proteins, the ribosome-membrane junction ‘opens’ or
‘breathes’ during translocational pausing before the next transmembrane domain is
reinitiated for insertion. Such a mechanism would allow the exit of the cytoplasmic
regions of the nascent chain into the cytosol (Beckmann et al., 2001; Hegde &
Lingappa, 1996; Menetret et al., 2000). This junction may then close during the
translocation of the next transmembrane domain. The alternate opening and closing of
the cytosolic and lumenal gates of the translocon during nascent chain translocation
maintain the permeability barrier of the ER membrane (as discussed in section 1.6).
Chapter 1 - Introduction
44
1.7.2 Lateral exit of TM domains into the ER membrane
Not only is the translocon dynamic in regulating the cytosolic and lumenal gates, it must
also allow the transmembrane regions of membrane proteins to exit laterally into the
lipid bilayer. Hydrophobic regions of the stop-transfer or signal-anchor sequences of
nascent membrane proteins could be cross-linked to lipids, implying that the ER
translocon must provide access to the lipid bilayer (Heinrich & Rapoport, 2003).
Alternatively, this observation could mean that the hydrophobic transmembrane
domains can simply ‘partition’ into the lipid bilayer thereby exiting the channel
completely. It has been suggested that a role for the Sec61 complex may be to overcome
any barrier posed by the charged head groups of the phospholipids during such
‘partitioning’ of the hydrophobic transmembrane domains into the lipid bilayer
(Heinrich et al., 2000).
On the other hand, photo-crosslinking experiments have revealed that in at least one
case, a transmembrane domain of a single-spanning membrane protein goes through
three different proteinaceous environments as it exits the aqueous pore and enters the
lipid phase. Thus, the integration of a transmembrane region is not necessarily a one-
step process and it may involve several protein mediated and potentially regulated steps
(Do et al., 1996). Although this observation was seen with a single-spanning membrane
protein, the integration of TM domains of a polytopic membrane protein is likely to
occur in a similar manner. In fact, the analyses of model polytopic proteins indicate that
some TM domains remain adjacent to Sec61 components and/or TRAM for a long
period during nascent chain synthesis, even though the polypeptide chain tether from
the ribosome is long enough to allow TM diffusion out of the translocon (Meacock et
al., 2002; Sauri et al., 2005). The prolonged retention of the TM domains within the
translocon environment suggests an active regulation of TM exit by the Sec61 complex.
In addition, specific interactions between the nascent chain and the translocon are
implied by the observation that TM domains of several substrates are positioned in a
non-random manner within the translocon with respect to Sec61α and TRAM
(McCormick et al., 2003).
Chapter 1 - Introduction
45
1.7.3 Models of polytopic membrane protein integration into the ER membrane
The unique feature of a polytopic protein is that several segments of the polypeptide
must at some point laterally exit the translocon, enter the lipid bilayer, and assemble to
form a functional protein. The observation that a transmembrane domain could be cross-
linked to lipids during early stages of protein biogenesis indicated that it had lateral
access to the membrane before the entire polypeptide chain had been synthesized
(Heinrich & Rapoport, 2003). The resistance of incomplete nascent chains to alkali and
urea extraction suggested that such transmembrane regions were already integrated into
the lipid bilayer before translation was terminated (Mothes et al., 1997). These data
support the view that the transmembrane segments of a polytopic protein can exit the
translocon sequentially before the entire protein has been synthesized (Fig. 1.11).
Figure 1. 11 Schematic representation of (a) sequential integration of polytopic membrane proteins, and (b) integration of polytopic membrane proteins upon completion of translation. In model (a), the transmembrane domains may exit one by one into the lipid bilayer even before translation of the full polypeptide chain has been completed. On the other hand, in model (b), integration of all the transmembrane domains occurs only after the entire polypeptide has been synthesised by the ribosome.
Chapter 1 - Introduction
46
Whilst this may be true for the transmembrane domains of some membrane proteins,
other transmembrane domains appear to remain in close proximity to each other within
the environment of the translocon until translation terminates. In fact, a nascent chain
with up to five transmembrane regions could be extracted from the membrane by urea
treatment in the presence of moderate salt concentrations (Borel & Simon, 1996). This
suggests that the transmembrane regions were not integrated into the lipid bilayer, but
rather that they were stabilised by salt-sensitive, electrostatic bonds within an aqueous
pore (refer to model in Fig. 1.11(b)). It is thus possible that the translocon is able to
release transmembrane segments either sequentially, or en masse, perhaps depending on
the specific properties of the nascent chain being synthesised (High & Laird, 1997) (c.f.
Fig. 1.11(a) and (b)).
1.8 Membrane chaperones and polytopic proteins
Several ER chaperones are known to interact with secretory proteins and the lumenal
regions of membrane proteins, for example BiP and calnexin (Helenius & Aebi, 2004).
These chaperones are involved in the folding and assembly of the nascent polypeptides.
However, it is less clear whether molecular chaperones also interact specifically with
the hydrophobic domains of integral membrane proteins during their biosynthesis. To
date, a generic molecular chaperone which binds the transmembrane domains of nascent
membrane proteins has not been identified. It is possible that the exit of the
transmembrane domains of a polytopic membrane protein from the translocon is
regulated by such chaperones, and an association of this type may even define whether
their exit occurs in a sequential or en masse fashion. It could be that the Sec61 complex
and/or TRAM provide a ‘chaperone’-like function, regulating the movement of
transmembrane domains during protein biosynthesis (Do et al., 1996).
Despite the lack of evidence for a generic chaperone of transmembrane domains,
several chaperones specific for certain membrane proteins or membrane protein families
are known. One example is Shr3p, an integral membrane protein that has been shown to
be necessary for the proper folding of amino acid permeases (AAPs) which have 12 TM
domains. The insertion of AAPs into the ER membrane does not require Shr3p
(Gilstring et al., 1999), but deletion of Shr3p resulted in the aggregation of AAPs in the
Chapter 1 - Introduction
47
ER (Kota & Ljungdahl, 2005), implying that Shr3p behaves as a chaperone in the
folding of AAPs. The TM domains of Shr3p are likely to be important for binding with
AAPs since a point mutation in its first TM domain abolishes this interaction (Gilstring
et al., 1999).
Other chaperones are also known to be involved in the assembly or transport of
membrane proteins rather than their integration into the ER membrane. One example is
the Vma12p and Vma22p in S. cerevisiae which form a membrane-associated complex
in the ER (Graham et al., 1998). The Vma12p/Vma22p complex interacts transiently
with a 100-kDa integral membrane protein, Vph1p, and allows it to assemble into the
Vo subcomplex of the vacuolar ATPase in the ER membrane. The Vo subcomplex later
interacts with other components to form the V-ATPase complex in vacuoles. Another
integral membrane protein which functions as a chaperone is DRiP78. DRiP78 binds to
the dopamine D1 receptor at a C-terminal hydrophobic motif, FxxxFxxxF, which is
conserved among several G protein-coupled receptors (GPCRs) (Bermak et al., 2001).
DRiP78 seems to regulate the transport of the D1 receptor away from the ER and the
export of the D1 receptor is sensitive to the levels of DRiP78 expression.
In the absence of functional Vma12p/Vma22p complex, Shr3p, and DRiP78, their
polytopic membrane protein substrates are prevented from exiting the ER, thus, these
factors play important roles in regulating protein assembly. The ‘quality control’
function of the ER ensures that only proteins which are folded and assembled correctly
may leave the ER to later compartments in the secretory pathway. Incorrectly folded
and unassembled proteins will ultimately be degraded via the ERAD pathway (Ahner &
Brodsky, 2004).
1.9 This study
1.9.1 Opsin as a model polytopic membrane protein
Opsin is a member of the guanidine-nucleotide binding protein (G-protein)-coupled
receptor (GPCR) family (Khorana, 1992) which consists of a diverse range of proteins
whose main function is to transduce an intracellular signal from an external stimuli.
Opsin has the typical topology of a GPCR; that is seven transmembrane domains with
an extracellular N-terminus and an intracellular C-terminus (Fig. 1.12). In bovine opsin,
Chapter 1 - Introduction
48
several of its 348 amino acids undergo covalent modification during its biosynthesis.
Two asparagine residues near the N-terminus, Asn-2 and Asn-15, are glycosylated
(Hargrave, 1977), while two cysteines near the C-terminus, Cys-322 and Cys-323, are
palmitoylated (Ovchinnikov Yu et al., 1988). A disulphide bridge is also present
between cysteines 110 and 187 (Karnik & Khorana, 1990). Additionally, the functional
form of opsin, rhodopsin, is only generated when the chromophore 11-cis-retinal (a
derivative of vitamin A) is linked to lysine 296 of opsin (Hargrave et al., 1983).
Rhodopsin is one of the best characterised GPCRs and is currently the only GPCR with
a high resolution crystal structure, solved to 2.8 Å (Palczewski et al., 2000).
Rhodopsin is found in the plasma membrane and disc membranes of rod photoreceptor
cells (Fig. 1.13) in the retina of the eye and is responsible for peripheral and dim light
vision. A large number of mutations, most being point mutations, in the rhodopsin gene
result in an autosomal dominant disease known as retinitis pigmentosa, characterised by
the patients having tunnel vision and night-blindness. Approximately 100 mutations in
the rhodopsin gene have been identified to cause this disease and most of these
mutations affect the folding or trafficking of opsin (Kennan et al., 2005). Studies using
rhodopsin mutants expressed in cultured mammalian cells have allowed these mutants
to be categorised into two broad classes based on the intracellular fates of these mutant
rhodopsin molecules (Sung et al., 1991; Sung et al., 1993). Class I mutants are
trafficked to the plasma membrane and are functional when reconstituted with 11-cis-
retinal (Sung et al., 1991), but these mutants are defective in transport to or retention in
the rod outer segment (Sung et al., 1994). These class I mutations tend to be clustered in
the first TM domain or at the C-terminus of opsin (Deretic et al., 1996; Sung et al.,
1993). Most of the rhodopsin mutations belong to class II in which the folding or
stability of the mutant proteins are affected. As a consequence, these mutants tend to
accumulate in the ER and fail to associate with 11-cis-retinal (Sung et al., 1991; Sung et
al., 1993). Unlike class I mutations, class II mutations are generally found in TM and
extracellular domains of opsin.
During the biosynthesis of opsin at the ER membrane, the first TM domain functions as
an uncleaved signal anchor sequence which directs targeting and translocation of the N-
terminus into the ER lumen. SRP is required to direct nascent opsin chains to the ER
membrane (Friedlander & Blobel, 1985), and it was shown that some of the opsin TM
Chapter 1 - Introduction
49
domains can behave as independent signal sequences and stop transfer sequences
(Audigier et al., 1987; Friedlander & Blobel, 1985). In Drosophila, the biosynthesis of a
subclass of rhodopsin isoforms involves a peptidyl-prolyl cis-trans isomerase, NinaA,
which is necessary for the proper folding and/or export of rhodopsin from the ER
(Baker et al., 1994; Colley et al., 1991; Shieh et al., 1989). NinaA forms a stable
complex with rhodopsin in vivo and its C-terminal tail is likely to be involved in this
interaction (Baker et al., 1994). Mutations in the ninaA gene result in the accumulation
of opsin at the ER and prevent opsin export to the plasma membrane (Colley et al.,
1991).
Figure 1. 12 A diagrammatic representation of bovine opsin sequence. Asparagine-linked glycan groups at residues 2 and 15 are indicated with ‘Y’, while the disulphide bond between cysteines 110 and 87 is shown as a blue dotted line. Palmitoyl groups linked to cysteines 322 and 323 are shown in magenta.
Chapter 1 - Introduction
50
Figure 1. 13 A schematic diagram of a rod photoreceptor cell. The regions of the rod photoreceptor cell are marked as indicated.
1.9.2 Use of site-specific cross-linking in the analysis of opsin integration
In this study, site-specific, thiol-mediated cross-linking was utilised to examine the
proteinaceous environment of specific regions of opsin. This approach has been
extensively used to examine the translocation of polypeptide chains at the ER
membrane (Abell et al., 2003; Laird & High, 1997; Meacock et al., 2002), most often
using nascent chains which are still attached to the ribosome to provide a ‘snapshot’ of
the insertion process (Martoglio & Dobberstein, 1996). Translation of the opsin
polypeptide chain was halted at defined chain lengths by the truncation of the encoding
mRNA causing the resulting nascent chain to remain attached to the ribosome as a
peptidyl-tRNA species. When such truncated mRNAs are translated in the presence of
ER membranes, the resulting ribosome-nascent chain complexes are targeted to the ER
and become trapped in the translocon as stable ‘integration intermediate’ which are
believed to represent a particular stage of opsin biosynthesis (Gilmore et al., 1991).
When a suitable cross-linking reagent is added at this stage, the integration
intermediates can be covalently attached to adjacent ER components and subsequently
analysed by immunoprecipitation and SDS-PAGE.
The cross-linking reactions used during this study rely exclusively on the use of a
homobifunctional cross-linking reagent, bismaleimidohexane (BMH), which has two
maleimide groups that can react with two free sulphydryl groups of cysteine side chains
(Fig 1.14). BMH is able to diffuse into the lipid bilayer of the ER membrane, thus
Chapter 1 - Introduction
51
allowing cross-linking of nascent chains with adjacent proteins even when the reactive
groups are located within a hydrophobic environment. BMH has a flexible spacer arm
of 16 Å, and thus can only react with available sulphydryl groups that are within a
limited proximity of each other. In order to confer site-specificity to the cross-linking
reaction, it is important that the opsin integration intermediate contains only one
cysteine residue which acts as the sole target for the reaction. In this way, any cross-
linking products formed can be attributed to a particular region of the nascent chain.
Cross-linking partners of each integration intermediate were identified by
immunoprecipitation with antisera specific for components of the ER translocon. By
performing site-specific cross-linking with opsin integration intermediates of increasing
nascent chain lengths, the proteinaceous environment of opsin could be determined at
each stage of opsin biosynthesis, allowing a model of opsin integration to be
constructed.
Figure 1. 14 Structure and chemical reaction of the homobifunctional cross-linking reagent, bismaleimidohexane (BMH).
Chapter 1 - Introduction
52
1.9.3 Overview
The aim of this study is to examine the molecular environment of specific regions of
opsin, in particular, its TM domains, during its biosynthesis at the ER. This information
will be used to try to understand the mechanism by which polytopic membrane proteins
are integrated into the phospholipid bilayer of the ER membrane. Opsin is a suitable
model protein because of the availability of a high resolution crystal structure for the
wild type molecule and a good monoclonal antibody specific to its N-terminus. The
molecular environment of all seven TM domains of opsin was characterised during the
course of this study, with specific focus on opsin TM3 to TM7 which had not been
studied before in any detail (Meacock et al., 2002). The influence of later TM domains
on the lateral exit of TM1 and TM3 at the ER translocon was investigated, and the
environment of TM3 during its movement from the ribosomal exit tunnel to the ER
translocon was also probed. These data were used to build a model describing the
molecular mechanisms that underlie opsin integration.
Chapter 2 – Materials & Methods
53
CHAPTER 2 Materials and Methods
Chapter 2 – Materials & Methods
54
2.1 Materials
The plasmid encoding a cysteine-null version of the opsin coding region in the
pGEM3Z vector was previously constructed by Suzanna Meacock (Meacock, 1999).
The cross-linking reagent, bismaleimidohexane (BMH), was purchased from Perbio
Science (Northumberland, UK). The sulphydryl specific modification reagents, 4-
acetamido-4’-maleimidylstilbene-2-2’-disulfonic acid (AMS) and QSY® 9 C5-
maleimide (QSY), were purchased from Molecular Probes (Leiden, The Netherlands).
Restriction ezymes and endoglycosidase H were obtained from New England BioLabs
(Hitchin, UK). The QuikChange mutagenesis kit and the competent XL1-Blue E. coli
cells were from Stratagene (Cambridge, UK). The QIAprep Spin Miniprep kit, the
QIAquick PCR purification kit and the RNeasy mini kit were obtained from QIAGEN
(Crawley, UK). The BigDye terminator cycle sequencing ready reaction was purchased
from Applied Biosystems (Warrington, UK).
T7 RNA polymerase, SP6 RNA polymerase, transcription buffers, rNTPs, RNasin
ribonuclease inhibitor, amino acids, RNase A and nuclease-treated rabbit reticulose
lysate were obtained from Promega (Herts, UK). The cap analogue m7G(5’)ppp(5’)G
was obtained from New England BioLabs (Hitchin, UK). The aurintricarboxylic acid
(ATCA), cycloheximide and phenylmethylsulfonyl fluoride (PMSF) were purchased
from Sigma (Gillingham, UK). Easytag L-[35S]-methionine was from NEN Du Pont
(Stevenage, UK). All reagents for cell culture were obtained from Invitrogen (Paisley,
UK). Digitonin was purchased from Merck Biosciences (Nottingham, UK). All other
chemicals were analytical grade or better and were obtained from BDH/Merck (Poole,
UK) and Sigma (Gillingham, UK).
The mouse monoclonal anti-haemagglutinin (HA) antibody was a gift from Dr. I.
Hagan, Paterson Laboratories, Manchester, and rabbit polyclonal antibodies specific for
the Sec61α and Sec61β subunits were kindly provided by Prof. R. Zimmermann,
University of the Saarland, Germany. The mouse monoclonal antibody specific for the
N-terminal region of opsin was purified from a hybridoma line originally supplied by
Dr. P. Hargrave, University of Florida, USA (Adamus et al., 1991).
Chapter 2 – Materials & Methods
55
2.2 Site-directed mutagenesis
The plasmid containing a cysteine-null version of the opsin-coding region in the
pGEM3Z vector has been previously described (Meacock et al., 2002). This construct
was the starting point for the introduction of single cysteine codons by site-directed
mutagenesis. Unique cysteine residues were introduced at positions 107, 115, 124, 132,
140, 154, 165, 204, 217, 229, 254, 275, 287 and 308 of the resulting opsin chains. The
mutagenesis was performed using the PCR-based QuikChange mutagenesis kit
(Stratagene). A set of complementary primers was designed to incorporate a cysteine at
a designated amino acid position for each opsin mutant (Table 2.1). For the PCR
reaction, the following solutions were mixed: 5 µl of 10x reaction mixture (100 mM
KCl, 100 mM (NH4)2SO4, 200 mM Tris-HCl pH 8.8, 20 mM MgSO4, 1 % Triton X-
100, 1 mg/ml nuclease-free bovine serum albumin), ∼ 200 ng of DNA template, 125 ng
of forward and reverse primers, 1 µl of 2.5 mM dNTP mix, 2.5 U of Pfu Turbo DNA
polymerase and water to a final volume of 50 µl. The PCR conditions were: initial
denaturation (95 °C, 30 seconds) for 1 cycle, then denaturation (95 °C, 30 seconds),
annealing (55 °C, 1 minute) and extension (68 °C, 10 minutes) for 16 cycles.
After the PCR reaction, the mixture was incubated at 37 °C with the restriction enzyme
DpnI (10 U) for 2 hours, which specifically cleaves -GA↓TC- where the adenine
residue is methylated in parental DNA strands, leaving only non-methylated DNA with
the incorporated mutation. The remaining plasmid DNA was purified by ethanol
precipitation using ∼2.5 µg of glycogen as a carrier. The resulting DNA was then
transformed into competent XL1-Blue bacterial cells (Stratagene). Up to 5 µl of DNA
solution was added to 50 µl of competent XL1-Blue cells and the samples left on ice for
30 minutes. The cells were heat-shocked for 45 seconds at 42°C in a waterbath, and
transferred back to ice. Luria-Broth (LB) (500 µl) was then added to the mixture and the
tube was incubated at 37 °C for 30 minutes. The cells (100 µl) were plated onto LB
plates supplemented with ampicillin (100 µg/ml) and incubated at 37 °C overnight.
Random colonies were picked from the LB plate of transformed XL1-Blue cells and
cultured in LB (2 ml) with ampicillin (100 µg/ml) at 37 °C overnight. The cells were
harvested by centrifugation at full speed (∼16,000g) in a microfuge for 20 seconds at
Chapter 2 – Materials & Methods
56
room temperature and the plasmid DNA was extracted from the cells using a QIAprep
Spin Miniprep kit (QIAGEN). The resulting DNA was used for a PCR-based
sequencing reaction by mixing: 4 µl of BigDye terminator cycle sequencing ready
reaction version 1.1 (Applied Biosystems), 0.75 µl of forward and reverse primers (∼3.2
µM), 2 µl of plasmid DNA and water to a final volume of 15 µl. The forward primer
used was the pG3Z–160T7 primer which was complementary to the DNA sequence 160
bases upstream of the T7 promoter, while the reverse primer used was complimentary to
the SP6 promoter. The sequence of the pG3Z-160T7 primer is 5’-
GGGCCTCTTCGCTATTACGC-3’ whilst the sequence of the SP6 primer is 5’-
TATTTAGGTGACACTATAG-3’. The PCR conditions were: initial denaturation (96
°C, 2 minutes) for 1 cycle, then denaturation (96 °C, 30 seconds), annealing (50 °C, 15
seconds) and extension (60 °C, 4 minutes) for 29 cycles. The PCR products were then
subjected to ethanol precipitation and left in pellet form for sequencing (Sequencing
Facility, Faculty of Life Sciences, The University of Manchester). All point mutations
were confirmed by DNA sequence analysis prior to further use.
Other opsin point mutants used in this study had been previously constructed, including
OP[cys14], OP[cys56] and OP[cys87] (Meacock, 1999). The opsin-coding regions of
these mutants are present in the PGEM3Z vector, except for OP[cys14] which is present
in pZEOSV2(+) vector. For the construction of double cysteine opsin mutants, plasmids
containing OP[cys56] and OP[cys87] were used as the templates for the introduction of
a cysteine residue at position 115 by site-directed mutagenesis.
Chapter 2 – Materials & Methods
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Table 2.1 Primers used for the introduction of cysteine residues into opsin.
Mutagenic primer Sequence of primer (5’ to 3’)
OP107C.1 GGATACTTCGTCTTTGGGTGCACGGGCGGCAACCTG OP107C.2 CAGGTTGCCGCCCGTGCACCCAAAGACGAAGTATCC
OP115C.1 GGCAACCTGGAGGGCTGCTTTGCCACCCTGGGC OP115C.2 GCCCAGGGTGGCAAAGCAGCCCTCCAGGTTGCC
OP124C.1 CACCCTGGGCGGTGAAATTTGCCTGTGGTCCTTGGTGGTCCTG OP124C.2 CAGGACCACCAAGGACCACAGGCAAATTTCACCGCCCAGGGTG
OP132C.1 GTCCTTGGTGGTCCTGTGCATCGAGCGGTACGTG OP132C.2 CACGTACCGCTCGATGCACAGGACCACCAAGGAC
OP140C.1 GAGCGGTACGTGGTGGTGTGCAAGCCCATGAGCAACTTCCGC OP140C.2 GCGGAAGTTGCTCATGGGCTTGCACACCACCACGTACCGCTC
OP154C.1 GGGGAGAACCACGCCTGCATGGGCGTCGCCTTC OP154C.2 GAAGGCGACGCCCATGCAGGCGTGGTTCTCCCC
OP165C.1 CACCTGGGTCATGGCTTGCGCCGGTGCCGCGCCC OP165C.2 GGGCGCGGCACCGGCGCAAGCCATGACCCAGGTG
OP204C.1 CCAACAATGAGTCGTTCTGCATCTACATGTTCGTGG OP204C.2 CCACGAACATGTAGATGCAGAACGACTCATTGTTGG
OP217C.1 CATCATCCCCCTGTGTGTCATATTCTTCGGCTACGGG OP217C.2 CCCGTAGCCGAAGAATATGACACACAGGGGGATGATG
OP229C.1 GGGCAGCTGGTGTTCTGCGTCAAGGAGGCGGC OP229C.2 GCCGCCTCCTTGACGCAGAACACCAGCTGCCC
OP254C.1 GGAGGTCACCCGCATGTGCATCATCATGGTCATCGC OP254C.2 GCGATGACCATGATGATGCACATGCGGGTGACCTCC
OP275C.1 GGGGTGGCGTTCTACTGCTTCACCCATCAGGG OP275C.2 CCCTGATGGGTGAAGCAGTAGAACGCCACCCC
OP287C.1 CTTTGGCCCCATCTGCATGACCATCCCGGC OP287C.2 GCCGGGATGGTCATGCAGATGGGGCCAAAG
OP308C.1 CCCCGTCATCTACATCTGCATGAACAAGCAGTTCCGG OP308C.2 CCGGAACTGCTTGTTCATGCAGATGTAGATGACGGGG
The suffix ’.1’ indicates a forward primer, while ‘.2’ indicates a reverse primer.
Chapter 2 – Materials & Methods
58
2.3 Generation of OPTM1-3PPL[cys115] and OPN/5-7[cys-null] mutants
Regions coding for opsin and preprolactin were fused to form the OPTM1-3PPL
chimera with a cysteine residue at position 115. Double-stranded DNA fragments
coding for a region of opsin with cys115 (residues 1 to 142, OPTM1-3) were generated
by PCR using the forward primer OP56C(1-70)-FOR and the reverse primer
PPL/OPTM3 (see Table 2.2 for the sequences of the primers). Similarly, double-
stranded DNA fragments coding for a region of preprolactin (residues 31 to 229, PPL)
were generated by PCR using the forward primer OPTM3/PPL and the reverse primer
PPL(31-229)-REV (Table 2.2). The PCR conditions were: initial denaturation (95 °C, 1
minutes) for 1 cycle, then denaturation (95 °C, 45 seconds), annealing (50 °C, 45
seconds) and extension (72 °C, 2 minutes) for 30 cycles. A second round of PCR using
annealed fragments of OPTM1-3 and PPL as templates with OP56C(1-70)-FOR primer
and PPL(31-229)-REV primer, gave DNA products of the OPTM1-3PPL chimera that
were subsequently cloned into the pSPUTK vector. Site-directed mutagenesis (section
2.2) was employed to replace the first three cysteine residues of the PPL coding region
within OPTM1-3PPL[cys115] with glycine residues. Complementary primers used for
the removal of these cysteine residues are shown in Table 2.3. OPTM1PPL[cys56] was
kindly provided by Samuel Crawshaw.
An opsin mutant with residues 36 to 194 deleted was generated to form the OPN/5-7
construct using a one-step PCR approach. The reverse primer OP35-REV defines the 5’
end of the region to be deleted while the forward primer defines the 3’ end of the region.
The sequences of the primers are shown in Table 2.2. A PCR reaction using these
primers resulted in the amplification of the entire plasmid which lacks the coding region
of residues 36 to 194 of opsin. The restriction enzyme DpnI was added to digest the
methylated parental DNA strand. The linear PCR products were ligated using Rapid
DNA Ligation kit (Stratagene) according to the manufacturer’s manual and transformed
into competent XL1-Blue competent cells. Extracted DNA was analysed by sequencing
as before (section 2.2). Single cysteine residues were introduced into the opsin TM
domains by site-directed mutagenesis using complementary primers as shown in Table
2.1.
Chapter 2 – Materials & Methods
59
Table 2.2 PCR primers used in the generation of OPTM1-3PPL[cys115] and OPN/5-7 constructs.
PCR primer Sequence of primer (5’ to 3’)
OPTM3/PPL CGGTACGTGGTGGTGGGCAAGCCCACCCCCGTCTGTCCCAATGGGCC
PPL/OPTM3 GGCCCATTGGGACAGACGGGGGTGGGCTTGCCCACCACCACGTACCG
OP56C(1-70)-FOR GCGAGATCTACCATGAACGGGACCGAGGGC
PPL(31-229)-REV GCGAGATCTTTAGCAGTTGTTGTTGTAGATG
OP35-REV *P-CCATGGCTCCGCCAGGTAGTACTGCGGGGC
OP195-FOR *P-CACGAGGAGACCAACAATGAGTCGTTCGTC
* ‘P’ indicates that the primers are phosphorylated.
Table 2.3 Primers used for the removal of cysteine residues from the preprolactin coding sequence of OPTM1-3PPL[cys115] construct.
Mutagenic primer Sequence of primer (5’ to 3’)
OP3PPLC34G.1 CCCACCCCCGTCGGTCCCAATGGG
OP3PPLC34G.2 CCCATTGGGACCGACGGGGGTGGG
PPLC41G.1 GGGCCTGGCAACGGCCAGGTATCC
PPLC41G.2 GGATACCTGGCCGTTGCCAGGCCC
PPLC88G.1 GCCCTCAACAGCGGCCATACCTCCTCC
PPLC88G.2 GGAGGAGGTATGGCCGCTGTTGAGGGC
The suffix ’.1’ indicates a forward primer, while ‘.2’ indicates a reverse primer.
Chapter 2 – Materials & Methods
60
2.4 Synthesis of truncated transcription templates lacking a stop codon
PCR was used to generate truncated sections of both the opsin-coding region and its
derivatives so as to provide DNA templates for in vitro transcription (see Laird and
High, 1997). The following solutions were mixed: 10 µl of 2.5 mM dNTPs, 10 µl of 10x
PWO reaction buffer (100 mM Tris-HCl pH 8.85, 250 mM KCl, 50 mM (NH4)2SO4, 20
mM MgSO4), 100 pmol each of forward and reverse primers, 5 U PWO DNA
polymerase and ∼1 µg of plasmid DNA and water to a final volume of 100 µl. For
opsin-coding regions present in PGEM3Z vector, the forward primer used was always
the pG3Z–160T7 primer (refer to section 2.2 for sequence) while the reverse primer
used depended on the length of truncated mRNA required (Table 2.4). For the synthesis
of truncated OPTM1PPL[cys56] and OPTM1-3PPL[cys115] transcription templates
from the pSPUTK vector, the forward primer used was SK-SP6, while the reverse
primer used depended on the length desired (shown in Table 2.5). The synthesis of
OP96[cys14] (in vector pZEOSV2(+)) uses the forward primer pZEO-160T7, which is
complementary to the sequence 160 bases upstream the T7 promoter in the
pZEOSV2(+) vector, and the reverse primer OP87HA-REV (Table 2.4). The sequence
of the SK-SP6 primer is 5’-CCAGAAACTCAGAAGGTTCG-3’ while the sequence of
the pZEO-160T7 primer is 5’-CCAGTTCCGCCCATTCTCCG-3’.
The PCR conditions used were: initial denaturation (94 °C, 4 minutes) for 1 cycle, then
denaturation (94 °C, 1 minute), annealing (60 °C, 1 minute) and extension (72 °C, 1
minute) for 35 cycles, and the final extension (72 °C, 10 min) for 1 cycle. The DNA
obtained was purified using QIAquick PCR purification kit (QIAGEN), and then treated
with 20 U of DpnI to remove the methylated parental DNA template. The QIAquick
PCR purification kit was again used to purify the DNA after the DpnI treatment.
Table 2.4 Primers used to generate truncated opsin transcription templates. Nascent
chain length
Truncation primer Sequence of primer (5’ to 3’)
96 OP87HA-REV
AGCGTAGTCTGGGACGTCGTATGGGTAGACCATGAAGAGGTCGGCCAC
130 OP121HA-REV
AGCGTAGTCTGGGACGTCGTATGGGTAACCGCCCAGGGTGGCAAAGAAGCC
Chapter 2 – Materials & Methods
61
140 OP131HA-REV
AGCGTAGTCTGGGACGTCGTATGGGTACAGGACCACCAAGGACCACAG
150 OP141HA-REV
AGCGTAGTCTGGGACGTCGTATGGGTACTTGCCCACCACCACGTACC
164 OP155HA-REV
AGCGTAGTCTGGGACGTCGTATGGGTACATGATGGCGTGGTTCTCCC
174 OP165HA-REV
AGCGTAGTCTGGGACGTCGTATGGGTACAGAGCCATGACCCAGGTG
204 OP195HA-REV
AGCGTAGTCTGGGACGTCGTATGGGTAGTGGGGCGTGTAGTAGTCAATC
259 OP250HA-REV
AGCGTAGTCTGGGACGTCGTATGGGTAGACCTCCTTCTCGGCCTTCTG
304 OP295HA-REV
AGCGTAGTCTGGGACGTCGTATGGGTAGGCAAAGAAAGCCGGGATGG
339 OP330HA-REV
AGCGTAGTCTGGGACGTCGTATGGGTAGTCACCCAGCGGGTTCTTGC
357 OP348HA-REV AGCGTAGTCTGGGACGTCGTATGGGTAGGCAGGCGCCACCTGGC
Table 2.5 Primers used to generate truncated OPTM1PPL[cys56] and OPTM1-3PPL[cys115] transcription templates. Nascent
chain length*
Truncation primer Sequence of primer (5’ to 3’)
TM1 109 OPPPL109HA-REV AGCGTAGTCTGGGACGTCGTATGGGTAATGGATGTAGT
GGGACACCATGACTGCCCG
TM1 130 OP1PPL121HA-REV AGCGTAGTCTGGGACGTCGTATGGGTAGGTAATGAACC
CTTTGCCCTGGG
TM1 150 OPPPL150HA-REV AGCGTAGTCTGGGACGTCGTATGGGTATTGTTCTTTATC
TTCCGGGGTAGG
TM1 164 OP1PPL155HA-REV AGCGTAGTCTGGGACGTCGTATGGGTAAAGAATCAAGC
TCATAAGGACTTC
TM1 204 OP1PPL195HA-REV AGCGTAGTCTGGGACGTCGTATGGGTAAAGTCGTTTGTT
TTCTTCCTCAATCTC
TM1 259 OP1PPL250HA-REV AGCGTAGTCTGGGACGTCGTATGGGTACTTGCTTGAATC
CCTGCGCAGGCC
TM3 164 OP3/PPL155HA-REV AGCGTAGTCTGGGACGTCGTATGGGTATACCTGGCCGTT
GCCAGGCCCATTGGG
TM3 204 OP3/PPL195HA-REV AGCGTAGTCTGGGACGTCGTATGGGTACATGGTAATGA
ACCCTTTGCC
* The prefix ‘TM1’ indicates truncation primers for OPTM1PPL[cys56] while ‘TM3’ indicates truncation primers for OPTM1-3PPL[cys115].
Chapter 2 – Materials & Methods
62
2.5 Preparation of semi-permeabilised cells
The human HT-1080 fibrosarcoma cell line (European Collection of Cell Cultures,
Salisbury, UK) was cultured with minimal essential medium (MEM) with Earle’s salts,
supplemented with 1/100 volume 200 mM L-glutamine, 1/100 volume 100 mM sodium
pyruvate, 1/100 volume MEM non-essential amino acids, 1/10 volume foetal calf serum
and 7/500 volume MEM vitamins solution. The cells were grown to ∼90 % confluence
in 75 cm2 flasks before they were used. The cells in the flasks were washed twice with
phosphate-buffered saline (PBS) and detached by incubation with 3 ml of Trypsin-
EDTA (0.05 % w/v Trypsin, 0.53 mM EDTA.4Na). The action of trypsin was inhibited
by the addition of 4 ml of KHM buffer (110 mM potassium acetate, 2 mM magnesium
acetate, 20 mM HEPES pH 7.2) with Soybean Trypsin Inhibitor at 100 µg/ml.
The cells were semi-permeabilised as previously described by Wilson et al (1995) using
the same procedure. Cells were pelleted in 15 ml polypropylene tubes at 240g for 3
minutes at 4 °C. The cell pellet was resuspended in 4 ml of KHM buffer containing
digitonin (40 µg/ml). Samples were left on ice for 5 minutes to allow permeabilisation
of the cells, 10 ml of KHM buffer was added to the tube to dilute the digitonin, and the
cells pelleted again at 240g for 3 minutes at 4 °C. The pellet was resuspended in 5 ml of
HEPES buffer (50 mM potassium acetate, 90 mM HEPES pH 7.2) and the cells left on
ice for 5 minutes to recover. The tube was spun as before and the permeabilised cells
were resuspended in 500 µl of KHM buffer. The suspension was transferred to a
microcentrifuge tube and spun at ∼16,000g for 10 seconds before the pellet was
resuspended in 100 µl of KHM buffer. 1 µl of 0.1 M calcium chloride was added and
0.1 U of calcium-dependent micrococcal nuclease was added to the cell suspension and
the sample incubated at room temperature for 12 minutes. The action of micrococcal
nuclease was then inhibited by the addition of 1 µl of 0.4 M EGTA. The sample was
spun again at ∼16,000g for 10 seconds and the cell pellet resuspended in KHM buffer to
give a concentration of ∼0.5 x 105 cells/µl.
Chapter 2 – Materials & Methods
63
2.6 In vitro transcription and translation
For in vitro transcription of opsin constructs in PGEM3Z vector, the following solutions
were mixed in a tube: 20 µl of 5x TSC buffer (200 mM Tris-HCl pH 7.9, 30 mM
MgCl2, 10 mM spermidine, 50 mM NaCl), 10 µl of 100 mM DTT, 4 µl of 25 mM
rNTPs, 5 µl of 10 mM m7G(5’)ppp(5’)G cap analogue, 55 µl of DNA template (from
PCR), 80U of RNAsin and 80U of T7 RNA polymerase. In vitro transcription of opsin
coding regions in pSPUTK vector uses 80U of SP6 RNA polymerase with the addition
of the following: 10 µl of 10x SP6 RNA polymerase buffer (400 mM Tris-HCl pH 7.9,
60 mM MgCl2, 100 mM dithiothreitol, 20 mM spermidine), 4 µl of 25 mM rNTPs, 5 µl
of 10 mM m7G(5’)ppp(5’)G cap analogue, 55 µl of DNA template (from PCR) and 80U
of RNAsin and H2O to a final volume of 100 µl. The sample was incubated at 37 °C for
2 hours and the RNA obtained was purified using an RNeasy mini kit (QIAGEN) and
stored at -80°C.
All in vitro translations were performed in the presence of semi-permeabilised cells,
unless otherwise specified. An initial mixture of 14 µl nuclease-treated rabbit reticulose
lysate, 0.5 µl 19 amino acid mix without methionine (each at 1mM), 1.5 µl 35S-
methionine (11µCi/µl) and 4 µl semi-intact cells, were incubated at 30 °C for 3 minutes.
2 µl of RNA transcript was added and the mixture incubated at 30 °C for 15 minutes.
Further initiation of translation was inhibited by the addition of aurintricarboxylic acid
(ATCA) to 0.1 mM with an incubation of 10 minutes at 30 °C.
For the generation of stable ribosome-nascent chain complexes, the translation mixture
was incubated with 1 µl of 50 mM cycloheximide for 5 minutes on ice to terminate
translation without the release of nascent chains. Alternatively, in cases where the
release of nascent chains from the ribosome was necessary, 1.5 µl of 20 mM puromycin
and 0.5 µl of 250 mM EDTA were added, and the sample was incubated for 10 minutes
at 30 °C. The membrane fraction was recovered by centrifugation at ∼16,000g for 10
seconds. The resulting pellet was washed twice in 20 µl of KHM buffer and then
resuspended in 20 µl of KHM buffer before cross-linking reactions.
Chapter 2 – Materials & Methods
64
2.7 Isolation of ribosome-nascent chain complexes
For the synthesis of nascent chains in the absence of membranes, in vitro translations
were carried out as described above without the addition of semi-permeabilised cells.
The translation was halted by incubation with cycloheximide at the final concentration
of 2 mM for 5 minutes on ice. Isolation of the ribosome-nascent chain complexes was
achieved by layering the sample over 3 volumes of high salt/sucrose cushion (250 mM
sucrose, 500 mM KOAc, 5 mM Mg(OAc)2, 50 mM HEPES.KOH pH 7.9) and
centrifugation at 213,000g (70,000 rpm in a TLA120.2 rotor using a Beckmann Optima
TLX benchtop ultracentrifuge) for 20 minutes at 4 °C. The supernatant was discarded
and the pellet was resuspended in low salt/sucrose buffer (250 mM sucrose, 100 mM
KOAc, 5 mM Mg(OAc)2, 50 mM HEPES.KOH pH 7.9) for modification reactions.
2.8 Cross-linking and modifications of nascent chains with sulphydryl specific
reagents
All cross-linking and modification reagents were prepared in dimethylsulphoxide
(DMSO) as 20 mM stock solutions. For cross-linking, the membrane fraction
resuspended in KHM was incubated with the cross-linking reagent, BMH, at a final
concentration of 1 mM for 10 minutes at 30 °C. Similarly, for modification reactions,
sulphydryl specific reagents, AMS and QSY, were added to a final concentration of 1
mM and the sample was incubated for 10 minutes at 30 °C. β-mercaptoethanol was then
added to a final concentration of 5 mM to quench the cross-linking or modification
reaction. For control experiments, DMSO (5 % v/v) was added instead of the sulphydryl
specific reagent. RNase A (250 µg/ml) was added to all the samples and they were
incubated at 37 °C for 5 minutes in order to destroy any remaining peptidyl tRNA
species and the samples were then prepared for SDS-PAGE or subjected to
immunoprecipitation.
2.9 Solubilisation of ribosome-nascent chain complexes in C12E8
In vitro translations were carried out in the presence of semi-permeabilised cells and
halted either with cycloheximide or puromycin as described in section 2.6. The
membrane fraction was isolated by centrifugation through 3 volumes of high
Chapter 2 – Materials & Methods
65
salt/sucrose cushion at 130,000g for 10 minutes at 4 °C and resuspended in low
salt/sucrose buffer. C12E8 detergent was added to a final concentration of 1% (w/v) and
the sample was incubated on ice for 10 minutes. Ribosomal subunits and associated
nascent chains were recovered by centrifugation through a high salt/sucrose cushion
containing 0.1% (w/v) C12E8 at 213,000g for 20 minutes at 4 °C. The pelleted material
was solubilised in SDS sample buffer for analysis on SDS PAGE. The products in the
supernatant were subjected to trichloroacetic acid (TCA) precipitation. Hence, an equal
volume of a mixture of 20 % TCA (w/v) / 50 % acetone (v/v) was added to the
supernatant and incubated for 15 minutes on ice. The sample was spun at ∼16,000g for
20 minutes and the pellet was washed with 1 ml of 50 % acetone. The sample was spun
at ∼16,000g for an additional 20 minutes and the pellet was dried at 37 °C in a heat
block. The pellet was resuspended in 50 µl of SDS sample buffer and prepared for
loading onto an SDS-polyacrylamide gel (section 2.12).
2.10 Immunoprecipitation
After cross-linking or modification reaction, SDS was added to a final concentration of
1 % v/v and samples incubated at 37 °C for 30 minutes. At least 4 volumes of Triton IP
buffer (10 mM Tris-HCl pH 7.6, 140 mM NaCl, 1 mM EDTA, 1 % Triton X-100) was
added, together with 0.2 mg/ml protease inhibitor phenylmethyl sulphonyl fluoride, 1
mM methionine and 10 µl of a pansorbin suspension. The samples were left at 4 °C for
30 minutes on a rolling platform to preclear, spun at ∼16,000g for 20 minutes at 4 °C
and the supernatant transferred to fresh tubes. The supernatant was incubated with the
appropriate anti-serum (1 µl) and Protein A Sepharose (20 µl) at 4 °C overnight on a
rolling platform. The beads were spun down for 10 seconds at ∼16,000g and the
supernatant was discarded. The beads were washed three times by resuspending them in
IP buffer (800 µl) and spinning them down again. The samples were then prepared for
loading onto an SDS- polyacrylamide gel as described in section 2.12.
2.11 Endoglycosidase H digestion
Endoglycosidase H (Endo H) removes asparagine-linked oligosaccharide units by
cleaving between the two N-acetylglucosamine (GlcNAc) residues. Following washing
in IP buffer, immunoprecipitated samples on Protein A beads were denatured in Endo H
Chapter 2 – Materials & Methods
66
denaturing buffer (0.5% SDS, 1% β-mercaptoethanol) for 30 minutes at 37 °C. 3 µl of
0.5M sodium citrate (pH 5.5) buffer, 0.5 µl of 100 mM phenylmethyl sulphonyl
fluoride, 5.5 µl of H2O and 500 U Endo H were added and the samples were incubated
for an additional 30 minutes at 37 °C. The samples were then solubilised in an equal
volume of 2x concentration SDS sample buffer and resolved by SDS PAGE.
2.12 SDS PAGE and sample analysis
The samples were first denatured in 50 µl of SDS sample buffer (0.1 M Tris-HCl pH
6.8, 5 mM EDTA, 0.5 M sucrose, 0.5 % L-methionine, 0.01 % bromophenol blue, 0.5
M DTT, 20% SDS) by incubation at 37 °C for 30 minutes. Cross-linking samples were
loaded onto a denaturing 14% polyacrylamide gel, while samples from modification
reactions with AMS or QSY were loaded onto a denaturing 18% polyacrylamide gel.
These samples were run in the presence of SDS and resolved at 150 V. The gels were
then fixed in acetic acid: methanol: water solution (1:2:7 v/v) for at least 5 minutes
before they were dried. They were then exposed to a phosphoimaging plate for three to
seven days and the results analysed on a Fuji BAStation phosphoimager using the
AIDA software. Where specified, quantification of the products was also performed
using the AIDA software.
Chapter 3 – The use of a site-specific cross-linking approach
67
CHAPTER 3 Results
The use of a site-specific cross-
linking approach to examine opsin
integration
Chapter 3 – The use of site-specific cross-linking approach
68
3.1 Introduction
An essential step during membrane protein biosynthesis at the ER is the integration of
its one or more transmembrane (TM) domains. This process involves the lateral
movement of the TM domain from the proteinaceous environment of the Sec61
translocon to the phospholipid bilayer of the ER membrane. Although the crystal
structure of an archeal Sec61 complex has been elucidated (Van den Berg et al., 2004),
the mechanism by which integration occurs is unknown. The insertion of a polytopic
membrane protein is a particularly complex process since several TM domains have to
be correctly integrated in order to enable the proper assembly of a functional
polypeptide. The aim of this study is to use the seven transmembrane domain protein,
opsin, as a model to examine the biosynthesis of polytopic membrane proteins and to
establish how the integration of multiple TM domains is co-ordinated.
Opsin has several characteristics that makes it well-suited to the study of polytopic
membrane protein biogenesis: 1) it belongs to a well-characterised family of seven
transmembrane domain receptor proteins; 2) extensive structural data is available from a
crystal structure (Palczewski et al., 2000); 3) a good monoclonal antibody to its N-
terminal region is available; 4) the two N-glycosylation sites near its N-terminus serve
as ideal marker for the authentic targeting and insertion of nascent opsin chains into the
ER membrane (c.f. Laird & High, 1997).
In this study, the principal approach employed to examine opsin integration is to
analyse the molecular environment of each TM domain at different stages of opsin
biosynthesis by using a site-specific cross-linking approach. To this end, all the natural
cysteines in the wild type opsin sequence were replaced (Meacock et al., 2002), and a
single cysteine residue was introduced into the particular TM domain of interest. This
approach ensures that the cross-linking reaction can only occur from the single cysteine
residue introduced into the nascent chain. In order to mimic specific stages of opsin
biosynthesis, nascent chains of specific lengths were generated by the translation of
truncated mRNAs. Since these mRNAs lack a stop codon, the nascent chains are not
released from the ribosome, but remain lodged at the ER translocon, creating artificial
‘integration intermediates’ (Gilmore et al., 1991). The addition of the homo-
bifunctional thiol-reactive reagent, bismaleimidohexane (BMH), at this stage allows the
Chapter 3 – The use of site-specific cross-linking approach
69
single cysteine probe within the nascent chain to be cross-linked to any available
cysteine residues present in adjacent proteins. By performing the cross-linking reaction
with integration intermediates of increasing nascent chain lengths, the molecular
environment of a specific TM domain could be examined at different stages of opsin
biosynthesis.
3.2 Optimisation of the experimental system
The analysis of opsin biogenesis using cross-linking techniques is well-established
(Laird & High, 1997) and has been extensively used to examine the environment of
TM1 and TM2 of opsin (Meacock et al., 2002). In this approach, an integration
intermediate of a particular chain length is taken to reflect a particular stage of
membrane integration (Thrift et al., 1991). However, previous studies of opsin have
shown that the population of nascent chains generated by this approach may not be
homogeneous, thus, the presence of nascent chains of differing lengths within a single
translation reaction can result in complex cross-linking patterns that may be difficult to
interpret unambiguously (Meacock et al., 2002).
One obvious factor that might contribute to the heterogeneity of the nascent chain
population is ribosome stacking. Multiple ribosomes often translate a single mRNA and
under normal circumstances, this leads to multiple copies of the full length protein.
However, in the case of the truncated mRNAs that are used in vitro, the ribosomes are
not released from the mRNA and may stack up at its 3’ end generating different length
nascent opsin chains, some of which lack the C-terminal region (Fig. 3.1). For
meaningful cross-linking experiments, it is imperative that cross-linking adducts formed
from authentic integration intermediates can be distinguished from any adducts that
might be formed from such shorter nascent chains. For this purpose, a haemagglutinin
(HA) epitope tag was introduced at the C-terminus of each integration intermediate. By
performing immunoprecipitation with an α-HA antiserum, only nascent chains with an
intact C-terminus (i.e. nascent chains of the correct length) will be recovered, thus
allowing ‘authentic’ cross-linking adducts to be identified (Fig. 3.1).
In order to establish whether the C-terminal HA-tagging of integration intermediates
allows the efficient selection of authentic nascent chains, two opsin constructs of
Chapter 3 – The use of site-specific cross-linking approach
70
different lengths were used. OP164[cys56] has a cysteine residue at position 56 with the
first 155 residues of opsin followed by the 9 residues which make up the HA tag at the
C-terminus (total 164 residues). The longer construct, OP357[cys56], also has a
cysteine residue at position 56 with the complete 348 residues of the opsin coding
region plus a 9-residue HA tag (total 357 residues).
Figure 3. 1 Rationale for HA tagging of nascent opsin chains. Opsin chains of different lengths may arise as a consequence of ribosome stacking where several ribosomes are synthesizing polypeptide chains from a single truncated mRNA chain (in blue). These shorter chains will lack the C-terminal HA tag (in green) but will still be immunoprecipitated by the α-opsin monoclonal antibody which recognises the N-terminus of the polypeptide. Adducts with these shorter chains will be recognised by sera specific for subunits of the ER translocon. However, only authentic opsin chains with an intact C-terminal HA tag will be immunoprecipitated by the α-opsin, α-Sec61 subunit and α-HA antisera.
mRNA representing each integration intermediate was translated in vitro in the presence
of semi-permeabilised mammalian cells and incubated with either BMH or a solvent
(DMSO) only control. After denaturation with 1% SDS, the nascent chains were
immunoprecipitated with the α-opsin antisera which recognises the N-terminus of opsin,
and the α-HA antisera which recognizes the C-terminal HA tag (Fig. 3.2).
The proper integration of the nascent opsin chains into the ER membrane of the semi-
permeabilised cells was confirmed by the presence of doubly N-glycosylated chains
(Fig. 3.2, denoted by (ii)). The lower product observed in all the samples represents the
fraction of opsin chains that are not glycosylated (Fig. 3.2, (i)). Several additional
Chapter 3 – The use of site-specific cross-linking approach
71
products were also present following immunoprecipitation with the α-opsin antisera, but
were absent after immunoprecipitation with the α-HA antisera (Fig. 3.2, c.f. lanes 1 and
2 to 3 and 4, and lanes 5 and 6 to 7 and 8, denoted by a bracket). The lack of the HA tag
confirms that these polypeptide chains were truncated at their C-termini and may have
been generated by ribosome stacking or by the degradation of longer opsin chains.
These truncated products were especially prevalent for the longer polypeptide,
OP357[cys56] (Fig. 3.2, c.f. lanes 1 and 2 to 5 and 6, (])), thus highlighting the
importance of C-terminal HA-tagging particularly for long nascent chains.
In the presence of BMH, two distinct cross-linking adducts were observed for
OP164[cys56] while one adduct was seen for OP357[cys56] (Fig. 3.2, lanes 2, 4, 6 and
8, (X)). These adducts were immunoprecipitated with the α-HA antisera (Fig. 3.2, lanes
4 and 8, (X)), indicating that the cross-linking products were formed with nascent
chains of the appropriate lengths. In this way, the inclusion of the HA tag at the C-
terminus of the integration intermediate allows authentic cross-linking adducts to be
distinguished from any other adducts that may be formed with any shorter nascent
chains present in the translation reaction.
3.3 Cross-linking adducts are formed with glycosylated opsin chains
In order to examine the molecular environment of opsin TM domains during nascent
chain biogenesis by cross-linking, it is important that cross-linking products are formed
with nascent chains that are properly integrated into the membrane. N-glycosylation of
the N-terminal region of opsin serves as a useful marker for efficient nascent chain
integration into the ER membrane. One way to ascertain that the BMH-dependent cross-
linking adducts are formed with glycosylated nascent opsin chains is by treating the
samples with endoglycosidase H. Endoglycosidase H cleaves the glycan group attached
to the opsin chain between the two N-acetylglucosamine (GlcNAc) residues, resulting
in faster migration in SDS-PAGE due to a reduction in molecular weight.
The construct, OP109[cys56], has a single cysteine residue located within TM1 of opsin
and a chain length of 109 residues, including the HA tag. mRNA encoding
OP109[cys56] was translated in the presence of semi-permeabilised cells, treated with
either BMH or DMSO and subjected to immunoprecipitation using α-opsin, α-HA, α-
Chapter 3 – The use of site-specific cross-linking approach
72
Sec61α and α-Sec61β antisera. A duplicate set of samples were additionally treated with
endoglycosidase H.
Figure 3. 2 Immunoprecipitation with α-HA antisera allows selection of authentic opsin chains. mRNA representing OP164[cys56] and OP357[cys56] were translated in vitro in a rabbit reticulocyte translation system in the presence of semi-permeabilised mammalian cells and radiolabelled methionine. The membrane fraction was isolated by centrifugation and treated with either BMH or DMSO (solvent control). The cross-linking reaction was then quenched with β-mercaptoethanol. The samples were denatured in 1% SDS and immunoprecipitations using α-opsin and α-HA antisera were carried out. Uncross-linked doubly-glycosylated opsin chains resulting from both constructs are denoted by (ii), while uncross-linked non-glycosylated opsin chains are indicated with (i). Truncated opsin chains which lack an intact C-terminal HA tag are present in immunoprecipitations with the α-opsin antibody (denoted by brackets (])), but were absent in immunoprecipitations with the α-HA antibody. BMH-dependent cross-linking adducts were also observed for both integration intermediates and are marked by a cross (X).
Chapter 3 – The use of site-specific cross-linking approach
73
Figure 3. 3 Cross-linking products are formed with glycosylated opsin chains. OP109[cys56] was synthesised in a rabbit reticulocyte translation system in the presence of digitonin-permeabilised mammalian cells. The membrane fraction was incubated either with BMH (+) or mock-treated with solvent only (-). Denaturing immunoprecipitation was then carried out using α-opsin, α-HA, α-Sec61α and α-Sec61β antisera. Duplicate samples were treated with endoglycosidase H (+ EndoH) after immunoprecipitation. Distinct cross-linking adducts obtained in the presence of BMH were marked with a cross (X). Products that were identified as adducts with Sec61α and Sec61β are indicated with ‘α’ and ‘β’ respectively. Other symbols are as previously defined in the legend to Figure 3.2.
Chapter 3 – The use of site-specific cross-linking approach
74
Both glycosylated and non-glycosylated OP109[cys56] nascent chains were observed
for samples which were not treated with endoglycosidase H (Fig. 3.3, lanes 1-3, denoted
by (ii) and (i) respectively). As before, immunoprecipitation with the α-HA antisera
identified authentic nascent chains and their corresponding BMH-dependent cross-
linking adducts (Fig. 3.3, lane 3, (i), (ii), (X)). The adducts were identified by
immunoprecipitation as nascent chains cross-linked to Sec61α and Sec61β (Fig. 3.3,
lanes 4 and 5, (α), (β)). Treatment of the samples with endoglycosidase H resulted in the
collapse of uncross-linked glycosylated opsin chains to a single lower molecular weight
band which represented the de-glycosylated nascent chains (Fig. 3.3, lanes 6-8, (i)).
The OP109[cys56] adducts with Sec61α and Sec61β also migrated faster following
treatment with endoglycosidase H, as shown by the shift of these products upon SDS-
PAGE, indicating a reduction in molecular weight (Fig. 3.3, c.f. lanes 4 and 5 to 9 and
10, (α), (β)). The sensitivity of the adducts to endoglycosidase H treatment indicated
that both Sec61α and Sec61β were cross-linked to opsin chains which were N-
glycosylated. Thus, these cross-linking products were formed with nascent chains which
have been correctly targeted and integrated into the ER membrane.
3.4 Cross-linking adduct formation is cysteine-dependent
The ‘site-specificity’ of the cross-linking reaction is pivotal to the use of cross-linking
techniques in the analysis of the environment of different TM domains during their
integration into the ER membrane. The use of a homo-bifunctional, cysteine reactive
cross-linking reagent such as BMH ensures that adducts are formed only from the target
cysteine probe present in the TM domain. However, previous studies have shown that
spontaneous non-specific cross-linking reactions may still occur in the absence of a
cross-linking reagent (Oliver et al., 1996). In order to establish that the adducts
observed in the previous experiments were cysteine dependent, cross-linking was
performed with opsin constructs which lack a cysteine probe.
OP164[cys-null] and OP204[cys-null] are two integration intermediates with chain
lengths 164 and 204 respectively including the C-terminal HA tag, which do not contain
any cysteine probes. In vitro translation and the cross-linking reaction were performed
as before and the products were immunoprecipitated using α-opsin, α-HA, α-Sec61α
Chapter 3 – The use of site-specific cross-linking approach
75
and α-Sec61β antisera. No distinct cross-linking adducts were observed for both
integration intermediates, indicating that BMH dependent cross-linking reaction
requires the presence of a cysteine probe in the nascent chains (Fig. 3.4, lanes 1-3 and 6-
8).
However, immunoprecipitation with the α-Sec61α antibody did result in two products of
∼40 kDa and ∼80 kDa (Fig. 3.4, lanes 4 and 9, (•)). These products were not recognized
by the α-opsin or α-HA antisera, indicating that they do not contain any opsin chains
(Fig. 3.4, c.f lanes 2 and 3 to 4, c.f. lanes 7 and 8 to 9). On the basis of size alone, the 40
kDa band may be due to endogenous, radiolabelled Sec61α molecules. As BMH was
present in the sample, the larger band of ∼80 kDa may consequently be due to two
molecules of endogenous Sec61α cross-linked together.
In order to determine if these products were due to radiolabelled Sec61α molecules, the
translation reaction was repeated without the addition of any exogenous mRNA
template and BMH cross-linking was performed (Fig. 3.5). Immunoprecipitation with
the α-Sec61α antibody gave a single product of ∼40 kDa in the absence of BMH (Fig.
3.5, lane 3, (•)), while two species of ∼40 kDa and ∼80 kDa were observed in the
presence of BMH, reminiscent of the products seen in the previous experiment (c.f. Fig.
3.5, lane 4, to Fig. 3.4, lanes 4 and 9, (•)). This confirmed that the ∼40 kDa band was an
endogenous, radiolabelled Sec61α monomer while the ∼80 kDa band was most likely
due to two Sec61α molecules cross-linked together. Immunoprecipitation using the α-
opsin and α-Sec61β antisera did not give any products, indicating that no endogenous
radiolabelled opsin or Sec61β molecules were present (Fig. 3.5, lanes 1, 2, 5 and 6).
The radiolabelled Sec61α species seen in Figures 3.4 and 3.5 were generated when
endogenous Sec61α mRNA associated with the semi-intact cells was translated in the
presence of radiolabelled methionine. Although the semi-permeabilised cells used in
these assays were treated with micrococcal nuclease, the nuclease treatment was clearly
not completely effective in removing all endogenous mRNA chains, thus allowing
traces of other molecules to be radiolabelled during the translation reaction. The
presence of these radiolabelled Sec61α species in the sample further highlighted the
need for strict criteria in distinguishing true cross-linking adducts from other species.
Hence, only if a cross-linking product is present in both the α-HA and α-opsin samples,
Chapter 3 – The use of site-specific cross-linking approach
76
will it be considered as a bona fide adduct formed from an authentic opsin integration
intermediate.
Figure 3. 4 Formation of BMH cross-linking adducts is cysteine dependent. mRNA representing OP164[cys-null] and OP204[cys-null] was translated in the presence of semi-permeabilised cells and the membrane fraction was treated with either BMH (+) or DMSO (-). As before, immunoprecipitations with α-opsin, α-HA, α-Sec61α and α-Sec61β antisera were carried out. No distinct BMH-dependent cross-linking adducts were observed. However, immunoprecipitation with the α-Sec61α antibody gave two faint products (denoted with (•)).
Chapter 3 – The use of site-specific cross-linking approach
77
Figure 3. 5 Radiolabelled endogenous Sec61α molecules were generated during translation. In vitro translation was performed without the addition of exogenous mRNA templates in the presence of radiolabelled methionine and digitonin-permeabilised cells. The sample was then subjected to denaturing immunoprecipitations using α-opsin, α- Sec61α and α-Sec61β specific antisera. Sec61α specific products obtained by immunoprecipitation with the α-Sec61α antibody are indicated with (•).
Chapter 3 – The use of site-specific cross-linking approach
78
3.5 Summary
For cross-linking to be successfully exploited in the study of opsin integration, it is
necessary to have specific controls to allow an unambiguous interpretation of the
resulting data. Crucial factors which have been addressed are: 1) the cross-linking
reaction is cysteine dependent, i.e. the reaction is site-specific; 2) the cross-linking
adducts are formed with glycosylated (i.e. properly integrated) opsin chains; 3) adducts
formed with authentic opsin chains of the correct length may be distinguished from
adducts formed with shorter, truncated nascent chains by the addition of a C-terminal
HA tag. The inclusion of a C-terminal epitope tag is particularly important for long
chain lengths.
Conclusion
Having established the validity of this experimental system, I now set out to exploit it in
order to analyse the molecular environment of specific regions of opsin, in particular its
TM domains, during membrane integration.
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
79
CHAPTER 4 Results
The membrane integration of the
N-terminal region of opsin:
TM1 to TM3
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
80
4.1 Introduction
This study aims to analyse the molecular environment of each of the seven TM domains
of opsin during its membrane integration in order to gain insights into the mechanism of
polytopic membrane protein biogenesis at the ER. In the previous chapter, the cysteine
mediated site-specific cross-linking approach employed during this investigation was
validated. Thus, unique cysteine residues can be placed at strategic locations within the
nascent chain and their environment probed using the homo-bifunctional, sulphydryl-
specific cross-linking reagent, BMH. A second feature of this approach is the use of
defined integration intermediates that can be generated in vitro. It had previously been
suggested that the presence of incomplete intermediates, generated either by ribosome
stacking or degradation of longer opsin chains, may complicate the interpretation of
such studies (Meacock et al., 2002). I showed that the addition of a C-terminal HA tag
to opsin integration intermediates allowed authentic chains and their cross-linking
products to be distinguished from other ‘spurious’ adducts. For this reason, all
integration intermediates generated in this study included the 9 residue HA epitope tag
at the C-terminus.
Previous studies of opsin fragments suggest it is comprised of two or more independent
folding domains (Ridge et al., 1995). The aim of the work presented in this chapter was
to investigate the molecular environment of the N-terminal region of opsin, comprising
TM1 to TM3 during membrane integration of the nascent polypeptide. Thus, a single
cysteine residue was introduced into a particular TM domain of a cysteine-null opsin
mutant to allow site-specific cross-linking from the target cysteine to ER components in
close proximity. In order to follow the location of a particular TM domain during its
integration, a range of integration intermediates of increasing chain lengths was
generated to represent different stages of opsin biosynthesis (as depicted in Figure 4.1).
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
81
Figure 4. 1 A diagrammatic representation of artificial opsin integration intermediates generated for site-specific cross-linking analysis of distinct TM domains. All cysteine residues of wild type opsin were replaced with glycine (Meacock et al., 2002) and a single cysteine probe was introduced into TM1, TM2 or TM3 (for simplicity, the cysteine residue is not shown in this diagram). Transmembrane domains are represented with a dashed line while the C-terminal HA tag is indicated in grey. The numbering shows the location of the TM domains (derived from the crystal structure, see Palczewski et al., 2000) in the context of the hypothetical topologies of the various integration intermediates. The two N-linked glycosylation sites at residues 2 and 15 are indicated with a ‘Y’.
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
82
4.2 TM1 is adjacent to discrete sets of ER components during its integration
Whilst the integration of opsin TM1 has been previously characterised by a cysteine-
mediated cross-linking study, the heterogeneity of the nascent chain population present
in this study complicated the interpretation of the data (Meacock et al., 2002). In order
to obtain a more accurate analysis of TM1 integration, nascent opsin chains of the same
lengths as those previously examined but including the HA epitope tag at their C-
terminus, were generated. These integration intermediates possess a single cysteine at
residue 56 within TM1 of opsin which acts as the sole target for the subsequent cross-
linking reaction.
mRNA transcripts representing integration intermediates of OP[cys56] were translated
in vitro in the presence of digitonin-permeabilised mammalian HT1080 cells. The
membrane fraction was isolated by centrifugation and was then either treated with the
sulphydryl-specific cross-linking reagent, BMH, or mock-treated with solvent alone
(DMSO). Following quenching of any unreacted maleimides and the denaturation of the
samples using 1% SDS, specific products were recovered by immunoprecipitation using
α-opsin, α-HA, α-Sec61α and α-Sec61β antisera. As previously observed (see Chapter
3), there was clear heterogeneity in the population of nascent chains that were
membrane-integrated (Fig. 4.2). This was largely restricted to longer intermediates, i.e.
OP150[cys56] to OP259[cys56], resulting in several products that were recognised by
the α-opsin but not the α-HA antibody (Fig. 4.2, c.f. lanes 17 and 18, 22 and 23, 27 and
28, 32 and 33, see *). In some cases, these incomplete intermediates gave adducts to ER
translocon components, but such adducts could be distinguished from those formed with
authentic integration intermediates since they were not immunoprecipitated with the α-
HA antibody (Fig. 4.2, c.f. adducts to Sec61α in lanes 13 and 14, 28 and 29).
In contrast, a number of cross-linking products were found to be efficiently
immunoprecipitated with both the α-opsin and α-HA antisera, indicating these adducts
resulted from authentic integration intermediates cross-linked to ER components (Fig.
4.2, lanes 2 and 3, 7 and 8, 12 and 13, 17 and 18, 22 and 23, 27 and 28, 32 and 33).
Several of these products were identified as adducts with either subunits of the Sec61
complex, or a previously defined novel 10 kDa protein PAT-10 (a protein associated
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
83
with the translocon of ~10 kDa) (Meacock et al., 2002) (Fig. 4.2, lanes 2-5, 7-10, 12-13,
17-19, 22-24, 27-28, 32-33, products denoted by α, β and P respectively).
Figure 4. 2 BMH-mediated cross-linking of OP[cys56] integration intermediates of increasing chain length to ER components. Truncated mRNA chains representing the integration intermediates indicated were translated in rabbit reticulocyte lysate supplemented with digitonin-permeabilised mammalian cells. Membrane-integrated products were isolated by centrifugation and incubated with either BMH (+) or DMSO (-). The samples were quenched with β-mercaptoethanol, denatured in 1% SDS and specific products were recovered by immunoprecipitations using α-opsin, α-HA, α-Sec61α and α-Sec61β antisera. Uncross-linked doubly-glycosylated and non-glycosylated opsin chains are denoted with (ii) and (i) respectively, while truncated opsin chains which lack an intact C-terminus are marked with asterisks. Distinct adducts to Sec61α, Sec61β and PAT-10 are indicated with ‘α’, ‘β’ and ‘P’ respectively.
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
84
It was striking that cross-linking to Sec61β was seen only with short opsin chains, OP96
and OP109 (Fig. 4.2, β), while cross-linking to PAT-10 was apparent with OP130 and
beyond (Fig. 4.2, P). Whilst the exact identity of PAT-10 remains unclear, it has been
extensively characterised during the previous analysis of TM1 integration (Meacock et
al., 2002).
Surprisingly, the cross-linking of opsin TM1 to Sec61α displayed a previously
unrecognised level of complexity (c.f. Meacock et al., 2002). Thus, TM1 formed
distinct adducts to Sec61α with the shorter opsin chains, OP96 and OP109, but adduct
formation was barely detectable when the nascent chain length was increased to 130
residues (Fig. 4.2, lanes 4, 9 and 14). Remarkably, extending the nascent chain further
to 150 and 164 residues resulted in a second association with Sec61α that was then
diminished again upon further chain extension to OP204 and OP259 (Fig. 4.2, lanes 19,
24, 29 and 34, α, see also Fig. 4.4 for quantification). These results indicated that TM1
experiences at least two distinct Sec61 mediated environments during opsin
biosynthesis; one in which TM1 may be cross-linked to both Sec61α and Sec61β, and
another where TM1 is adjacent to Sec61α and PAT-10. Taken together, these data
suggest TM1 engages the translocon as soon as it emerges from the ribosome, moves
away upon chain extension, but then transiently re-associates with the translocon in an
environment close to PAT-10.
4.3 TM1 environment is influenced by subsequent TM domains
The alteration in the environment of TM1 observed by cross-linking of different
integration intermediates is most likely a consequence of TM1 relocation during nascent
chain extension. If so, then the environment of TM1 may alter either as a result of
‘active displacement’ by the subsequent TM domains that are synthesised as the nascent
opsin chain gets longer, or alternatively, the change may simply result from having a
longer polypeptide ‘tether’ to the ribosome thereby allowing TM1 to relocate to a new
environment. In order to distinguish between these two possibilities and determine
whether the subsequent TM domains have an influence on TM1 relocation, TM2 to
TM7 of opsin were replaced with a hydrophilic region of the secretory protein
preprolactin to produce the OPTM1PPL[cys56] polypeptide (see Fig. 4.3A). The
environment of TM1 during nascent chain extension was then analysed using
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
85
integration intermediates of OPTM1PPL[cys56] with lengths identical to those
previously used to analyse the opsin nascent chain (c.f. Fig. 4.2).
The site-specific cross-linking products of a range of OPTM1PPL[cys56] integration
intermediates were analysed by immunoprecipitation as previously described. Authentic
adducts, i.e. recognised by both α-opsin and α-HA antisera, were observed between the
short OPTM1PPL109[cys56] nascent chain and both Sec61α and Sec61β (Fig. 4.3B,
lanes 2-5). These adducts were found to persist at longer chain lengths of 130, 150, 164
and 204 residues (Fig. 4.3B, lanes 7-10, 12-15, 17-20, 22-25, α and β) and this
prolonged association of TM1 with the Sec61 complex when present in
OPTM1PPL[cys56] was clearly distinct from its previous behaviour (c.f. Fig. 4.2).
Whilst immunoprecipition using antisera recognising Sec61α and Sec61β also
recovered products with the OPTM1PPL259 intermediate, these adducts were not
significantly immunoprecipitated with the α-HA antibody, indicating that they were
adducts to shorter nascent chains lacking the C-terminal HA epitope (Fig. 4.3B, c.f.
lanes 29-30). It was also apparent that TM1 of the OPTM1PPL construct was cross-
linked to PAT-10 from a chain length of 130 residues through to 259 residues, although
the efficiency of cross-linking was substantially reduced in comparison to the opsin
chains (c.f. Fig. 4.2 and 4.3B, lanes 8, 13, 18, 23, 28, P). Adducts with subunits of the
Sec61 complex and PAT-10 were not observed when OPTM1PPL integration
intermediates were released with puromycin prior to BMH cross-linking (data not
shown).
In order to allow a better comparison between the behaviour of TM1 in the context of
both OP[cys56] and OPTM1PPL[cys56], the relative cross-linking efficiency of Sec61α
was determined in both cases (Fig. 4.4). This reaffirmed that, whilst TM1 experiences
two distinct Sec61 based environments when present in the opsin chain, this periodicity
is not observed when it is present in OPTM1PPL. In OPTM1PPL, TM1 cross-linking to
Sec61α remained fairly constant until the nascent chain was 259 residues long. Taken
together, with the distinct difference in Sec61β adduct formation between the two
constructs, these results suggest that the synthesis of the subsequent TM domains (TM2
to TM7) influences TM1 relocation from the Sec61 complex and that the absence of
these TM domains causes a delay in its exit from the ER translocon.
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
86
Figure 4. 3 A) A schematic representation of the OPTM1PPL[cys56] polypeptide. The first 70 residues of opsin including TM1 (in black) were fused to a region of the secretory protein, preprolactin (PPL, residues 31-229, in grey) to form OPTM1PPL. A single cysteine probe was present at position 56 within TM1 (indicated by a white star). B) BMH cross-linking of OPTM1PPL[cys56] integration intermediates of to translocon associated components. Integration intermediates of different lengths were synthesised in a rabbit reticulocyte translation system in the presence of semi-permeabilised cells and treated as described in the legend to Figure 4.2. Uncross-linked doubly-glycosylated, non-glycosylated and truncated nascent opsin chains are denoted with (ii), (i) and asterisks respectively, while cross-linking adducts to Sec61α, Sec61β and PAT-10 are labelled ‘α’, ‘β’ and ‘P’ respectively.
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
87
Figure 4. 4 A plot of the relative efficiency of cross-linking to Sec61α versus the chain length of the integration intermediate. For relevant integration intermediates, quantification of the uncross-linked doubly-glycosylated opsin chains (e.g. Fig. 4.3B, lane 1, (ii)) and its adducts with Sec61α (e.g. Fig. 4.3B, lane 2, α) uses the products immunoprecipitated with the α-opsin antibody and was carried out using the AIDA software (see appendix for raw data). The fraction of nascent chains cross-linked to Sec61α was initially calculated by dividing the amount of the Sec61α adduct by the total amount of glycosylated opsin chains present in the cross-linking reaction. The integration intermediate for which the highest fraction of nascent chain was cross-linked to Sec61α was then set as the nominal value of 1.0 and other levels of adduct formation were expressed relative to the highest level. These values (y-axis) were then plotted against the length of the nascent chain (x-axis). In the case of normal opsin (represented by solid line), OP96 was set as the reference point, while in the case of OPTM1PPL (represented by dashed line), OPTM1PPL164 was set as the reference point.
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
88
4.4 TM2 has exited the translocon in the OP204 integration intermediate
An extensive analysis of TM2 integration has been carried out in a previous study of
opsin biosynthesis (Meacock et al., 2002), therefore less emphasis was placed on
investigating the molecular environment of TM2 in this study. However, the alteration
of the behaviour of TM1 when examined in the context of the OPTM1PPL raises the
question of the relative position of the other TM domains with respect to TM1 when
present in the normal opsin construct with multiple TM domains. The environment of
integration intermediates with cys89 located within TM2 of three different chain
lengths, OP140, OP164 and OP204, was therefore analysed to examine the location of
TM2.
OP140[cys89], OP164[cys89] and OP204[cys89] chains were treated with BMH and
recovered by immunoprecipitation as previously described. Adducts with Sec61α and
Sec61β were observed with OP140 and OP164 opsin chains (Fig. 4.5, lanes 2-5, 7-10, α
and β), but when the nascent chain is extended to 204 residues, no distinct adducts to
translocon components were seen (Fig. 4.5, lanes 12-15). These results indicate that
TM2 is engaged with the translocon when the nascent chain is 140 or 164 residues long,
but TM2 is presumed to have exited the translocon when the nascent chain is 204
residues.
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
89
Figure 4. 5 BMH cross-linking of integration intermediates with cys89. OP140[cys89], OP164[cys89] and OP204[cys89] were synthesised, treated with BMH and recovered by immunoprecipitation as described in the legend of Figure 4.2. Symbols used are as previously defined. This figure was provided by Samuel Crawshaw.
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
90
4.5 TM3 is associated with the ER translocon in the OP164 integration
intermediate
OP164 represents an important stage in opsin biosynthesis where changes in the
environment of TM1 seem to occur, where TM1 was seen to regain association with
Sec61α (Fig. 4.2). On the basis of chain length, one might expect opsin TM3 to have
exited the ribosome (see Fig. 4.1) in the OP164 integration intermediate and hence the
environment of TM3 was investigated at this point. As opsin TM3 is a long TM domain
which consists of residues 107 to 139 (Palczewski et al., 2000), its molecular
environment was examined using probes introduced at three different locations. Thus,
single cysteine probes were placed towards the extracellular end of TM3 (cys115), in
the middle of TM3 (cys124) or towards the intracellular end of TM3 (cys132).
Integration intermediates of OP164[cys115], OP164[cys124] and OP164[cys132] were
cross-linked to adjacent ER proteins using BMH and adducts identified by
immunoprecipitation. The resulting adducts were quite distinct for the three different
cysteine residues used, and whilst discrete adducts with both Sec61α and Sec61β were
observed using OP164[cys115] (Fig. 4.6, lanes 2-5, α and β), only an adduct to Sec61β
was seen with OP164[cys124] (Fig. 4.6, lanes 7-10, β). On the other hand, no
significant adducts to any of these Sec61 subunits were seen with OP164[cys132] (Fig.
4.6, lanes 12-15). It is quite possible that cys132 has not fully exited the ribosome at a
chain length of 164 residues and is therefore not sufficiently close to the ER translocon
for efficient cross-linking to occur. On the basis of adduct formation between cysteine
probes 115 and 124, and subunits of the Sec61 complex, I conclude that TM3 has begun
to engage the translocon at its N-terminal end when the opsin chain has reached 164
residues in length.
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
91
Figure 4. 6 BMH cross-linking of OP164 integration intermediates with cysteine probes in three different locations within TM3. mRNA chains representing OP164[cys115], OP164[cys124] and OP164[cys132] were translated in vitro in the presence of semi-permeabilised cells and treated as described in the legend of Figure 4.2. As before, uncross-linked doubly-glycosylated and non-glycosylated opsin chains are indicated with (ii) and (i) respectively, while truncated opsin chains lacking a C-terminal HA tag are denoted by asterisks. Distinct cross-linking adducts to Sec61α and Sec61β are indicated with ‘α’ and ‘β’.
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
92
4.6 TM3 exits the translocon upon chain extension
Since cys115 of TM3 gave clear adducts to both the Sec61α and Sec61β subunits in
OP164, the effect of altering chain length upon the cross-linking partners of TM3 was
investigated by using integration intermediates of different lengths, all containing a
cysteine probe at position 115. In this case, integration intermediates ranging from
OP150[cys115] to OP357[cys115] were subjected to BMH mediated cross-linking and
the resulting products identified by immunoprecipitation.
At a chain length of 150 residues, the shortest analysed in this case, adducts with the
Sec61β subunit (Fig. 4.7, lanes 2-5, β) and a ∼21 kDa component that was similar to a
previously identified ribosomal protein were observed (Fig. 4.7, lanes 2-3, R) (Laird &
High, 1997). Thus, at this stage, opsin TM3 appears to be in the process of leaving the
ribosomal exit site and engaging the ER translocon. Distinct adducts with both Sec61α
and Sec61β were seen for the longer integration intermediates, OP164[cys115] and
OP174[cys115], indicating that cys115 of TM3 has now fully engaged the ER
translocon (Fig. 4.7, lanes 7-10, 12-15, α and β). A higher molecular weight product
representing species containing HA-tagged opsin chains and both the α and β subunits
of the Sec61 complex were also observed with OP164[cys115] and OP174[cys115]
(Fig. 4.7, lanes 9-10, 14-15, αβ).
On the other hand, no authentic adducts to subunits of the Sec61 complex were seen for
nascent chains longer than 174 residues (Fig. 4.7, lanes 17-21, 23-27, 29-33, 35-39, 41-
45). In some cases, endogenous radiolabelled Sec61α molecules were
immunoprecipitated with the Sec61α antibody (Fig. 4.7, •, see also Chapter 3.4), while
products immunoprecipitated with α-Sec61β were adducts formed with incomplete
opsin chains which lacked the C-terminal HA tag (Fig. 4.7, c.f. 18 and 21, 24 and 27, 30
and 33, 36 and 39, 42 and 45, ). On this basis, I conclude that TM3 fully engages the
translocon from a nascent chain length of 164 residues and has exited the translocon
when the nascent chain is 204 residues long. Since the extension of the nascent chain
from 164 residues to 204 residues results in opsin TM4 (residues 151 to 173)
(Palczewski et al., 2000) being synthesised in full and predicted to have largely exited
the ribosome, TM3 is likely to have left the Sec61 complex when TM4 begins to engage
the translocon (see Chapter 5).
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
93
Figure 4. 7 BMH mediated cross-linking of integration intermediates containing TM3 specific cysteine probes to ER translocon components. Integration intermediates of the chain lengths indicated and containing cys115 probes were synthesised in a rabbit reticulocyte translation system in the presence of digitonin-permeabilised cells and treated with either BMH (+) or DMSO (-). Samples were denatured in 1% SDS and subjected to immunoprecipitation using α-opsin, α-HA, α-Sec61α, α-Sec61β and a non-related antisera (NS). Uncross-linked doubly- and non-glycosylated opsin chains are denoted with (ii) and (i) respectively. Endogenous, radiolabelled Sec61α molecules which were immunoprecipitated by the Sec61α antibody are marked a filled circle while Sec61β adducts to truncated opsin chains are denoted with an empty circle. Cross-linking products to Sec61α and Sec61β are indicated by ‘α’ and ‘β’ while nascent chains cross-linked to both Sec61α and Sec61β are marked with ‘αβ’. Adducts with a putative ∼21 kDa ribosomal protein are also indicated, see lanes 2 and 3, R (see also Laird and High, 1997).
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
94
4.7 TM3 relocation is independent of the presence of subsequent TM domains
TM3 appeared to be fully engaged with the Sec61 translocon when the opsin chain was
164 residues long and to have left the Sec61 complex by the time the nascent chain
reached 204 residues. As with the TM1 analysis in Section 4.3, the influence of
subsequent TM domains (i.e. TM4 to TM7) on the relocation of TM3 was investigated.
The region of opsin C-terminal of TM3, including TM4 to TM7, was replaced with a
hydrophilic region from the secretory protein, preprolactin, to give the OPTM1-
3PPL[cys115] construct (Fig. 4.8A). Integration intermediates of 164 and 204 residues,
comparable to those examined during the analysis of TM3, were generated.
The integration intermediates of this chimera, OPTM1-3PPL164[cys115] and OPTM1-
3PPL204[cys115], were then analysed by BMH mediated cross-linking as before (c.f.
section 4.2). OPTM1-3PPL164[cys115] gave a very similar cross-linking pattern to
OP164[cys115] displaying clear adducts with Sec61α and Sec61β (Fig. 4.8B, lanes 2-5,
c.f. Fig. 4.7, lanes 7-10, α and β). Extension of the OPTM1-3PPL nascent chain to 204
residues resulted in a loss of any authentic cross-linking to these translocon
components, implying that TM3 has moved out of the translocon by this chain length
(Fig. 4.8B, lanes 7-10). I therefore conclude that the relocation of TM3 from the ER
translocon does not require the presence of the later TM domains (TM4 to TM7) and
appears to be driven solely by chain extension.
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
95
Figure 4. 8 A) A schematic representation of OPTM1-3PPL[cys115] polypeptide chain. Residues 1 to 142 of opsin which contained TM1 to TM3 (in black) were fused to residues 31 to 229 of preprolactin (PPL) (in grey) to generate OPTM1-3PPL. A cysteine probe was located within TM3 at position 115 and is indicated with a white star. B) BMH cross-linking of OPTM1-3PPL[cys115] integration intermediates to translocon components. mRNA encoding OPTM1-3PPL164[cys115] and OPTM1-3PPL204[cys115] was translated in the presence of semi-permeabilised cells and treated as described in the legend of Figure 4.2. Glycosylated and non-glycosylated uncross-linked opsin chains are indicated with (ii) and (i) while truncated nascent chains are marked with asterisks. Authentic adducts to Sec61α and Sec61β are indicated with ‘α’ and ‘β’.
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
96
4.8 Nascent opsin chains are associated with a single copy of the Sec61 complex
during integration
An implicit assumption of site-specific cross-linking analysis exploited above is that the
environment of the nascent chain as a whole is independent of probe location. This
assumption allows a composite model to be generated by combining the results obtained
from the analyses of single cross-linking probes placed at different locations within the
same polypeptide chain. In order to test this assumption, and also to further characterise
the composition of the translocon, opsin nascent chains containing two cysteine probes
were generated. In OP[cys56,115], one cysteine probe is placed at position 56 within
TM1 while the second cysteine probe is at position 115 within TM3 of opsin.
Integration intermediates of OP[cys56,115] of two chain lengths, 164 and 204 residues,
were first synthesised in the presence of semi-permeabilised cells. BMH cross-linking
and immunoprecipitations were then carried out as before (described in section 4.2).
The cross-linking patterns of OP164[cys56,115] and OP204[cys56,115] reflected the
results observed earlier for integration intermediates with only a single cysteine probe
(c.f. Fig. 4.2, lanes 22-25, Fig. 4.7, lanes 7-10, Fig. 4.9, lanes 2-5). Clear adducts were
seen with Sec61α, Sec61β and PAT-10 (Fig. 4.9, lanes 2-5, α, β and P). In addition, a
higher molecular weight adduct of ∼40 kDa was also immunoprecipitated with the α-
Sec61β antibody (Fig. 4.9, lane 5, β+P). This adduct is likely to represent a single
OP164 nascent chain simultaneously cross-linked to both Sec61β (from cys115) and
PAT-10 (from cys56). When the nascent chain is extended to 204 residues, only a single
distinct adduct to PAT-10, most likely from cys56, was observed (c.f. Fig. 4.2, lanes 27-
28, Fig. 4.7, lanes 17-18, Fig. 4.9, lanes 7-8). Therefore, the analysis of nascent chains
with double cysteine probes confirmed the results obtained from the analysis of
integration intermediates with a single cysteine probe, indicating that the nascent chains
as a whole occupy the same environment independent of the cysteine probe location.
In the single cysteine probe analysis of the OP164 integration intermediate, both cys56
and cys115 can individually form adducts with Sec61α (Fig. 4.2, lane 24, Fig. 4.7, lane
9), therefore, if the nascent chain OP164 is adjacent to multiple copies of Sec61α,
having both cys56 and cys115 within a single nascent chain would in principle allow
the integration intermediate to cross-link two copies of the Sec61α subunit at the same
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
97
time. A glycosylated OP164 nascent chain cross-linked to two molecules of Sec61α is
expected to have a mobility of ∼100 kDa. In fact, no evidence of such a species
containing two copies of the Sec61α subunit cross-linked to a single opsin chain was
observed (Fig. 4.9, lane 4). An adduct of ∼70 kDa was seen, but this adduct contained
both Sec61α and Sec61β, and was also observed in the earlier analysis of
OP164[cys115] (c.f. Fig. 4.9, lanes 4-5, Fig. 4.7, lanes 9-10, (αβ)). These results
indicate that the TM domains of nascent opsin chains are most likely adjacent to only a
single copy of the Sec61α molecule at this stage of biosynthesis. The double probe
studies also showed that PAT-10 is adjacent to the functional Sec61 complex since the
OP164 integration intermediate can be simultaneously cross-linked to both Sec61β and
PAT-10.
Figure 4. 9 BMH cross-linking with OP164 and OP204 integration intermediates containing double cysteine probes. OP164 and OP204 integration intermediates of [cys56,115] were translated in the presence of semi-permeabilised cells and then treated as described in the legend of Figure 4.2. Doubly-glycosylated and non-glycosylated opsin chains which were not cross-linked were indicated with (ii) and (i) respectively. Asterisks denote opsin chains which lack the C-terminal HA tag. Cross-linking products to Sec61α, Sec61β, PAT-10 and Sec61α-Sec61β dimer are indicated with ‘α’, ‘β’, ‘P’ and ‘αβ’ respectively. Nascent chains which were simultaneously cross-linked to Sec61β and PAT-10 were marked with ‘β+P’.
Chapter 4 – The analysis of TM1, TM2 and TM3 of opsin
98
4.9 Summary
The analysis of the membrane integration of the N-terminal region of opsin revealed
that TM1 interacts with the ER translocon in two discrete phases, each generating a
distinct set of cross-linking partners. In one phase, TM1 is adjacent to both Sec61α and
Sec61β, while in the other phase, TM1 is adjacent to Sec61α and PAT-10. The absence
of TM2 to TM7 of opsin affects TM1 relocation through these phases, delaying the
relocation from the former. The analysis of TM3 integration showed that TM3 engages
the Sec61 complex from a chain length of ∼150 residues and leaves the translocon when
the nascent chain reaches ∼204 residues in length. However, TM3 exit from the
translocon is independent of the presence of TM4 to TM7. Results obtained from the
double probe studies confirmed the observations made with single probes and suggested
that the TM domains of opsin nascent chains associate with only one copy of the Sec61
complex. In addition, analysis of integration intermediates with double cysteine probes
showed that the novel PAT-10 component is in close proximity to a functional Sec61
complex.
Principal conclusions
1) Opsin TM1 experiences at least two distinct phases of Sec61 mediated
environments during opsin biosynthesis. The movement of TM1 through these
phases is dependent on the presence of TM2 and/or TM3.
2) Opsin TM3 exits the translocon as soon as TM4 has been synthesised, but unlike
TM1, relocation of TM3 is independent of the presence of TM4.
3) The TM domains of the nascent opsin chains engage with only a single copy of
the Sec61 complex.
4) PAT-10 is adjacent to a functional Sec61 translocon.
Chapter 5 – The analysis of TM4 to TM7 of opsin
99
CHAPTER 5 Results
The integration of the C-terminal
region of opsin:
TM4 to TM7
Chapter 5 – The analysis of TM4 to TM7 of opsin
100
5.1 Introduction
My examination of the molecular environment of the N-terminal region of opsin,
specifically TM1 to TM3, revealed the distinct behaviour of different TM domains
during their integration at the ER. In this chapter, my study of opsin integration is
further extended to incorporate the remaining opsin TM domains, namely TM4 to TM7.
The prior analysis of TM3 integration had indicated that the ‘gross’ location of a
cysteine probe within the TM domain may influence the pattern of its cross-linking to
translocon components. With this in mind, the molecular environment of at least two
different cysteine probes within each TM domain was initially examined. A unique
cysteine residue was introduced at a specific position within a particular TM domain of
a cysteine-null opsin mutant and a range of integration intermediates of increasing
nascent chain lengths was generated as before (refer to Figure 4.1 in Chapter 4). This
was combined with the use of the sulphydryl-specific homobifunctional cross-linking
reagent BMH to enable a site-specific cross-linking analysis of the proteinaceous
environment of each integration intermediate.
5.2 TM4 exits the translocon upon chain extension
In comparison to some of its other TM domains which are atypically long, opsin TM4 is
a fairly typical transmembrane span, comprising residues 151 to 173 (Palczewski et al.,
2000). Single cysteine probes were placed towards the N-terminus of TM4, at residue
154, and more centrally at residue 165. In the first instance, an integration intermediate
of 204 residues was generated since this was predicted to provide insights into the
environment of TM4 as it is emerging from the ribosome. OP204[cys154] and
OP204[cys165] chains were synthesised in a rabbit reticulocyte translation system in the
presence of digitonin-permeabilised mammalian cells and membrane-integrated
intermediates were isolated by centrifugation. Cross-linking was carried out by
incubation with BMH, with control samples being mock-treated with solvent (DMSO).
The resulting adducts were analysed by immunoprecipitation as previously described.
OP204[cys154] gave a distinct adduct with Sec61β (Fig. 5.1, lanes 2-5, β), but produced
no authentic adducts with Sec61α that were immunoprecipitated with the α-HA
antibody (Fig. 5.1, c.f. lanes 3 and 4, ). A faint ~70 kDa adduct is recognised by both
Chapter 5 – The analysis of TM4 to TM7 of opsin
101
the α-opsin and α-HA antisera (Fig. 5.1, lanes 2 and 3, see X), but this product is not
recovered with the α-Sec61α antiserum (Fig. 5.1, lane 4) and its origin is presently
unknown. OP204[cys165] did not produce discrete adducts with any translocon
components (Fig. 5.1, lanes 7-10). On the basis that OP204[cys154] could be cross-
linked to Sec61β, I conclude the N-terminal region of TM4 has begun to engage the
Sec61 translocon in the 204-residue integration intermediate.
In order to examine the environment of TM4 at different stages during its integration,
BMH-mediated cross-linking was performed with increasing lengths of polypeptide
chains containing the cys154 probe. Samples were treated as before and
immunoprecipitations were carried out with α-opsin, α-HA, α-Sec61α, α-Sec61β and a
non-related (NS) antisera. As observed earlier, cys154 gave an adduct with Sec61β
when the nascent chain is 204 residues (Fig. 5.2, lanes 2-6, β), but upon chain extension
to 259 residues and beyond, no authentic adducts with any translocon components were
observed (Fig. 5.2, lanes 8-12, 14-18, 20-24 and 26-30). Some of the longer chains did
show cross-linking to Sec61β, but none of these species were recognised by the α-HA
antibody and are most likely adducts with shorter nascent chains (Fig. 5.2, c.f. lanes 9
and 12, 15 and 18, 21 and 24, 27 and 30, ). This is particularly apparent where the
cross-linking products are shorter than the nascent chains used for adduct formation
(Fig. 5.2, lanes 24 and 30, see also Meacock et al., 2002).
Products detected with the α-Sec61α antibody were due to the presence of radiolabelled
Sec61α molecules resulting from the translation of endogenous mRNA and these
species do not represent true adducts with Sec61α (Fig. 5.2, •, see Chapter 3.4).
Interestingly, adducts to a ∼10 kDa molecule were observed with the long integration
intermediates, from OP259 to OP357 (Fig. 5.2, lanes 8-9, 14-15, 20-21, 26-27, P?).
Although these adducts were weaker, they resemble the adducts observed between TM1
and PAT-10 (see Chapter 4, Fig. 4.2), implying that TM4 may be adjacent to a PAT-10
like molecule. Taken together, these results imply that TM4 engages the Sec61 complex
at a chain length of 204 residues and moves away from the translocon by the point at
which a chain length of 259 residues has been reached - a stage at which TM5 has been
synthesised. The absence of any adducts with the Sec61 subunits for any of the longer
integration intermediates, OP304 to OP357, indicates that TM4 does not re-associate
Chapter 5 – The analysis of TM4 to TM7 of opsin
102
with the translocon during the later stages of opsin biosynthesis (Fig. 5.2, lanes 14-18,
20-24, 26-30).
Figure 5. 1 BMH cross-linking of OP204 integration intermediates from two different single cysteine probes within TM4. OP204[cys154] and OP204[cys165] were synthesised in a rabbit reticulocyte translation system and membrane-integrated products isolated by centrifugation. The samples were treated with BMH (+) or DMSO (-), quenched, and denatured with 1% SDS. Immunoprecipitations using α-opsin, α-HA, α-Sec61α and α-Sec61β antisera were carried out. Uncross-linked doubly-glycosylated and non-glycosylated nascent opsin chains are indicated with (ii) and (i) respectively, while truncated opsin chains which lack the C-terminal HA tag are marked with asterisks. The adduct with Sec61β is indicated with ‘β’. A species containing Sec61α that does not align with the products recognised by the α-HA serum is indicated by ‘ ’ and an adduct with an unknown cross-linking partner by ‘X’.
Chapter 5 – The analysis of TM4 to TM7 of opsin
103
Figure 5. 2 Cross-linking of OP[cys154] integration intermediates of increasing chain lengths to translocon components. Integration intermediates of OP[cys154] were synthesised by in vitro translation and subjected to BMH cross-linking and immunoprecipitation as described in the legend to Figure 5.1. Endogenous, radiolabelled Sec61α molecules are indicated with ‘•’, while Sec61β-containing adducts to truncated opsin chains lacking the C-terminal HA tag are marked with ‘ ’. Putative adducts to a PAT-10 like molecule are labelled ‘P?’. Other symbols are as previously defined in the legend to Figure 5.1.
Chapter 5 – The analysis of TM4 to TM7 of opsin
104
5.3 Opsin TM5 engages the translocon at 259 residues
In order to examine the integration of opsin TM5 (residues 200 to 225, Palczewski et
al., 2000) into the lipid bilayer by a similar cross-linking approach, single cysteine
probes were introduced at three different locations within TM5, namely residue 204
near its N-terminus, residue 217 near its centre, and residue 229 at the C-terminal
boundary of TM5. Integration intermediates of 259 residues, a length at which TM5 is
predicted to be largely out of the ribosome, were generated and their proximity to
components of the ER translocon examined by cross-linking as previously described.
Interestingly, in this case, only the nascent chain with a cysteine probe at the boundary
of TM5, OP259[cys229], generated authentic cross-linking adducts with Sec61
components, i.e. the Sec61β subunit (Fig. 5.3, see lanes 12-15, β). Whilst there may also
be some cross-linking to Sec61α from the OP259[cys229], this was certainly not
unambiguous and there was no compelling evidence of any such product in the α-HA
recovered material (Fig. 5.2, lane 13, ∼66 kDa range). In contrast, the OP259[cys204]
and OP259[cys217] showed no authentic adducts with subunits of the Sec61 complex
(Fig. 5.3, lanes 3-5 and 8-10), and only endogenously encoded Sec61α (Fig. 5.3, lanes 4
and 9, •) and incomplete chains cross-linked to Sec61β (Fig. 5.3, lane 10, ) were seen.
Since cys229 is adjacent to the Sec61β subunit in the 259 residue integration
intermediate, I conclude that opsin TM5 has engaged the Sec61 translocon at this stage
of biosynthesis.
Chapter 5 – The analysis of TM4 to TM7 of opsin
105
Figure 5. 3 BMH cross-linking of OP259 integration intermediates using different TM5 specific probes. mRNA chains encoding OP259[cys204], OP259[cys217] and OP259[229] were translated in vitro in the presence of semi-permeabilised cells. BMH cross-linking and immunoprecipitations were performed as described in the legend to Figure 5.1. Uncross-linked doubly-glycosylated and non-glycosylated opsin chains are indicated with (ii) and (i) respectively. Truncated opsin chains which lack the C-terminal HA tag are marked with asterisks. Endogenous radiolabelled Sec61α molecules are denoted with ‘•’ and a Sec61β-containing adduct to truncated nascent opsin chains is marked with ‘ ’. Adducts with Sec61β are indicated with ‘β’.
Chapter 5 – The analysis of TM4 to TM7 of opsin
106
5.4 Opsin TM5 is adjacent to a PAT-10-like molecule during its integration
The analysis of the C-terminal region of opsin requires long integration intermediates to
be generated for cross-linking, since the bulk of the polypeptide must be synthesised to
expose this part of the protein. As is readily apparent from the analysis of TM5 shown
above, adducts obtained with long nascent chains are often very weak and diffuse (Fig.
5.3, see lanes 3, 8 and 13). In addition, significantly more products lacking the C-
terminal HA tag were present (Fig. 5.3, lanes 2, 7 and 12, *, see also Chapter 3.2). As a
consequence, the interpretation of the resulting cross-linking products can be difficult.
In order to facilitate the analysis of TM5 to TM7 of opsin, a truncated version of the
opsin polypeptide chain was generated in which the first 35 residues of opsin, which
contains the two N-glycosylation sites, were fused to a C-terminal region of opsin
containing TM5 to TM7 (residues 195 to 348) to form the OPN/5-7 polypeptide chain
(Fig. 5.4A). In vivo expression has shown that opsin TM5 to TM7 (residues 195 to 348)
is one of several fragments that behaves as an ‘independent folding domain’ (Ridge et
al., 1996). The potential advantage of this deletion mutant is that shorter integration
intermediates can be used for the cross-linking analysis of TM5 to TM7.
In the first instance, the topology of the OPN/5-7 polypeptide chains was ascertained by
determining if cross-linking products were formed with doubly glycosylated nascent
chains. An integration intermediate of OPN/5-7 with a cysteine probe in the first TM
domain (OPN/5-7 259[cys229]) was subjected to BMH cross-linking and
immunoprecipitation, followed by treatment with endoglycosidase H to remove the
asparagine-linked glycan groups. BMH-dependent adducts to Sec61α and a ∼20 kDa
unidentified molecule were observed (Fig. 5.4C, lanes 2-4, α and X) and these adducts
were endoglycosidase H sensitive, as shown by the faster migration of the products
(Fig. 5.4C, lanes 7-9, α and X), implying that these adducts were formed with OPN/5-7
nascent chains which were glycosylated. This indicated that OPN/5-7 polypeptide
chains have been inserted in the correct orientation into the ER membrane.
Chapter 5 – The analysis of TM4 to TM7 of opsin
107
Figure 5. 4 (A) A schematic diagram of the OPN/5-7 polypeptide chain. The first 35 residues of opsin was fused to a C-terminal region of opsin (residues 195 to 348) which contains TM5, TM6 and TM7 (TM domains are shown in black). (B) Predicted topology of OPN/5-7 polypeptide chain. TM domains are shown as grey boxes while the numbers indicate the position of TM5 to TM7 in context of the OPN/5-7 polypeptide chain. N-glycosylation sites are marked with ‘Y’ and the C-terminal HA tag is represented with a grey line. (C) Cross-linking adducts are formed with glycosylated OPN/5-7 nascent chains. An integration intermediate of OPN/5-7 with a single cysteine probe in TM5 was subjected to BMH mediated cross-linking and immunoprecipitation as described in legend 5.1. Duplicate samples were additionally treated with endoglycosidase H to cleave the asparagine-linked glycan groups (+ Endo H). Adducts with Sec61α are labelled ‘α’ and other distinct BMH dependent cross-linking adducts are marked with ‘X’. Other symbols are as previously defined in Figure 5.1.
Chapter 5 – The analysis of TM4 to TM7 of opsin
108
However, whilst the topology of TM5 to TM7 should be the same as in the wild type
protein (Fig. 5.4B), the relative position of TM5, TM6 and TM7 in the context of the
polypeptide chain has changed (i.e. TM5 is now the first TM domain and so on). For
this reason, a cross-linking analysis with the normal opsin polypeptide chain was
performed concurrently with that of OPN/5-7 to compare the two species. Although the
analysis of TM5 integration should ideally utilise a cysteine probe located within the
TM domain, earlier analysis of cys204 and cys217 introduced into TM5 failed to
produce any adducts even at the shortest chain length (OP259) (see section 5.3). Hence,
further analysis of TM5 was carried out using integration intermediates with a cysteine
residue at position 229. Cys229 is located in the hydrophilic loop just at the C-terminal
boundary of TM5, thus, cross-linking performed with integration intermediates of the
cys229 mutant should reflect the environment of at least the C-terminal end of the TM
domain.
Four integration intermediates of full length opsin, OP259[cys229], OP304[cys229],
OP339[cys229] and OP357[cys229], were generated by in vitro translation and
subjected to BMH-mediated cross-linking and immunoprecipitation as before. As
described earlier in section 5.3, OP259[cys229] formed adducts with Sec61β (Fig. 5.5A,
lanes 2-6, β). In this experiment, it also appeared that there may be an authentic adduct
with Sec61α (Fig. 5.5A, c.f. lanes 3 and 5, α). At the longer chain lengths of 304, 339
and 357 residues, Sec61α- and Sec61β-containing adducts were present to some degree
but it was impossible to determine whether these adducts were immunoprecipitated with
the α-HA antibody (Fig. 5.5A, c.f. lanes 9 and 11, 15 and 17, 21 and 23, α? and β?).
What was clear was that distinct adducts with a ∼10 kDa protein were observed with
each of these longer nascent chains (Fig. 5.5A, lanes 8-9, 14-15, 20-21, P). This adduct
is remarkably similar to the species observed between a TM1 cysteine probe and PAT-
10 (see Chapter 4.2, Fig. 4.2), implying that TM5 is adjacent to a PAT-10-like
molecule.
When a similar cross-linking analysis was performed with the equivalent OPN/5-7
intermediates, adducts with Sec61 subunits were more distinct. Hence, the analysis of
OPN/5-7 259[cys229] revealed a prominent adduct to Sec61α, which was clearly
recognised by the α-HA antibody (c.f. Fig. 5.5A, lanes 3 and 5 to Fig. 5.5B, lanes 3 and
4, α). Similarly, when longer OPN/5-7 integration intermediates were examined, distinct
Chapter 5 – The analysis of TM4 to TM7 of opsin
109
adducts to Sec61α were observed even until the longest chain length (Fig. 5.5B, lanes 7-
9, 12-14, 17-19, α). Cross-linking to a ∼10 kDa protein from cys229 was also seen from
OPN/5-7 304 until OPN/5-7 357 (Fig. 5.5B, lanes 7-8, 12-13, 17-18, P).
Figure 5. 5 BMH cross-linking of cys229 integration intermediates of A) normal-length opsin polypeptide chain, and B) OPN/5-7 polypeptide chain. OP[cys229] nascent chains of 259, 304, 339 and 357, and OPN/5-7 integration intermediates of equivalent lengths, were synthesised by in vitro translation and subjected to BMH cross-linking and immunoprecipitations as described in the legend to Figure 5.1. Putative adducts to Sec61α and Sec61β are denoted with ‘α?’ and ‘β?’ respectively, while clear adducts to Sec61α and Sec61β are indicated with ‘α’ and ‘β’. PAT-10 adducts are marked with ‘P’ and putative adducts with ribosomal proteins are labelled ‘R’ (see Laird and High, 1997). Other symbols are as defined in the legend to Figure 5.3.
Chapter 5 – The analysis of TM4 to TM7 of opsin
110
OPN/5-7 259[cys229] gave a discrete adduct to Sec61β (Fig. 5.5B, lanes 2-5, β).
However, for the longer OPN/5-7[cys229] nascent chains, it is difficult to ascertain
whether the faint Sec61β adducts that were still observed, were formed with authentic
integration intermediates, since the PAT-10 adducts mask the presence of any Sec61β
adducts due to their similar molecular weights (Fig. 5.5B, lanes 10, 15, 20, β?). On the
basis of adduct formation with Sec61α, I concluded that TM5 engages the Sec61
complex at 259 residues and remains associated with the translocon until the entire
opsin polypeptide chain has been synthesised. TM5 is also adjacent to a PAT-10-like
molecule from 304 residues and remains in close proximity to it throughout the
remainder of nascent chain synthesis.
5.5 Opsin TM1 and TM5 are adjacent to a single copy of PAT-10
One of the striking features of the behaviour of opsin TM1 is that it associates with the
novel ∼10 kDa protein, PAT-10, as soon as the nascent chain is long enough to allow
the next TM domain (TM2) to emerge from the ribosome, and this interaction lasts for a
prolonged period of opsin synthesis (see Fig. 4.2) and probably until the completion of
opsin biosynthesis and chain termination (Meacock et al., 2002). Surprisingly, TM5 is
also adjacent to a ∼10 kDa protein at a stage when the subsequent TM domain (TM6)
has been synthesised, and adduct formation is observed for all the longer chains
analysed as integration intermediates. As the identity of PAT-10 is unknown, it is
difficult to ascertain if TM5 is interacting with PAT-10 or a distinct ∼10 kDa molecule
with similar properties. In order to better establish whether TM5 is adjacent to PAT-10,
and to determine whether the nascent opsin chain associates with multiple copies of
identical or different ∼10 kDa proteins, an OP304 integration intermediate with one
cysteine probe in TM1 (cys56) and a second cysteine probe flanking TM5 (cys229) was
generated. A chain length of 304 residues was chosen because it is the shortest
integration intermediate which would allow both TM1 and TM5 to cross-link the ∼10
kDa protein (see Fig. 5.5A, P).
BMH-mediated cross-linking of OP304[cys56,229] produced only one major adduct
which corresponded to the nascent chain being cross-linked to a single copy of a ∼10
kDa protein (Fig. 5.6, lanes 2-3, P). If the OP304 integration intermediate were to cross-
link two molecules of the ∼10 kDa protein from both TM1 and TM5, then a higher
Chapter 5 – The analysis of TM4 to TM7 of opsin
111
molecular weight adduct of ∼50 kDa would be observed. Since only one copy of the
∼10 kDa protein is adjacent to OP304[cys56,229], the protein found adjacent to TM5 is
presumably the same molecule that is adjacent to TM1. Hence, I conclude that TM5 is
most likely associated with PAT-10, and that there is only one copy of PAT-10 adjacent
to the functional Sec61 translocon.
Figure 5. 6 Double probe analysis of the OP304 integration intermediate. mRNA transcripts encoding OP304[cys56,229] nascent chains were translated in vitro in the presence of semi-permeabilised cells. Membrane-integrated products were subjected to BMH cross-linking and immunoprecipitations as described in the legend to Figure 5.1. Uncross-linked doubly-glycosylated and non-glycosylated nascent opsin chains are labelled (ii) and (i), while truncated nascent chains are marked with an asterisk. The presumptive adduct to PAT-10 is indicated with ‘P’.
Chapter 5 – The analysis of TM4 to TM7 of opsin
112
5.6 Opsin TM6 engages the translocon throughout opsin biosynthesis
The molecular environment of TM6 during opsin biosynthesis was initially examined
by introducing a unique cysteine probe at two separate locations within the TM domain,
namely residue 254 in the middle of TM6 or residue 275 near the C-terminal end of the
TM domain. For both cysteine positions, integration intermediates of 304 residues in
which TM6 is presumed to have fully emerged from the ribosome, were generated by in
vitro translation. BMH-mediated cross-linking and immunoprecipitations were carried
out as previously described in section 5.2.
Whilst OP304[cys275] gave a distinct adduct with Sec61β that was also recognised by
the α-HA antibody (Fig. 5.7, lanes 7-10, β), it was not clear that the Sec61β adduct seen
with OP304[cys254] was recognised by the α-HA antibody and hence uncertain as to
whether it was formed with the 304 residue long chain in this case (Fig. 5.7, lanes 2-4,
β?). Likewise, whilst products were immunoprecipitated with the α-Sec61α serum in
both cases (Fig. 5.7, lanes 4 and 9, •), no obvious equivalent was seen in the
accompanying α-HA immunoprecipitation (Fig. 5.7, lanes 3 and 8). Thus, with such
long intermediates, only evidence of proximity between the C-terminal region of TM6
and the Sec61β subunit could be unequivocally determined.
Since cys275 gave clear and unambiguous data, integration intermediates of three
different lengths, 304, 339 and 357, each with a single cysteine at residue 275, were
analysed by BMH-mediated cross-linking. As before, OP304[cys275] gave a distinct
adduct with Sec61β, indicating that TM6 has engaged the Sec61 complex (Fig. 5.8A,
lane 2-6, β). Extending the nascent chain to 339 residues allowed TM6 to form discrete
adducts with both Sec61α and Sec61β (Fig. 5.8A, lanes 8-12, α and β). Upon further
extension to OP357, an authentic adduct with Sec61α appeared to be maintained (Fig.
5.8A, lanes 14, 15 and 17, α) whilst the authenticity of a potential Sec61β adduct was
less obvious (Fig. 5.8A, lanes 14, 15 and 18). As seen for the analysis of TM5 (section
5.4), adducts with long nascent chain lengths were diffuse and difficult to substantiate
by parallel immunoprecipitation using the α-HA sera. In order to try to resolve some of
the ambiguities resulting from the cross-linking of these longer chains, a parallel
analysis was performed using the opsin deletion mutant, OPN/5-7. This analysis
revealed a similar, albeit more clear cut, pattern of adduct formation with Sec61
Chapter 5 – The analysis of TM4 to TM7 of opsin
113
subunits. Thus, an adduct with Sec61β was seen for the shortest integration
intermediate, OPN/5-7 304[cys275], while adducts with both Sec61α and Sec61β were
observed with OPN/5-7 339[cys275] and OPN/5-7 357[cys275] (Fig. 5.8B, lanes 2-5, 7-
10, 12-15, α and β). Taken together, these results suggest that TM6 engages the Sec61
complex when the nascent chain is 304 residues long and remains associated with the
translocon throughout the remainder of opsin synthesis. Since the cross-linking pattern
to translocon components changes at increasing chain lengths, it is plausible that TM6
relocates from its initial location within the Sec61 complex during the extension of the
nascent chain, along the lines of the process previously described for TM1.
Figure 5. 7 BMH dependent cross-linking from single cysteine probes in opsin TM6. In vitro translation of OP304[cys254] and OP304[cys275], BMH cross-linking and immunoprecipitation were carried out as described in the legend of Figure 5.1. Uncross-linked doubly-glycosylated, non-glycosylated and truncated opsin chains are indicated with (ii), (i) and ‘ ’ respectively. Distinct and putative adducts to Sec61β are labelled ‘β’ and ‘β?’ respectively. Presumptive endogenous radiolabelled Sec61α molecules are marked ‘•’.
Chapter 5 – The analysis of TM4 to TM7 of opsin
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Figure 5. 8 BMH cross-linking of cys275 integration intermediates of A) normal-length opsin polypeptide chains, and B) OPN/5-7 polypeptide chains. OP304[cys275], OP339[cys275], OP357[cys275] and their respective OPN/5-7 equivalents, were synthesised by in vitro translation in the presence of semi-intact cells and subjected to BMH cross-linking and immunoprecipitations as described in the legend of Figure 5.1. Adducts to Sec61α and Sec61β are labelled ‘α’ and ‘β’, while nascent chains simultaneously cross-linked to both Sec61α and Sec61β are indicated with ‘αβ’. Unidentified adducts are marked with ‘X’. Other symbols used were previously defined in the legend of Figure 5.3.
Chapter 5 – The analysis of TM4 to TM7 of opsin
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5.7 Opsin TM7 is associated with the translocon throughout opsin biosynthesis
In the analysis of the final TM domain, opsin TM7, the cross-linking patterns of
cysteine probes at two different positions were again analysed. One cysteine probe was
located near the N-terminus of TM7 (residue 287) and the other near its C-terminus
(residue 308). Integration intermediates with a nascent chain length of 339 residues,
OP339[cys287] and OP339[cys308], were then generated and subjected to BMH cross-
linking and immunoprecipitation.
Both OP339[cys287] and OP339[cys308] gave only distinct adducts with Sec61β that
also appeared to be recognised by the α-HA serum (Fig. 5.9, lanes 3 and 5, 8 and 10, see
β). The behaviour of the two TM7 probes with respect to adduct formation with Sec61α
was more difficult to assess (Fig. 5.9, lanes 3 and 4, 8 and 9). Probe 287 was selected
for further analysis, and initially two different intermediates investigated, namely
OP339[cys287] and OP357[cys287]. In both cases, adducts with Sec61β were observed
and appeared to be authentic by the criteria of α-HA immunoprecipitation (Fig. 5.10A,
lanes 3 and 5, 8 and 10, β). As before, the presence of adducts with Sec61α could not be
unambiguously distinguished (Fig. 5.10A, lanes 3 and 4, 8 and 9, α?). In order to better
understand TM7 integration, equivalent integration intermediates were analysed for the
OPN/5-7 polypeptide. As with the normal length chains, authentic adducts with Sec61β
were seen (Fig. 5.10B, lanes 3 and 5, 8 and 10, β). However, in this case, adducts with
Sec61α could also be definitely identified (Fig. 5.10B, lanes 3 and 4, 8 and 9, α). Taken
together, these data indicate TM7 engages the Sec61 translocon after emerging from the
ribosome and, under normal circumstances, remains associated with the ER translocon
for the remainder of opsin biogenesis.
Chapter 5 – The analysis of TM4 to TM7 of opsin
116
Figure 5. 9 BMH mediated cross-linking from distinct cysteine probes in opsin TM7. In vitro translation of OP339[cys287] and OP339[cys308], BMH cross-linking and immunoprecipitations were carried out as described in the legend of Figure 5.1. Symbols indicated were as previously defined in the legend of Figure 5.3.
Chapter 5 – The analysis of TM4 to TM7 of opsin
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Figure 5. 10 BMH cross-linking of cys287 integration intermediates of A) normal-length opsin polypeptide chain, and B) OPN/5-7 polypeptide chain. OP339[cys287], OP357[cys287] and their OPN/5-7 equivalents, were synthesised using a rabbit reticulocyte translation system in the presence of semi-permeabilised cells and treated with BMH as described in the legend of Figure 5.1. Symbols used were defined in the legend of Figure 5.8.
Chapter 5 – The analysis of TM4 to TM7 of opsin
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5.8 Summary
The C-terminal region of opsin, comprising TM4 to TM7, experiences distinct
molecular environments during their integration into the ER membrane. TM4 engages
the Sec61 complex as TM3 departs and is itself then ‘displaced’ as TM5 emerges from
the ribosome. In contrast, TM5, TM6 and TM7 all appear to remain in close proximity
to the ER translocon until the entire opsin polypeptide chain has been synthesised.
Interestingly, like TM1, TM5 is found adjacent to a component assumed to be PAT-10
during opsin biosynthesis. A double probe analysis indicated that only one copy of
PAT-10 is in close proximity to an integrating opsin polypeptide chain. The analysis of
the C-terminal TM domains of opsin was facilitated by the use of a shorter version of
opsin, OPN/5-7, which allowed unambiguous interpretation of the cross-linking data.
Conclusion
1) Opsin TM4 enters the Sec61 complex as TM3 departs, and then exits the
translocon itself once TM5 emerges from the ribosome.
2) Opsin TM5, TM6 and TM7 remain associated with the translocon throughout
polypeptide chain synthesis.
3) Both TM1 and TM5 of a single opsin chain are adjacent to a single copy of
PAT-10, suggesting that only one PAT-10 molecule is associated with a
functional ER translocon.
Chapter 6 – Probing the environment of a translocating opsin chain
119
CHAPTER 6 Results
Probing the environment of a
translocating nascent opsin chain
Chapter 6 – Probing the environment of a translocating opsin chain
120
6.1 Introduction
The previous chapters have focused on the identity of the ER translocon components
that are adjacent to specific TM domains of opsin during their integration into the
membrane. The distinct behaviour of each TM domain was demonstrated by their
different cross-linking patterns to subunits of the Sec61 complex and other ER
components, particularly PAT-10. One limitation of this approach was that the
relationship of TM domains to the lipid bilayer was not directly analysed. Hence, loss of
cross-linking to Sec61 subunits was taken to reflect a transfer to the phospholipid phase
in line with several previous studies (Martoglio et al., 1995; McCormick et al., 2003;
Mothes et al., 1997). In order to obtain an alternative view of TM environment during
opsin polypeptide chain elongation, a distinct approach to analyse the location of single
cysteine probes in opsin integration intermediates was explored.
Figure 6. 1 Structures of (a) 4-acetamido-4′-maleimidylstilbene-2-2′-disulfonic acid (AMS) and (b) QSY 9 C5-maleimide (QSY). Both AMS and QSY are sulphydryl specific modification reagents. Their molecular weight (MW) and their solubility properties are indicated.
AMS MW: 536.44
Water solubility: High
Lipid solubility: Low
QSY MW: 1083.3
Water solubility: Moderate
Lipid solubility: High
(a)
(b)
Chapter 6 – Probing the environment of a translocating opsin chain
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In this chapter, two monofunctional sulphydryl-specific reagents were used to examine
the ‘environment’ of TM3 during its movement from the ribosome into the ER
translocon. 4-acetamido-4′-maleimidylstilbene-2-2′-disulfonic acid (AMS) is a
hydrophilic molecule (Fig. 6.1), and thus reacts preferentially with free sulphydryl
groups in a hydrophilic environment, such as the cytosol, the aqueous translocon pore or
the lumen of the ER (Krishnasastry et al., 1994). On the other hand, QSY 9 C5-
maleimide (QSY) is a more hydrophobic molecule (Fig. 6.1) that would be predicted to
react with free sulphydryl groups in both hydrophilic and hydrophobic environments
including the phospholipid bilayer of the ER membrane. The rationale for this work was
to explore whether there was any correlation between the accessibility of a particular
cysteine residue to these two probes and the stage of opsin biogenesis that was explored.
As with the site-specific cross-linking analysis, this ‘probe accessibility’ study took
advantage of single cysteine residues located within the opsin chain. In this instance, I
focused on analysing opsin TM3 which I had previously found to exit the ER translocon
independently of TM4 to TM7 (see Chapter 4). Thus, integration intermediates of
increasing chain lengths were generated to mimic different stages of TM3 insertion and
the access of AMS and QSY to different cysteine probes present in these nascent chains
was determined.
6.2 AMS and QSY can modify cysteine probes in the ER lumen
Previous studies have shown that small molecules of ∼0.5 kDa can readily cross the ER
membrane and access the lumen (Le Gall et al., 2004). The movement of these
molecules is passive, although their mode of entry is not known. Since AMS and QSY
have molecular weights of ∼0.5 kDa and ∼1 kDa respectively, the permeability of the
ER membrane to AMS and QSY was first determined. An opsin integration
intermediate of 96 residues with a single cysteine probe at position 14 near its N-
terminus was generated for this purpose. At 96 residues, the nascent chain is long
enough to allow the full translocation of the opsin N-terminus into the ER lumen and it
is therefore N-glycosylated at both available sites (c.f. Fig. 4.2). Hence, cys14 can only
be modified by AMS and QSY if these reagents traverse the ER membrane.
Modification of the cysteine probe with AMS or QSY should result in a small increase
in molecular weight of the nascent chain, producing an alteration in the migration of the
product up on SDS PAGE.
Chapter 6 – Probing the environment of a translocating opsin chain
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mRNA transcripts encoding OP96[cys14] were translated in vitro in a rabbit
reticulocyte translation system supplemented with digitonin-permeabilised mammalian
cells, and the membrane fraction was isolated by centrifugation. The membrane-
integrated products were incubated with either AMS or QSY, or mock treated with
solvent (DMSO) only. The reaction was quenched by the addition of β-mercaptoethanol
and the samples were denatured with 1% SDS. The products were then recovered by
immunoprecipitation with the α-HA antiserum which recognised the C-terminal epitope
tag present in all the intermediates studied. Since modification with AMS or QSY was
expected to produce only small shifts in mobility on SDS PAGE, duplicate samples
were treated with endoglycosidase H to cleave the glycan groups attached to correctly
integrated opsin chains and increase any alteration in apparent mobility due to
modification.
This preliminary experiment showed that a shift in mobility could be observed for both
the AMS- and QSY-treated samples (Fig. 6.2a and b, c.f. lanes 1 and 2, 3 and 4). Most
importantly, these shifts were observed for the doubly-glycosylated opsin chains,
indicating that properly integrated nascent chains have been modified by AMS and
QSY (Fig. 6.2a, lanes 2 and 4, (ii)). Treatment of the nascent chains with
endoglycosidase H produced a more apparent change in the mobility of modified
polypeptide chains (Fig. 6.2b, c.f. lanes 1 and 2, 3 and 4). A comparison of the opsin
chains after endoglycosidase H treatment also suggested that the majority of the
polypeptides were modified by AMS and QSY, indicating that the environment of the
bulk of the nascent chains would be reported by this approach. Since cys14 is present in
the ER lumen, these results indicate that both AMS and QSY can cross the ER
membrane and react with sulphydryl groups present in the lumen. This behaviour
should allow ready access of both AMS and QSY to cysteine residues present in regions
of the polypeptide that are located in an aqueous environment in the context of an
integration intermediate (c.f. cartoon to the side of Figure 6.2).
Chapter 6 – Probing the environment of a translocating opsin chain
123
Figure 6. 2 AMS and QSY modification of OP96[cys14] integration intermediates. A schematic representation of the ribosome-nascent chain complex and the translocon is shown in the right panel. The translocon is represented by dark grey boxes, the TM domain is represented by a black box and the position of the cysteine residue is indicated by a star. The two glycosylation sites on the opsin chain are marked with ‘Y’. Membrane-associated OP96[cys14] integration intermediates were isolated as previously described (Chapters 4 and 5) and treated with AMS (+) or QSY (+), or mock-treated with DMSO (-). The reactions were quenched with β-mercaptoethanol, the samples were denatured with 1% SDS, and reaction products recovered by immunoprecipitation with the α-HA antiserum. Panel A shows samples as recovered, panel B shows samples after endoglycosidase H treatment to remove N-linked glycans. Doubly-glycosylated and non-glycosylated opsin chains are indicated with (ii) and (i) respectively.
Chapter 6 – Probing the environment of a translocating opsin chain
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6.3 Opsin nascent chains are modified by AMS in the absence of membranes
Having established proof of principle and shown that both AMS and QSY have ready
access to a cysteine probe in the lumenal side of the ER membrane, I next established
whether these reagents were suitable for the analysis of cysteine probes located in TM3.
To this end, single cysteine probes were introduced at three different locations relative
to TM3. The first probe was located at residue 107 within the hydrophilic loop
preceding TM3. A second unique cysteine was introduced at residue 115 to allow the
environment of the N-terminal region of TM3 to be monitored, while a third cysteine
residue was introduced at residue 124 to report the middle of TM3.
In the first instance, I established whether these different cysteine probes could be
modified in the absence of ER membranes such that a visible shift in mobility upon
SDS PAGE could be seen. Thus, polypeptide chains of four different lengths, OP130,
OP140, OP150 and OP164 were examined for each cysteine mutant. The exception is
cys124, where an OP130 chain could not be generated because the cysteine probe is so
close to the C-terminus that it is replaced by the 9 residue HA tag. The position of TM3
and the cysteine probes relative to the ribosome for each nascent chain length is
represented schematically in Figure 6.3. At the shortest chain length examined (130
residues), TM3 is only partly synthesised and the cysteine probes are predicted to be
still buried in the ribosome (Kowarik et al., 2002). As the nascent chains become
progressively longer, TM3 is expected to move out from the ribosome and by 164
residues, TM3 should have largely emerged. The generation of polypeptide chains with
these precise lengths allows the ‘environment’ of the single cysteine probes to be
monitored during nascent chain elongation. As a further control, cysteine-null chains for
each of the intermediates were also generated and subjected to identical treatments.
Chapter 6 – Probing the environment of a translocating opsin chain
125
Figure 6. 3 AMS modification of various OP130 to OP164 chains. In the schematic diagrams (top and right), TM3 is represented by a black box while the numbers indicate the relative positions of the single cysteine probes (star). Regions of the nascent chain which are not shown in the diagram are indicated with dotted lines. Nascent chains of 130 (a), 140 (b), 150 (c) or 164 residues (d) with single cysteine probes as indicated, were synthesised in vitro in the absence of membranes. Ribosome-nascent chain complexes were isolated and treated with either AMS (+) or DMSO (-). The reaction was quenched with β-mercaptoethanol and products recovered by immunoprecipitation using α-HA antiserum.
Chapter 6 – Probing the environment of a translocating opsin chain
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Cysteine-null polypeptide chains that contain no thiol groups showed no changes in
apparent molecular mass after incubation with AMS, indicating that the AMS reaction
is cysteine-specific (Fig. 6.3 panels a to d, lanes 7 and 8). When the nascent chain is 130
residues long, a small proportion of the OP130[cys115] intermediates showed a reduced
mobility (Fig. 6.3a, lanes 3 and 4). This implies that AMS can enter the ribosome tunnel
since cys115 is expected to be within the ribosome at 130 residues. However, in this
location, the majority of cysteine residues were not affected. A larger proportion of
OP130 chains were modified when the cysteine probe is placed at position 107,
implying greater AMS accessibility to residues nearer to the exit site of the ribosomal
tunnel (Fig. 6.3a, lanes 5 and 6). When longer polypeptide chains, OP140, OP150 and
OP164, were analysed, all the nascent chains exhibit a slower mobility in the presence
of AMS, irrespective of the location of the cysteine probe (Fig. 6.3b, c, d, lanes 1-6).
This shows that the single cysteine probes in the nascent chains can be modified by
AMS when present in a hydrophilic environment located near to or beyond the
ribosomal exit site.
6.4 The environment of cys124 in the OP150 and OP164 integration intermediates
is altered in the presence of the ER translocon
During the integration of opsin TM3 into the ER membrane, this region of polypeptide
must leave the ribosomal exit tunnel, enter the ER translocon, and finally relocate into
the lipid bilayer. In order to better understand this process, the access of AMS to TM3
was now re-investigated in the context of ribosome bound membrane integration
intermediates identical to those previously used for cross-linking studies. Different
stages of biosynthesis were represented by integration intermediates of increasing
lengths and the location of the cysteine probes was analysed as previously established
(c.f. Fig. 6.3). Experimental details were as previously described except that nascent
chains were synthesised in the presence of ER derived membranes allowing N-
glycosylation. For this reason, duplicate samples were treated with endoglycosidase H
to remove the glycans and enhance small mobility shifts (c.f. Fig. 6.2).
No shifts in mobility were observed with any of the cysteine-null nascent chains
confirming the specificity of AMS modification (Fig. 6.4a, b, c and d, lanes 7 and 8). As
before (section 6.3), only a small proportion of OP130 chains with a cys115 probe gave
Chapter 6 – Probing the environment of a translocating opsin chain
127
a molecular weight shift consistent with this residue being deep within the ribosome
(Fig. 6.4a, lanes 3 and 4). A much larger proportion of OP130 chains with a cys107
probe were modified, consistent with greater AMS accessibility as the probe is located
closer to the exit site (Fig. 6.4a, lanes 5 and 6).
At 140 residues, cysteine probes in all locations gave a shift in molecular weight due to
AMS modification (Fig. 6.4b, lanes 1-6). This was especially obvious after the
endoglycosidase H treatment and indicates that all of the cysteines are in a hydrophilic
environment. At this stage, cys107 may have emerged from the ribosome and have
entered the aqueous environment of the translocon pore, while cys115 and cys124 are
most likely still within the ribosomal tunnel. Clearly, by whatever route, AMS has ready
access to all three cysteine probes in the OP140 integration intermediate. Strikingly,
when the nascent chains were extended to 150 and 164 residues, only polypeptide
chains with cys107 and cys115 probes now produced an AMS-mediated mobility shift,
while nascent chains with cys124 displayed no change in apparent molecular weight
(Fig. 6.4c and d, c.f. lanes 1 and 2, to lanes 3-6). The simplest interpretation of this
result is that at these chain lengths, cys107 and cys115 have remained in an aqueous
environment, while cys124 has relocated to an environment that is not accessible to
AMS (c.f. cartoons to the right of Figure 6.4).
Chapter 6 – Probing the environment of a translocating opsin chain
128
Figure 6. 4 AMS modification of various OP130 to OP164 integration intermediates. The integration intermediates were synthesised and treated with AMS as described in the legend to Figure 6.3. Duplicate samples were treated with endoglycosidase H (+ EndoH) to remove N-linked glycans.
Chapter 6 – Probing the environment of a translocating opsin chain
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6.5 Cys124 in OP150 and OP164 integration intermediate is in a hydrophobic
environment
The absence of AMS modification for OP150[cys124] and OP164[cys124] suggests that
cys124 may be in a location that cannot be modified by AMS, for example, a
hydrophobic environment. Alternatively, the extension of the nascent chain from 140 to
150 or 164 residues may alter the nascent chain to fold into a conformation that
prohibits any modification of cys124. In order to distinguish between such possibilities,
the integration intermediates were analysed with a second sulphydryl specific reagent,
QSY. Since QSY is moderately hydrophobic, it should react with cysteine residues in
both hydrophobic and hydrophilic environments. If QSY can react with cys124 in
OP150 and OP164, this would suggest that it is the environment of the cysteine probe
that prevents its AMS modification and not the conformation of the nascent chain.
Identical integration intermediates to those shown in Figure 6.4 were treated with QSY
and the products analysed as before. As for AMS, the cysteine-null integration
intermediates were not modified with QSY, showing that QSY reaction is cysteine
specific (Fig. 6.5a, b, c and d, lanes 7 and 8). For OP130, a molecular weight shift was
observed for both cys107 and cys115, indicating that QSY can enter the ribosome (Fig.
6.5a, lanes 3-6). QSY modifications were also seen for all three cysteine residues in the
OP140 integration intermediate as indicated by changes in mobility similar to those
previously observed with AMS (Fig. 6.5b, lanes 1-6, see also Fig. 6.4b, lanes 1-6). As
previously observed, the shifts were more apparent after endoglycosidase H treatment
(Fig. 6.5b, +Endo H, lanes 1-6).
Chapter 6 – Probing the environment of a translocating opsin chain
130
Figure 6. 5 QSY modification of various OP130 to OP164 integration intermediates. The integration intermediates were synthesised and treated with QSY as described in the legend to Figure 6.4.
Chapter 6 – Probing the environment of a translocating opsin chain
131
When the OP150 and OP164 intermediates were analysed, cys107 and cys115
continued to be modified by treatment with QSY (Fig. 6.5c and d, lanes 3-6). Most
significantly however, QSY was found to react with cys124 in these longer integration
intermediates (Fig. 6.5c and d, lanes 1 and 2). This indicates that at nascent chain
lengths of 150 and 164 residues, cys124 is most likely in a hydrophobic environment
which permits QSY access to the cysteine probe but excludes the hydrophilic AMS.
Since cys124 is in the middle of TM3, it is possible that this region of the nascent chain
either contacts phospholipids or is embedded in a hydrophobic region of the Sec61
complex (refer to the schematic diagrams of the ribosome-nascent chain complex at the
translocon in Fig. 6.5c and d).
6.6 The loop region C-terminal to TM3 is in a hydrophilic environment at 164
residues
An alternative explanation of the inaccessibility of cys124 to AMS in the OP150 and
OP164 intermediates is that some type of ‘gating’ event occurs at these chain lengths
(Alder & Johnson, 2004) and prevents access of AMS via closure of the Sec61
translocon and/or the tight binding of the ribosome to the Sec61 complex. Such gating
would also prevent access to probes C-terminal of cys124 that were in a hydrophilic
environment, and this possibility was investigated further to better establish the basis for
the specific absence of AMS modification. To this end, a unique cysteine was
introduced at residue 140 of opsin to determine the environment of the hydrophilic
region of polypeptide C-terminal of TM3. Since in OP164, cys140 is predicted to be
near the ribosomal exit site, an additional integration intermediate of 174 residues was
also generated to examine the environment of cys140 when it was predicted to have
emerged from the ribosome.
In the first instance, OP164[cys140] and OP174[cys140] were synthesised in the
absence of membranes to ascertain that cys140 can be modified by AMS and QSY
when in a freely accessible aqueous environment. Both OP164[cys140] and
OP174[cys140] displayed shifts in molecular weight after treatment with AMS or QSY,
indicating that cys140 can be modified by both reagents at both chain lengths (Fig. 6.6a
and b, lanes 1 and 2, 3 and 4). Having established that cys140 could be modified in the
Chapter 6 – Probing the environment of a translocating opsin chain
132
context of OP164 and OP174 chains, the analysis was repeated using membrane
integration intermediates where the nascent chains were trapped in the ER translocon.
Figure 6. 6 AMS and QSY modification of cys140 in (a) OP164 and (b) OP174 chains. mRNA chains representing OP164[cys140] and OP174[cys174] were translated in vitro in a rabbit reticulocyte translation system without the addition of membranes and subjected to AMS or QSY modification as described in the legend of Figure 6.3.
Chapter 6 – Probing the environment of a translocating opsin chain
133
Figure 6. 7 AMS and QSY modification of (a) OP164[cys140] and (b) OP174[cys140], in the presence of semi-permeabilised mammalian cells. OP164[cys140] and OP174[cys174] were synthesised in a rabbit reticulocyte translation system in the presence of digitonin-permeabilised cells and subjected to AMS or QSY modification as described in the legend of Figure 6.2.
Chapter 6 – Probing the environment of a translocating opsin chain
134
In the presence of the ER translocon, OP164[cys140] was shown to be modified by both
AMS and QSY (Fig. 6.7a, lanes 1 and 2, 3 and 4), and when the nascent chain was
extended to 174 residues, both AMS and QSY were still able to react with cys140 (Fig.
6.7b, lanes 1 and 2, 3 and 4). Taken together, this analysis shows that the loop region
following TM3 remains in a hydrophilic environment as it moves out of the ribosome
and confirms that the failure of AMS to modify cys124 in the OP164 intermediate is not
due to a ‘gating’ phenomenon.
6.7 The OP164 nascent chain represents a true integration intermediate that is
attached to the ribosome
The analysis of the nascent chain environment during integration by using thiol-specific
probes relies on the assumption that the ribosome bound integration intermediates are
stable and reflect the environment of the nascent chain within the ER translocon. In
order to test this assumption, an established assay was utilised to verify the association
of nascent opsin chains with the ribosome (Wilson et al., 2005). This is of paramount
importance for OP164[cys124] since it is important to establish that the lack of AMS
modification (section 6.4) is not simply a consequence of the specific release of these
opsin chains from the ribosome resulting in their release into the phospholipid bilayer
which would preclude AMS modification.
In this case, OP164[cys124] integration intermediates were synthesised in the presence
of semi-permeabilised mammalian cells. The translation reaction was then halted with
the addition of either cycloheximide, which stabilises ribosome-nascent chain
complexes, or puromycin, which releases nascent chains from the ribosome. The
membrane fraction was isolated by centrifugation and then treated with a non-ionic
detergent, C12E8, which efficiently solubilises the membranes without disrupting any
ribosome-nascent chain complexes (Wilson et al., 2005). The ribosomes and any
associated nascent chains were recovered by centrifugation, and products in the pellet
and the supernatant were subsequently resolved by SDS PAGE.
Chapter 6 – Probing the environment of a translocating opsin chain
135
Figure 6. 8 Isolation of ribosomes and associated OP164[cys124] chains. OP164[cys124] was synthesised in a rabbit reticulocyte translation system in the presence of semi-permeabilised cells. The translation reaction was halted either with cycloheximide (CHX) or puromycin. The membrane fraction was isolated by centrifugation and was solubilised in 1% C12E8. An aliquot was removed and analysed on SDS PAGE as total products (T). Ribosomes and any associated nascent chains were pelleted by centrifugation and proteins in the supernatant were precipitated using trichloroacetic acid (TCA). Products in the pellet (P) and supernatant (S) were denatured in sample buffer and resolved by SDS PAGE. Doubly-glycosylated opsin chains are indicated with (ii).
Chapter 6 – Probing the environment of a translocating opsin chain
136
This experiment showed that the OP164[cys124] chains were almost exclusively in the
pellet fraction when the samples were cycloheximide treated, confirming that the
nascent chains were not released from the ribosome under these conditions and were
therefore authentic integration intermediates (Fig. 6.8, lanes 1-3). On the other hand,
treatment of the nascent chains with puromycin resulted in the release of a substantial
fraction of the nascent opsin chains from the ribosome and these were found in the
supernatant fraction (Fig. 6.8, lane 6). The puromycin treatment was not completely
effective since a proportion of nascent chains was still found associated with the
ribosomes in the pellet fraction (Fig. 6.8, lane 5). Nonetheless, since the AMS and QSY
modification reactions are performed on cycloheximide treated nascent chains, it can be
concluded that the earlier observations with AMS and QSY detailed above reflect the
true environment of the opsin chain during its insertion at the ER translocon.
6.8 Summary
Distinct regions of opsin TM3 experience different environments during membrane
integration via the ER translocon, as evidenced by the use of sulphydryl specific
reagents with distinct biophysical properties. The hydrophilic loop preceding TM3, and
the N-terminal region of TM3, remain in a hydrophilic location, presumably the
aqueous environment of the ribosomal exit tunnel and the translocon pore, during
nascent chain extension from 130 to 164 residues. On the other hand, the central region
of TM3, as represented by cys124, moves from a hydrophilic to a hydrophobic
environment when the nascent chain is extended from 140 to 150 residues where it
remains at a chain length of 164 residues. This environment relates directly to the
precise segment of polypeptide within TM3, since a probe located C-terminal of TM3
(cys140) is in a hydrophilic environment.
Conclusion
Extension of the nascent opsin chain from 140 to 150 residues and beyond results in the
central region of opsin TM3 relocating from a hydrophilic to a hydrophobic
environment within the context of the ribosomal exit tunnel and/or the ER translocon.
Chapter 7 - Discussion
137
CHAPTER 7 Discussion
Chapter 7 - Discussion
138
7.1 Introduction
The aim of this project was to investigate the molecular environment of the seven TM
domain protein, opsin, during its biosynthesis at the ER, so as to better understand the
mechanism by which the multiple TM domains of a polytopic protein are integrated into
the ER membrane. Opsin was chosen as a suitable model because a high resolution
structure of the fully folded protein is available (Palczewski et al., 2000). In addition,
the N-glycosylation of its N-terminal region serves as a useful marker for the correct
targeting and insertion of the truncated integration intermediates used to study its
biogenesis.
In this study, a cysteine-mediated site-specific cross-linking approach was primarily
employed to examine the environment of the TM domains of nascent opsin during
synthesis at the ER. Cross-linking using the sulphydryl specific reagent, BMH, was
carried out with integration intermediates of defined lengths to represent different stages
of nascent chain elongation. By performing immunoprecipitations using specific
antisera, molecules adjacent to the TM domain of the nascent chain at a distinct stage
during its biosynthesis could be identified, thus allowing alterations in the environment
of the TM domain to be followed. Since a heterogeneous population of nascent chains
could be generated during in vitro translation, a HA epitope tag was added to the C-
terminus of each nascent opsin chain analysed, allowing the selection of authentic
integration intermediates and their cross-linking partners by immunoprecipitation using
an α-HA antiserum. This approach facilitated the accurate interpretation of the cross-
linking data obtained during this work.
My study focused on the association of each opsin TM domain with components of the
Sec61 complex. I made the assumption that a loss of cross-linking to the Sec61α and
Sec61β subunits reflected the lateral movement of a TM domain from the protein lined
translocon and into a primarily phospholipid environment on the basis of previous
photocross-linking studies (Martoglio et al., 1995; McCormick et al., 2003; Mothes et
al., 1997). In addition, this cross-linking approach was complemented by the analysis of
TM ‘environment’ during insertion into the ER translocon using two sulphydryl specific
modification reagents with different physical properties. On the basis of the data
Chapter 7 - Discussion
139
obtained during this study, a working model of the integration process for opsin from
the translocon into the ER membrane has been generated (Fig. 7.1).
Figure 7. 1 A working model for the integration of opsin into the ER membrane. Schematic representation of Sec61α and Sec61β are shown in orange, opsin TM domains are shown in blue, while PAT-10 is shown in purple. TM1 is fully engaged with the translocon when the nascent chain is ∼96 residues long. The insertion of TM2 causes TM1 to relocate to the periphery of the translocon where it associates with PAT-10 (OP130 and OP140). When the nascent chain is ∼150 residues, TM3 is now engaged with the translocon pore while TM1 re-associates with Sec61α. Extension of the nascent chain to ∼204 residues results in the insertion of TM4 into the pore and the simultaneous integration of TM1 to TM3 into the membrane. At 259 residues, TM4 exits the translocon as soon as TM5 is inserted into the pore. When the nascent chain is ∼304 residues, TM5 associates with PAT-10 but still remains close to the Sec61 complex, while TM6 is engaged with the translocon. Lengthening of the nascent chain to ∼339 and ∼357 residues allow the insertion of TM7 into the translocon.
Chapter 7 - Discussion
140
7.2 The integration of TM1, TM2 and TM3 is a ‘co-ordinated’ process
To analyse the environment of TM1, cross-linking was performed using a previously
characterised cysteine probe located at residue 56 (Meacock et al., 2002) using
integration intermediates ranging from 96 to 259 residues in length. This study revealed
an unexpected level of complexity in the behaviour of TM1 which had not been
apparent during the previous analysis (Meacock et al., 2002). At the shortest chain
length examined (OP96), TM1 has fully emerged from the ribosome and engaged the
Sec61 translocon. TM1 remains at the translocon at 109 residues but moves away once
the nascent chain is extended to 130 residues, only to re-associate with the translocon at
150 and 164 residues (Fig. 7.1 and 7.2). Further extension of the nascent chain beyond
164 residues then allows TM1 to exit the translocon again. The association of TM1 with
the Sec61 complex for a second time at a chain length of 150 and 164 residues
generated adducts with Sec61α, but not Sec61β, implying this re-association may occur
at a different location to that occupied when TM1 first engages the Sec61 translocon at
a nascent chain length of 96 residues, when both Sec61α and Sec61β adducts are seen.
In addition, TM1 is in close proximity to PAT-10 when the nascent chain is 150 and
164 residues. For simplicity, I have termed the first location where cross-links to both
Sec61α and Sec61β were observed as a ‘Phase I’ environment, while the second
location, where only adducts with Sec61α was seen, as ‘Phase II’ environment (denoted
as ‘I’ and ‘II’ in Fig. 7.1 and 7.2).
Interestingly, this periodicity in the interaction of TM1 with the Sec61 subunits is only
observed when the subsequent TM domains (TM2 to TM7) are present. When TM2 to
TM7 of opsin are replaced with a hydrophilic region, TM1 relocation from the phase I
Sec61 environment is significantly delayed. Unlike the behaviour of TM1 in the normal
opsin polypeptide chains, TM1 in the context of chimeric OPTM1PPL chains remains
adjacent to Sec61α and Sec61β for a prolonged period of nascent chain synthesis. Thus,
TM1 only appears to exit the translocon when the nascent chain is longer than 204
residues (Fig. 7.2). This implies that the synthesis of additional TM domains is
necessary for authentic TM1 movement out of the translocon. The simplest explanation
for this observation is that the subsequent TM domains promote TM1 exit by displacing
it from the core of the translocon. This possibility is substantiated by the observation
Chapter 7 - Discussion
141
that TM2 has entered a phase I-like environment when TM1 is out of the translocon
(Fig. 7.2, OP140).
Figure 7. 2 The roles of TM1 and TM3 during opsin integration. The Sec61 complex, opsin TM domains and PAT-10 are represented in orange, blue and purple respectively. See text for details.
The OP164 integration intermediate represents an important stage in opsin biosynthesis
where TM1 ‘returns’ to the Sec61 complex whilst TM2 is still associated with the
translocon and TM3 is located in the phase I environment (Fig. 7.2, OP164). Extension
of the nascent chain to 204 residues resulted in the loss of Sec61 adducts from all three
TM domains, implying the concurrent release of TM1, TM2 and TM3 from their
association with the translocon (Fig. 7.2, OP204). This suggests that, following its
initial exit, TM1 re-associates with the Sec61 complex to allow an association with
TM2 and/or TM3, resulting in the synchronised integration of TM1 to TM3 into the ER
membrane. In support of this model, previous studies of opsin point mutants indicated
that the stability of the full length protein is influenced by interactions between TM1
and TM2 (Bosch et al., 2003). Furthermore, the expression of opsin fragments in vivo
showed that stable membrane integration occurs only when at least TM1 and TM2 are
present in a single polypeptide chain (Heymann & Subramaniam, 1997). This particular
behaviour of opsin TM1 appears similar to that observed in studies of model proteins
Chapter 7 - Discussion
142
with two transmembrane domains where the integration of TM1 and TM2 appeared to
be co-ordinated (Heinrich & Rapoport, 2003; Sauri et al., 2005).
The observation that TM3 exit from the ER translocon is not dependent upon the
presence of later TM domains (TM4 to TM7), as evidenced by the OPTM1-3PPL
polypeptide, is also consistent with the view that TM1, TM2 and TM3 interact with one
another to form a folding domain that can independently exit the ER translocon (Fig.
7.2). This notion is consistent with an in vivo expression study in which two separate
opsin fragments, TM1-3 and TM4-7, could be co-expressed to form a ‘functional’ opsin
molecule with spectral properties similar to the wild type protein (Ridge et al., 1995).
Thus, the integration of TM1, TM2 and TM3 into the ER membrane is likely to be a
‘co-ordinated’ process where mutual stabilisation of TM1, TM2 and TM3 acts to
facilitate the lateral exit of these TM domains from the translocon. Mutual stabilisation
between TM domains during nascent chain integration into the ER membrane is not
unique to opsin and appears to contribute to the integration of other polytopic
membrane proteins such as Neurospora plasma membrane H+-ATPase and human P-
glycoprotein (Lin & Addison, 1995; Skach & Lingappa, 1993).
7.3 An alternative analysis of TM3 environment during opsin integration
In order to further define the integration of opsin TM3, monofunctional sulphydryl-
specific reagents of different hydrophilicity, AMS and QSY, were utilised to examine
the environment of single cysteine residues introduced at different locations within
TM3. The exclusion of AMS from hydrophobic environments meant that only cysteine
probes present in an aqueous surrounding should be AMS modified, while QSY should
modify cysteine residues present in both hydrophilic and hydrophobic environments.
Using this approach, I found clear evidence that the presence of the ER translocon
specifically altered the local environment of a cysteine probe located in the middle of
opsin TM3. In particular, cys124 relocated from an aqueous environment in the OP140
intermediate into a hydrophobic environment in the OP150 and OP164 intermediates.
The most likely explanation for this observation is that the cysteine probe relocates from
the water lined exit tunnel of the ribosome to a hydrophobic region of the ER translocon
as the chain gets longer. In contrast, cys115 remains accessible to AMS at each chain
Chapter 7 - Discussion
143
length tested and hence does not undergo a similar ‘relocation’. Why should two
different cysteine residues display different properties in the same intermediate? Two
possibilities present themselves and both impact upon how we view the membrane
insertion process.
In the first scenario, TM3 would have formed an α-helix within the ER translocon
where its orientation would be relatively fixed (c.f. McCormick et al., 2003). A helical
wheel plot predicts that cys115 and cys124 would be on different sides of such an α-
helix. Thus, one side of the TM domain could be partitioning into the hydrophobic lipid
bilayer (cys124) whilst the other remained in the more hydrophilic environment of the
Sec61 complex (cys115) (‘Partitioning’ model, Figure 7.3a).
Figure 7. 3 Possible models of TM3 integration into a hydrophobic environment. The nascent opsin chain is represented by a black solid line, while the numbers indicate the relative position of the cysteine residues. The hydrophilic and hydrophobic environments are shown in blue and brown respectively. See text for details.
The second scenario allows for the lateral gating of the Sec61 translocon to be in the
form of a ‘zipper’ (‘Zipper’ model, Figure 7.3b). In this case, the region towards the
cytosolic face of the ER membrane could open laterally and allow a portion of opsin
TM3, including cys124, to access the lipid bilayer. In contrast, in the OP164
intermediate, the region of the Sec61 complex towards the ER lumen could remain
closed thereby retaining the other part of TM3, including cys115, in a hydrophilic
environment. Thus, whilst my cross-linking data leads me to present a series of
Chapter 7 - Discussion
144
‘snapshots’ representing distinct stages of membrane integration, my preliminary use of
‘probe access’ implies there is most likely a more refined and complex behaviour for
individual TM domains than the composite models that I present are capable of
expressing.
It should be noted that an alternative explanation for the differences in probe access that
I observe is that patches of lipid molecules are present within the core of the Sec61
translocon (McCormick et al., 2003) which could not be observed in the crystal
structure (Van den Berg et al., 2004). Cys124 may be buried in one of these
hydrophobic patches while cys115 remains in the aqueous translocon pore. In order to
differentiate between these different models, an AMS accessibility ‘scan’ of TM3 could
be performed using nascent chains with single cysteine residues introduced at positions
along the entire length of the TM domain.
Interestingly, the different environments of cys124 and cys115 were also reflected in
their cross-linking partners. When the nascent opsin chain is 164 residues long, cys124
formed an adduct only with Sec61β while cys115 formed adducts with both Sec61β and
Sec61α. This observation implied that there may be a correlation between Sec61α
adduct formation and AMS accessibility, although further examination of nascent
chains with varying lengths will be necessary to confirm this. It is possible that adduct
formation with Sec61α occurs only when the cysteine probe is in the aqueous pore and
not when it is in a hydrophobic environment. Although the function of Sec61β is
currently unknown, the formation of Sec61β cross-links alone may reflect an ‘early’
stage in nascent chain translocation, especially since the single cysteine residue of
Sec61β is present in its cytosolic domain. In fact, the use of small molecular weight
inhibitors which block an early stage of translocation, enhanced the formation of
Sec61β cross-links with nascent chains (Besemer et al., 2005; Garrison et al., 2005).
Thus, Sec61β may have a role in facilitating the insertion of some TM domains into a
‘phase I’ like environment within the Sec61 translocon.
Chapter 7 - Discussion
145
7.4 Opsin TM4 exits the translocon upon nascent chain extension
Unlike the apparently synchronised integration of TMs 1-3, the integration of opsin
TM4 into the ER membrane is a relatively straightforward process. Hence, TM4 is
inserted into the core of the ER translocon when the nascent chain is 204 residues long
and exits the translocon upon chain extension to 259 residues, at which stage TM5 is in
the translocon pore (Fig. 7.1, OP204 and OP259). TM4 is fully integrated into the lipid
bilayer by the time the nascent chain is 259 residues since further extension of the
nascent chain does not result in any detectable re-association of TM4 with translocon
components. There was a suggestion that TM4 associates with a PAT-10 like
component that was observed when cross-linking was carried out with chains of 259
residues and longer. However, the efficiency of adduct formation was low and the
significance of this cross-linking product remains to be fully established.
7.5 Opsin TM5 is engaged with the ER translocon throughout opsin biosynthesis
One of the limitations of site-specific cross-linking is that the location/orientation of the
probe may influence the efficiency of the cross-linking reaction. In the study of opsin
TM5, two single cysteine probes located within TM5 did not generate any adducts to
Sec61 components, even at the shortest chain length in which the TM domain is
expected to engage the ER translocon. This may reflect the relative positions of the
cysteine residues within TM5 or a failure of this TM to engage the Sec61 complex.
However, when a cysteine probe (cys229) located in a hydrophilic loop region four
residues from the boundary of TM5 (residues 200 to 225) was analysed, discrete
adducts to Sec61 subunits were seen. This probe was therefore used for the remainder of
the analysis on the basis that it most likely reflects the associations of the nearby TM
region. Nevertheless, further studies with probes located within the hydrophobic TM
region will be needed to fully validate the conclusions based on the data obtained using
this probe location.
A further level of complexity with regard to the analysis of TM5 is that long integration
intermediates have to be analysed, and such chains generally give low efficiency
adducts that can be difficult to unambiguously confirm as being with the authentic
intermediate (see Chapter 5). In order to simplify the interpretation of such cross-linking
Chapter 7 - Discussion
146
data, a shortened opsin mutant containing only the N-terminal region and TM5 to TM7
of opsin (OPN/5-7) was generated. Since the relative position of the TM domains was
altered, i.e. TM5 is now the first TM domain and so on, the resulting data must be
interpreted with care and, as much as possible, any cross-linking data obtained using
OPN/5-7 nascent chains was compared with that from equivalent authentic opsin
intermediates.
Although TM5 is effectively the first TM domain in any OPN/5-7 nascent chains, TM5
does not behave like TM1 during opsin biosynthesis. When cross-linking patterns of
TM1 integration intermediates were compared to those of equivalent lengths of TM5
OPN/5-7 integration intermediates, differences in adduct formation to Sec61
components were observed. Whilst TM1 exhibits a periodic interaction with the Sec61
complex, TM5 in the context of OPN/5-7 chains is in constant association with Sec61
components throughout nascent chain synthesis. This suggests that, unlike TM1 to
TM4, TM5 remains associated with the translocon until the complete polypeptide chain
is synthesised (see Fig. 7.1). This is significant because it implies that the translocon is
able to distinguish between different TM domains and perhaps somehow regulate their
movement into the ER membrane. This is consistent with the observation that TM
domains from various precursors are specifically positioned within the translocon,
suggesting a sequence-dependent interaction between TM domains and the Sec61
complex (McCormick et al., 2003).
One of the most striking features of the cross-linking analysis of TM5 is the strong
adduct formation with PAT-10, akin to that observed with TM1. Although it is possible
that TM5 is cross-linked to a different ∼10 kDa molecule, this is highly unlikely since
only one adduct with a ∼10 kDa protein was observed when cross-linking was
performed with opsin chains containing two cysteine residues, one in TM1 and the other
in TM5. The formation of PAT-10 adducts with TM5 in the OPN/5-7 integration
intermediates is not simply due to the relative position of TM5 as the first TM domain
in the polypeptide chain. TM5 can still form adducts with PAT-10 when cross-linking
was performed using authentic opsin nascent chains. TM5 association with PAT-10
upon nascent chain extension suggests that TM5 may relocate with respect to the
translocon during opsin biosynthesis resulting in reduced adduct formation with Sec61β
(see Fig. 7.1).
Chapter 7 - Discussion
147
7.6 Opsin TM6 and TM7 are associated with the Sec61 complex throughout opsin
biosynthesis
As for the analysis of TM5, the environment of TM6 and TM7 was examined using the
shorter opsin mutant, OPN/5-7. In the context of both authentic opsin chains and
OPN/5-7 chains, TM6 was found associated with the Sec61 complex from the shortest
integration intermediate examined to the full length polypeptide chain, indicating that
TM6 engages the translocon for a prolonged period of time (Fig. 7.1). Like TM6, the
cross-linking pattern of TM7 in the context of both authentic opsin and OPN/5-7
derived chains also showed that TM7 remains in the translocon throughout nascent
chain synthesis (Fig. 7.1). Since TM6 and TM7 are near the C-terminal end of full
length opsin, it is unclear whether their prolonged association with the translocon is due
to specific interactions with the Sec61 complex, or because the polypeptide ‘tether’
from the ribosome is too short to allow TM6 and TM7 to be released from the proximity
of the translocon. Hence, although ∼49 residues are present between the
peptidyltransferase centre of the ribosome and the C-terminal boundary of TM7, up to
40 residues may be within the ribosome (Kowarik et al., 2002). One possible way of
determining whether the translocon plays an active role in retaining TM6 and TM7
would be to lengthen the C-terminal polypeptide tether and see if the additional chain
length allows TM6 and TM7 to move away from the translocon.
7.7 Nascent opsin chains engage a single copy of the Sec61 complex during opsin
biosynthesis
The crystal structure of an archeal Sec61 complex suggests that a single Sec61
heterotrimer is sufficient to form the central aqueous pore of the ER translocon (Van
den Berg et al., 2004), although low resolution EM structures indicate that the Sec61
complex is normally found in the form of an oligomer composed of three or four copies
of the heterotrimer (Beckmann et al., 1997; Hanein et al., 1996; Menetret et al., 2000;
Menetret et al., 2005). The recent observation that a cysteine residue located in the
centre of the related SecY complex could form a disulphide bond with a translocating
nascent chain supports the hypothesis that the pore of the translocon resides within a
single Sec61 complex (Cannon et al., 2005), and hence the functional relevance of the
oligomeric status of the Sec61 complexes is currently unclear. However, several
Chapter 7 - Discussion
148
potential reasons have been postulated; for example, it might be that an interaction
between Sec61 complexes is necessary to ‘activate’ one heterotrimer to form the
channel of the ER translocon. Alternatively, whilst one Sec61 heterotrimer forms the
active channel, the remaining complexes of the oligomer may recruit accessory
components involved in co- or post-translocational modifications such as the SPC and
OST complexes (Dobberstein & Sinning, 2004).
Since each Sec61 complex within the oligomer may be capable of forming an active
pore, different substrates or different regions of a single nascent chain might be
simultaneously translocated/integrated at separate pores within the same higher order
translocon. In the case of a polytopic membrane protein, it was suggested that different
TM domains could be translocated concurrently into the distinct pores of adjacent
Sec61 complexes within the same translocon (Dobberstein & Sinning, 2004). Double
probe analysis was employed during this study in order to investigate this possibility. I
found that the different TM domains of a single nascent opsin chain are adjacent to only
one copy of the Sec61α subunit, and conclude that it is unlikely that the different TM
domains of a polytopic membrane protein utilise separate Sec61 heterotrimers during
their integration and that only one Sec61 complex forms the active pore of the
translocon (c.f. Cannon et al., 2005).
7.8 The possible role of PAT-10 as a TM chaperone
An interesting feature observed during the analysis of opsin integration is the adduct
formation between specific TM domains and a novel ∼10 kDa protein, PAT-10. Cross-
linking between opsin and PAT-10 was first observed during an extensive analysis of
TM1 environment (Meacock et al., 2002). However, adduct formation with PAT-10 is
not specific to opsin since similar adducts were seen with the rat neurotensin receptor
(Meacock et al., 2002). In this study, PAT-10 adducts were also observed with opsin
TM4 (weakly) and TM5 (strongly), in addition to TM1. PAT-10 cross-linking to TM1,
TM4 and TM5 is characterised by an adduct which normally appears after the
subsequent TM domain has emerged from the ribosome and remains until the full length
opsin chain has been synthesised. Since nascent chain extension is necessary for the TM
domains to form adducts with PAT-10, this suggests that TM domain relocation is
required for association with PAT-10. Proximity is lost when the nascent chain is
Chapter 7 - Discussion
149
released from the ribosome, indicating that PAT-10 is most likely associated with the
translocon (Meacock et al., 2002). This idea is strongly supported by my double
cysteine probe analysis which showed that a single nascent chain could be cross-linked
to both Sec61β and PAT-10 at the same time. This analysis also indicated that, like the
Sec61 complex, only a single copy of the PAT-10 protein was adjacent to an opsin
chain during its membrane integration.
Although the identity of PAT-10 is currently unknown, PAT-10 has been postulated to
have a role as a TM chaperone (Meacock et al., 2002). It is possible that PAT-10
functions to regulate the ‘release’ of specific TM domains to allow their co-ordinated
integration into the ER membrane. For example, the interaction of PAT-10 with TM1
might facilitate its assembly with TM2 and TM3 for a synchronised membrane
integration. The novel observation that PAT-10 interacts specifically with TM1, TM4
and TM5 is interesting because these TM domains may represent the beginnings of
separate folding domains. Although distinct subregions are not obvious from the crystal
structure (Palczewski et al., 2000), in vivo expression of opsin fragments has shown that
TM1-3 and TM4-7 (Ridge et al., 1995), or TM1-4 and TM5-7 (Ridge et al., 1996), can
be correctly assembled to produce functional opsin molecules. The potential role of
PAT-10 as a TM chaperone further supports the idea that TM integration into the ER
membrane is a highly regulated event.
7.9 Conclusion
The use of opsin as a model polytopic membrane protein to study the integration of TM
domains into the ER membrane underscores the fact that the integration of multiple TM
domains is a highly complex process. The working model I have produced for opsin
integration (Fig. 7.1) does not precisely conform to either the ‘sequential’ or the ‘en
masse’ model (see Chapter 1), but rather combines elements of both models. During
integration, each TM domain of opsin behaves in a unique manner, with some TM
domains engaging the translocon for longer periods than others. Opsin TM1 displays a
complex behaviour where it initially moves away from the translocon after its insertion
only to return at a later stage, most likely to interact with TM2 and TM3 in order to
allow a co-ordinated integration of these three TM domains into the ER membrane.
TM4, on the other hand, exits the translocon in an independent manner almost as soon
Chapter 7 - Discussion
150
as the next TM domain has been inserted into the translocon pore. TM5, TM6 and TM7
appear to remain associated with the Sec61 complex throughout nascent chain synthesis
and presumably integrate into the membrane once the polypeptide chain is released
from the ribosome. This study has also provided further evidence that the integration of
the multiple TM domains of a polytopic membrane protein is a highly regulated process
that involves the interplay of various components such as the Sec61 complex and the
potential chaperone PAT-10.
Chapter 8 - Bibliography
151
CHAPTER 8 Bibliography
Chapter 8 - Bibliography
152
Abell, B. M., High, S. & Moloney, M. M. (2002). Membrane protein topology of oleosin is constrained by its long hydrophobic domain. J Biol Chem 277, 8602-8610.
Abell, B. M., Jung, M., Oliver, J. D., Knight, B. C., Tyedmers, J., Zimmermann, R. & High, S. (2003). Tail-anchored and signal-anchored proteins utilize overlapping pathways during membrane insertion. J Biol Chem 278, 5669-5678.
Adamus, G., Zam, Z. S., Arendt, A., Palczewski, K., McDowell, J. H. & Hargrave, P. A. (1991). Anti-rhodopsin monoclonal antibodies of defined specificity: characterization and application. Vision Res 31, 17-31.
Ahner, A. & Brodsky, J. L. (2004). Checkpoints in ER-associated degradation: excuse me, which way to the proteasome? Trends Cell Biol 14, 474-478.
Alder, N. N. & Johnson, A. E. (2004). Cotranslational membrane protein biogenesis at the endoplasmic reticulum. J Biol Chem 279, 22787-22790.
Alder, N. N., Shen, Y., Brodsky, J. L., Hendershot, L. M. & Johnson, A. E. (2005). The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum. J Cell Biol 168, 389-399.
Audigier, Y., Friedlander, M. & Blobel, G. (1987). Multiple topogenic sequences in bovine opsin. Proc Natl Acad Sci USA 84, 5783-5787.
Bacher, G., Lutcke, H., Jungnickel, B., Rapoport, T. A. & Dobberstein, B. (1996). Regulation by the ribosome of the GTPase of the signal-recognition particle during protein targeting. Nature 381, 248-251.
Baker, K. B., Colley, N. J. & Zucker, C. S. (1994). The cyclophilin homolog Nina A functions as a chaperone, forming a stable complex in vivo with its protein target rhodopsin. EMBO J 13, 4886-4895.
Bause, E. & Hettkamp, H. (1979). Primary structural requirements for N-glycosylation of peptides in rat liver. FEBS Lett 108, 341-344.
Beckmann, R., Bubeck, D., Grassucci, R., Penczek, P., Verschoor, A., Blobel, G. & Frank, J. (1997). Alignment of conduits for the nascent polypeptide chain in the ribosome-Sec61 complex. Science 278, 2123-2126.
Chapter 8 - Bibliography
153
Beckmann, R., Spahn, C. M., Eswar, N., Helmers, J., Penczek, P. A., Sali, A., Frank, J. & Blobel, G. (2001). Architecture of the protein-conducting channel associated with the translating 80S ribosome. Cell 107, 361-372.
Belin, D., Bost, S., Vassalli, J. D. & Strub, K. (1996). A two-step recognition of signal sequences determines the translocation efficiency of proteins. EMBO J 15, 468-478.
Bermak, J. C., Li, M., Bullock, C. & Zhou, Q. Y. (2001). Regulation of transport of the dopamine D1 receptor by a new membrane-associated ER protein. Nat Cell Biol 3, 492-498.
Besemer, J., Harant, H., Wang, S. & other authors (2005). Selective inhibition of cotranslational translocation of vascular cell adhesion molecule 1. Nature 436, 290-293.
Blobel, G. (1980). Intracellular protein topogenesis. Proc Natl Acad Sci USA 77, 1496-1500.
Bohni, P. C., Deshaies, R. J. & Schekman, R. W. (1988). SEC11 is required for signal peptide processing and yeast cell growth. J Cell Biol 106, 1035-1042.
Borel, A. C. & Simon, S. M. (1996). Biogenesis of polytopic membrane proteins: membrane segments assemble within translocation channels prior to membrane integration. Cell 85, 379-389.
Bosch, L., Ramon, E., Del Valle, L. J. & Garriga, P. (2003). Structural and functional role of helices I and II in rhodopsin. A novel interplay evidenced by mutations at Gly-51 and Gly-89 in the transmembrane domain. J Biol Chem 278, 20203-20209.
Breyton, C., Haase, W., Rapoport, T. A., Kuhlbrandt, W. & Collinson, I. (2002). Three-dimensional structure of the bacterial protein-translocation complex SecYEG. Nature 418, 662-665.
Cannon, K. S., Or, E., Clemons, W. M., Jr., Shibata, Y. & Rapoport, T. A. (2005). Disulfide bridge formation between SecY and a translocating polypeptide localizes the translocation pore to the center of SecY. J Cell Biol 169, 219-225.
Caplan, A. J., Cyr, D. M. & Douglas, M. G. (1992). YDJ1p facilitates polypeptide translocation across different intracellular membranes by a conserved mechanism. Cell 71, 1143-1155.
Chapter 8 - Bibliography
154
Chavan, M., Yan, A. & Lennarz, W. J. (2005). Subunits of the translocon interact with components of the oligosaccharyl transferase complex. J Biol Chem 280, 22917-22924.
Colley, N. J., Baker, E. K., Stamnes, M. A. & Zuker, C. S. (1991). The cyclophilin homolog ninaA is required in the secretory pathway. Cell 67, 255-263.
Crowley, K. S., Reinhart, G. D. & Johnson, A. E. (1993). The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation. Cell 73, 1101-1115.
Crowley, K. S., Liao, S., Worrell, V. E., Reinhart, G. D. & Johnson, A. E. (1994). Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore. Cell 78, 461-471.
Davila, S., Furu, L., Gharavi, A. G. & other authors (2004). Mutations in SEC63 cause autosomal dominant polycystic liver disease. Nat Genet 36, 575-577.
Deretic, D., Puleo-Scheppke, B. & Trippe, C. (1996). Cytoplasmic domain of rhodopsin is essential for post-Golgi vesicle formation in a retinal cell-free system. J Biol Chem 271, 2279-2286.
Deshaies, R. J. & Schekman, R. (1987). A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum. J Cell Biol 105, 633-645.
Deshaies, R. J. & Schekman, R. (1989). SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum. J Cell Biol 109, 2653-2644.
Deshaies, R. J., Eun, A., Koch, J. A., Rothblatt, J. A., Sanders, S., Stirling, C. & Schekman, R. (1990). Soluble and membrane-associated factors required for protein translocation into the yeast endoplasmic reticulum. In Dynamics and biogenesis of membranes, pp. 327-341. Edited by J. A. F. Op den Kamp. Heidelberg: Springer-Verlag.
Do, H., Falcone, D., Lin, J., Andrews, D. W. & Johnson, A. E. (1996). The cotranslational integration of membrane proteins into the phospholipid bilayer is a multistep process. Cell 85, 369-378.
Dobberstein, B. & Sinning, I. (2004). Structural biology. Surprising news from the PCC. Science 303, 320-322.
Chapter 8 - Bibliography
155
Dunlop, J., Jones, P. C. & Finbow, M. E. (1995). Membrane insertion and assembly of ductin: a polytopic channel with dual orientations. EMBO J 14, 3609-3616.
Ellgaard, L. & Ruddock, L. W. (2005). The human protein disulphide isomerase family: substrate interactions and functional properties. EMBO Rep 6, 28-32.
Emanuelsson, O. & von Heijne, G. (2001). Prediction of organellar targeting signals. Biochim Biophys Acta 1541, 114-119.
Esnault, Y., Blondel, M. O., Deshaies, R. J., Scheckman, R. & Kepes, F. (1993). The yeast SSS1 gene is essential for secretory protein translocation and encodes a conserved protein of the endoplasmic reticulum. EMBO J 12, 4083-4093.
Esnault, Y., Feldheim, D., Blondel, M. O., Schekman, R. & Kepes, F. (1994). SSS1 encodes a stabilizing component of the Sec61 subcomplex of the yeast protein translocation apparatus. J Biol Chem 269, 27478-27485.
Evans, E. A., Gilmore, R. & Blobel, G. (1986). Purification of microsomal signal peptidase as a complex. Proc Natl Acad Sci USA 83, 581-585.
Fang, H. & Green, N. (1994). Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae. Mol Biol Cell 5, 933-942.
Fang, H., Panzner, S., Mullins, C., Hartmann, E. & Green, N. (1996). The homologue of mammalian SPC12 is important for efficient signal peptidase activity in Saccharomyces cerevisiae. J Biol Chem 271, 16460-16465.
Feldheim, D. & Schekman, R. (1994). Sec72p contributes to selective recognition of signal peptides by the secretory polypeptide translocation complex. J Cell Biol 126, 935-943.
Finke, K., Plath, K., Panzner, S., Prehn, S., Rapoport, T. A., Hartmann, E. & Sommer, T. (1996). A second trimeric complex containing homologs of the Sec61p complex functions in protein transport across the ER membrane of S. cerevisiae. EMBO J 15, 1482-1494.
Fons, R. D., Bogert, B. A. & Hegde, R. S. (2003). Substrate-specific function of the translocon-associated protein complex during translocation across the ER membrane. J Cell Biol 160, 529-539.
Chapter 8 - Bibliography
156
Freedman, R. B., Hirst, T. R. & Tuite, M. F. (1994). Protein disulphide isomerase: building bridges in protein folding. Trends Biochem Sci 19, 331-336.
Friedlander, M. & Blobel, G. (1985). Bovine opsin has more than one signal sequence. Nature 318, 338-343.
Fu, J. & Kreibich, G. (2000). Retention of subunits of the oligosaccharyltransferase complex in the endoplasmic reticulum. J Biol Chem 275, 3984-3990.
Fulga, T. A., Sinning, I., Dobberstein, B. & Pool, M. R. (2001). SRbeta coordinates signal sequence release from SRP with ribosome binding to the translocon. EMBO J 20, 2338-2347.
Garrison, J. L., Kunkel, E. J., Hegde, R. S. & Taunton, J. (2005). A substrate-specific inhibitor of protein translocation into the endoplasmic reticulum. Nature 436, 285-289.
Gierasch, L. M. (1989). Signal sequences. Biochem 28, 923-930.
Gilmore, R., Collins, P., Johnson, J., Kellaris, K. & Rapiejko, P. (1991). Transcription of full-length and truncated mRNA transcripts to study protein translocation across the endoplasmic reticulum. In Methods in Cell Biology, pp. 223-239. Edited by A. M. Tartakoff: Academic Press.
Gilstring, C. F., Melin-Larsson, M. & Ljungdahl, P. O. (1999). Shr3p mediates specific COPII coatomer-cargo interactions required for the packaging of amino acid permeases into ER-derived transport vesicles. Mol Biol Cell 10, 3549-3565.
Goder, V., Bieri, C. & Spiess, M. (1999). Glycosylation can influence topogenesis of membrane proteins and reveals dynamic reorientation of nascent polypeptides within the translocon. J Cell Biol 147, 257-266.
Goder, V. & Spiess, M. (2001). Topogenesis of membrane proteins: determinants and dynamics. FEBS Lett 504, 87-93.
Goder, V. & Spiess, M. (2003). Molecular mechanism of signal sequence orientation in the endoplasmic reticulum. EMBO J 22, 3645-3653.
Chapter 8 - Bibliography
157
Goder, V., Junne, T. & Spiess, M. (2004). Sec61p contributes to signal sequence orientation according to the positive-inside rule. Mol Biol Cell 15, 1470-1478.
Gorlich, D., Prehn, S., Hartmann, E. & other authors (1990). The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by bifunctional reagents. J Cell Biol 111, 2283-2294.
Görlich, D., Hartmann, E., Prehn, S. & Rapoport, T. A. (1992a). A protein of the endoplasmic reticulum involved early in polypeptide translocation. Nature 357, 47-52.
Görlich, D., Prehn, S., Hartmann, E., Kalies, K.-U. & Rapoport, T. A. (1992b). A mammalian homolog of Sec61p and SecYp is associated with ribosomes and nascent polypeptides during translocation. Cell 71, 489-503.
Görlich, D. & Rapoport, T. A. (1993). Protein translocation into proteoliposomes from purified components of the ER membrane. Cell 75, 615-630.
Graham, L. A., Hill, K. J. & Stevens, T. H. (1998). Assembly of the yeast vacuolar H+-ATPase occurs in the endoplasmic reticulum and requires a Vma12p/Vma22p assembly complex. J Cell Biol 142, 39-49.
Haigh, N. G. & Johnson, A. E. (2002). A new role for BiP: closing the aqueous translocon pore during protein integration into the ER membrane. J Cell Biol 156, 261-270.
Halic, M., Becker, T., Pool, M. R., Spahn, C. M., Grassucci, R. A., Frank, J. & Beckmann, R. (2004). Structure of the signal recognition particle interacting with the elongation-arrested ribosome. Nature 427, 808-814.
Hamman, B. D., Chen, J. C., Johnson, E. E. & Johnson, A. E. (1997). The aqueous pore through the translocon has a diameter of 40-60 A during cotranslational protein translocation at the ER membrane. Cell 89, 535-544.
Hammond, C., Braakman, I. & Helenius, A. (1994). Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. Proc Natl Acad Sci USA 91, 913-917.
Hanein, D., Matlack, K. E., Jungnickel, B., Plath, K., Kalies, K. U., Miller, K. R., Rapoport, T. A. & Akey, C. W. (1996). Oligomeric rings of the Sec61p complex induced by ligands required for protein translocation. Cell 87, 721-732.
Chapter 8 - Bibliography
158
Hann, B. C., Poritz, M. A. & Walter, P. (1989). Saccharomyces cerevisiae and Schizosaccharomyces pombe contain a homologue to the 54-kD subunit of the signal recognition particle that in S. cerevisiae is essential for growth. J Cell Biol 109, 3223-3230.
Hann, B. C., Stirling, C. J. & Walter, P. (1992). SEC65 gene product is a subunit of the yeast signal recognition particle required for its integrity. Nature 356, 532-533.
Hargrave, P. A. (1977). The amino-terminal tryptic peptide of bovine rhodopsin. A glycopeptide containing two sites of oligosaccharide attachment. Biochim Biophys Acta 492, 83-94.
Hargrave, P. A., McDowell, J. H., Curtis, D. R., Wang, J. K., Juszczak, E., Fong, S. L., Rao, J. K. & Argos, P. (1983). The structure of bovine rhodopsin. Biophys Struct Mech 9, 235-244.
Harley, C. A., Holt, J. A., Turner, R. & Tipper, D. J. (1998). Transmembrane protein insertion orientation in yeast depends on the charge difference across transmembrane segments, their total hydrophobicity, and its distribution. J Biol Chem 273, 24963-24971.
Hartmann, E., Rapoport, T. A. & Lodish, H. F. (1989). Predicting the orientation of eukaryotic membrane-spanning proteins. Proc Natl Acad Sci U S A 86, 5786-5790.
Hartmann, E., Gorlich, D., Kostka, S., Otto, A., Kraft, R., Knespel, S., Burger, E., Rapoport, T. A. & Prehn, S. (1993). A tetrameric complex of membrane proteins in the endoplasmic reticulum. Eur J Biochem 214, 375-381.
Hartmann, E., Sommer, T., Prehn, S., Gorlich, D., Jentsch, S. & Rapoport, T. A. (1994). Evolutionary conservation of components of the protein translocation complex. Nature 367, 654-657.
Hebert, D. N., Garman, S. C. & Molinari, M. (2005). The glycan code of the endoplasmic reticulum: asparagine-linked carbohydrates as protein maturation and quality-control tags. Trends Cell Biol.
Hegde, R. S. & Lingappa, V. R. (1996). Sequence-specific alteration of the ribosome-membrane junction exposes nascent secretory proteins to the cytosol. Cell 85, 217-228.
Hegde, R. S., Voigt, S. & Lingappa, V. R. (1998). Regulation of protein topology by trans-acting factors at the endoplasmic reticulum. Mol Cell 2, 85-91.
Chapter 8 - Bibliography
159
Heinrich, S. U., Mothes, W., Brunner, J. & Rapoport, T. A. (2000). The Sec61p complex mediates the integration of a membrane protein by allowing lipid partitioning of the transmembrane domain. Cell 102, 233-244.
Heinrich, S. U. & Rapoport, T. A. (2003). Cooperation of transmembrane segments during the integration of a double-spanning protein into the ER membrane. EMBO J 22, 3654-3663.
Helenius, A. & Aebi, M. (2004). Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73, 1019-1049.
Hermansson, M., Monne, M. & von Heijne, G. (2001). Formation of helical hairpins during membrane protein integration into the endoplasmic reticulum membrane. Role of the N and C-terminal flanking regions. J Mol Biol 313, 1171-1179.
Hessa, T., Kim, H., Bihlmaier, K., Lundin, C., Boekel, J., Andersson, H., Nilsson, I., White, S. H. & von Heijne, G. (2005). Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433, 377-381.
Heymann, J. A. & Subramaniam, S. (1997). Expression, stability, and membrane integration of truncation mutants of bovine rhodopsin. Proc Natl Acad Sci U S A 94, 4966-4971.
High, S. & Dobberstein, B. (1991). The signal sequence of preprolactin interacts with the methionine-rich domain of the 54 kD protein of signal recognition particle. J Cell Biol 113, 229-233.
High, S., Görlich, D., Wiedmann, M., Rapoport, T. A. & Dobberstein, B. (1991). The identification of proteins in the proximity of signal-anchor sequences during their targeting to and insertion into the membrane of the ER. J Cell Biol 113, 35-44.
High, S. & Laird, V. (1997). Membrane protein biosynthesis - all sewn up? Trends Cell Biol 7, 206-210.
Iwasaki, K., McCarter, J., Francis, R. & Schedl, T. (1996). emo-1, a Caenorhabditis elegans Sec61p gamma homologue, is required for oocyte development and ovulation. J Cell Biol 134, 699-714.
Johnson, A. E. & van Waes, M. A. (1999). The translocon: a dynamic gateway at the ER membrane. Annu Rev Cell Dev Biol 15, 799-842.
Chapter 8 - Bibliography
160
Jungnickel, B. & Rapoport, T. A. (1995). A posttargeting signal sequence recognition event in the endoplasmic reticulum membrane. Cell 82, 261-270.
Kalies, K. U., Gorlich, D. & Rapoport, T. A. (1994). Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex. J Cell Biol 126, 925-934.
Kalies, K. U. & Hartmann, E. (1996). Membrane topology of the 12- and the 25-kDa subunits of the mammalian signal peptidase complex. J Biol Chem 271, 3925-3929.
Kalies, K. U., Rapoport, T. A. & Hartmann, E. (1998). The beta subunit of the Sec61 complex facilitates cotranslational protein transport and interacts with the signal peptidase during translocation. J Cell Biol 141, 887-894.
Karnik, S. S. & Khorana, H. G. (1990). Assembly of functional rhodopsin requires a disulfide bond between cysteine residues 110 and 187. J Biol Chem 265, 17520-17524.
Kaufmann, A., Manting, E. H., Veenendaal, A. K., Driessen, A. J. & van der Does, C. (1999). Cysteine-directed cross-linking demonstrates that helix 3 of SecE is close to helix 2 of SecY and helix 3 of a neighboring SecE. Biochemistry 38, 9115-9125.
Keenan, R. J., Freymann, D. M., Walter, P. & Stroud, R. M. (1998). Crystal structure of the signal sequence binding subunit of the signal recognition particle. Cell 94, 181-191.
Keenan, R. J., Freymann, D. M., Stroud, R. M. & Walter, P. (2001). The signal recognition particle. Annu Rev Biochem 70, 755-775.
Kellaris, K. V., Bowen, S. & Gilmore, R. (1991). ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein. J Cell Biol 114, 21-33.
Kelleher, D. J., Karaoglu, D., Mandon, E. C. & Gilmore, R. (2003). Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties. Mol Cell 12, 101-111.
Kennan, A., Aherne, A. & Humphries, P. (2005). Light in retinitis pigmentosa. Trends Genet 21, 103-110.
Khorana, H. G. (1992). Rhodopsin, photoreceptor of the rod cell. An emerging pattern for structure and function. J Biol Chem 267, 1-4.
Chapter 8 - Bibliography
161
Kim, S. J., Mitra, D., Salerno, J. R. & Hegde, R. S. (2002). Signal sequences control gating of the protein translocation channel in a substrate-specific manner. Dev Cell 2, 207-217.
Knauer, R. & Lehle, L. (1999). The oligosaccharyltransferase complex from yeast. Biochim Biophys Acta 1426, 259-273.
Knight, B. C. & High, S. (1998). Membrane integration of Sec61alpha: a core component of the endoplasmic reticulum translocation complex. Biochem J 331, 161-167.
Kota, J. & Ljungdahl, P. O. (2005). Specialized membrane-localized chaperones prevent aggregation of polytopic proteins in the ER. J Cell Biol 168, 79-88.
Kowarik, M., Kung, S., Martoglio, B. & Helenius, A. (2002). Protein folding during cotranslational translocation in the endoplasmic reticulum. Mol Cell 10, 769-778.
Krieg, U. C., Johnson, A. E. & Walter, P. (1989). Protein translocation across the endoplasmic reticulum membrane: Identification by photocross-linking of a 39 kD integral membrane glycoprotein as part of a putative translocation tunnel. J Cell Biol 109, 2033-2043.
Krishnasastry, M., Walker, B., Braha, O. & Bayley, H. (1994). Surface labeling of key residues during assembly of the transmembrane pore formed by staphylococcal alpha-hemolysin. FEBS Lett 356, 66-71.
Kuroiwa, T., Sakaguchi, M., Omura, T. & Mihara, K. (1996). Reinitiation of protein translocation across the endoplasmic reticulum membrane for the topogenesis of multispanning membrane proteins. J Biol Chem 271, 6423-6428.
Laird, V. & High, S. (1997). Discrete cross-linking products identified during membrane protein biosynthesis. J Biol Chem 272, 1983-1989.
Lauring, B., Kreibich, G. & Weidmann, M. (1995). The intrinsic ability of ribosomes to bind to endoplasmic reticulum membranes is regulated by signal recognition particle and nascent-polypeptide-associated complex. Proc Natl Acad Sci U S A 92, 9435-9439.
Le Gall, S., Neuhof, A. & Rapoport, T. (2004). The endoplasmic reticulum membrane is permeable to small molecules. Mol Biol Cell 15, 447-455.
Chapter 8 - Bibliography
162
Lecomte, F. J., Ismail, N. & High, S. (2003). Making membrane proteins at the mammalian endoplasmic reticulum. Biochem Soc Trans 31, 1248-1252.
Levy, D. (1996). Membrane proteins which exhibit multiple topological orientations. Essays Biochem 31, 49-60.
Levy, R., Wiedmann, M. & Kreibich, G. (2001). In vitro binding of ribosomes to the beta subunit of the Sec61p protein translocation complex. J Biol Chem 276, 2340-2346.
Liao, S., Lin, J., Do, H. & Johnson, A. E. (1997). Both lumenal and cytosolic gating of the aqueous ER translocon pore are regulated from inside the ribosome during membrane protein integration. Cell 90, 31-41.
Lin, J. & Addison, R. (1995). A novel integration signal that is composed of two transmembrane segments is required to integrate the Neurospora plasma membrane H(+)-ATPase into microsomes. J Biol Chem 270, 6935-6941.
Liu, L. P. & Deber, C. M. (1998). Uncoupling hydrophobicity and helicity in transmembrane segments. Alpha-helical propensities of the amino acids in non-polar environments. J Biol Chem 273, 23645-23648.
Luirink, J. & Sinning, I. (2004). SRP-mediated protein targeting: structure and function revisited. Biochim Biophys Acta 1694, 17-35.
Martoglio, B., Hofmann, M. W., Brunner, J. & Dobberstein, B. (1995). The protein-conducting channel in the membrane of the endoplasmic reticulum is open laterally toward the lipid bilayer. Cell 81, 207-214.
Martoglio, B. & Dobberstein, B. (1996). Snapshots of membrane-translocating proteins. Trends Cell Biol 6, 142-147.
Mason, N., Ciufo, L. F. & Brown, J. D. (2000). Elongation arrest is a physiologically important function of signal recognition particle. EMBO J 19, 4164-4174.
Matlack, K. E., Mothes, W. & Rapoport, T. A. (1998). Protein translocation: tunnel vision. Cell 92, 381-390.
Matoba, S. & Ogrydziak, D. M. (1998). Another factor besides hydrophobicity can affect signal peptide interaction with signal recognition particle. J Biol Chem 273, 18841-18847.
Chapter 8 - Bibliography
163
Mayor, S. & Riezman, H. (2004). Sorting GPI-anchored proteins. Nat Rev Mol Cell Biol 5, 110-120.
McCormick, P. J., Miao, Y., Shao, Y., Lin, J. & Johnson, A. E. (2003). Cotranslational protein integration into the ER membrane is mediated by the binding of nascent chains to translocon proteins. Mol Cell 12, 329-341.
Meacock, S. L. (1999). The biosynthesis of a polytopic membrane protein at the endoplasmic reticulum. PhD thesis The University of Manchester.
Meacock, S. L., Lecomte, F. J., Crawshaw, S. G. & High, S. (2002). Different Transmembrane Domains Associate with Distinct Endoplasmic Reticulum Components during Membrane Integration of a Polytopic Protein. Mol Biol Cell 13, 4114-4129.
Meacock, S. L. G., J. J. A.; High, S. (2000). Protein targeting and translocation at the endoplasmic reticulum membrane - through the eye of a needle? Essays Biochem: Molecular trafficking 36, 1-13.
Menetret, J. F., Neuhof, A., Morgan, D. G., Plath, K., Radermacher, M., Rapoport, T. A. & Akey, C. W. (2000). The structure of ribosome-channel complexes engaged in protein translocation. Mol Cell 6, 1219-1232.
Menetret, J. F., Hegde, R. S., Heinrich, S. U., Chandramouli, P., Ludtke, S. J., Rapoport, T. A. & Akey, C. W. (2005). Architecture of the ribosome-channel complex derived from native membranes. J Mol Biol 348, 445-457.
Meyer, H. A. & Hartmann, E. (1997). The yeast SPC22/23 homolog Spc3p is essential for signal peptidase activity. J Biol Chem 272, 13159-13164.
Meyer, H. A., Grau, H., Kraft, R., Kostka, S., Prehn, S., Kalies, K. U. & Hartmann, E. (2000). Mammalian Sec61 is associated with Sec62 and Sec63. J Biol Chem 275, 14550-14557.
Migliaccio, G., Nicchitta, C. V. & Blobel, G. (1991). The signal sequence receptor, unlike the signal recognition prticle receptor, is not essential for protein translocation. J Cell Biol 117, 15-25.
Millman, J. S. & Andrews, D. W. (1997). Switching the model: a concerted mechanism for GTPases in protein targeting. Cell 89, 673-676.
Chapter 8 - Bibliography
164
Molinari, M. & Helenius, A. (2000). Chaperone selection during glycoprotein translocation into the endoplasmic reticulum. Science 288, 331-333.
Molinari, M., Calanca, V., Galli, C., Lucca, P. & Paganetti, P. (2003). Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle. Science 299, 1397-1400.
Moss, K., Helm, A., Lu, Y., Bragin, A. & Skach, W. R. (1998). Coupled translocation events generate topological heterogeneity at the endoplasmic reticulum membrane. Mol Biol Cell 9, 2681-2697.
Mothes, W., Prehn, S. & Rapoport, T. A. (1994). Systematic probing of the environment of a translocating secretory protein during translocation through the ER membrane. EMBO J 13, 3973-3982.
Mothes, W., Heinrich, S. U., Graf, R., Nilsson, I., von Heijne, G., Brunner, J. & Rapoport, T. A. (1997). Molecular mechanism of membrane protein integration into the endoplasmic reticulum. Cell 89, 523-533.
Mullins, C., Meyer, H. A., Hartmann, E., Green, N. & Fang, H. (1996). Structurally related Spc1p and Spc2p of yeast signal peptidase complex are functionally distinct. J Biol Chem 271, 29094-29099.
Mutka, S. C. & Walter, P. (2001). Multifaceted physiological response allows yeast to adapt to the loss of the signal recognition particle-dependent protein-targeting pathway. Mol Biol Cell 12, 577-588.
Newitt, J. A. & Bernstein, H. D. (1997). The N-domain of the signal recognition particle 54-kDa subunit promotes efficient signal sequence binding. Eur J Biochem 245, 720-729.
Ng, D. T. W., Brown, J. D. & Walter, P. (1996). Signal sequences specify the targeting route to the endoplasmic reticulum membrane. J Cell Biol 134, 269-278.
Nilsson, I., Kelleher, D. J., Miao, Y., Shao, Y., Kreibich, G., Gilmore, R., von Heijne, G. & Johnson, A. E. (2003). Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex. J Cell Biol 161, 715-725.
Nothwehr, S. F. & Gordon, J. I. (1990). Targeting of proteins into the eukaryotic secretory pathway: signal peptide structure/function relationships. Bioessays 12, 479-484.
Chapter 8 - Bibliography
165
Oda, Y., Hosokawa, N., Wada, I. & Nagata, K. (2003). EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin. Science 299, 1394-1397.
Oliver, J., Jungnickel, B., Gorlich, D., Rapoport, T. & High, S. (1995). The Sec61 complex is essential for the insertion of proteins into the membrane of the endoplasmic reticulum. FEBS Lett 362, 126-130.
Oliver, J. D., Hresko, R. C., Mueckler, M. & High, S. (1996). The Glut 1 glucose transporter interacts with calnexin and calreticulin. J Biol Chem 271, 13691-13696.
Ota, K., Sakaguchi, M., Hamasaki, N. & Mihara, K. (2000). Membrane integration of the second transmembrane segment of band 3 requires a closely apposed preceding signal-anchor sequence. J Biol Chem 275, 29743-29748.
Ovchinnikov Yu, A., Abdulaev, N. G. & Bogachuk, A. S. (1988). Two adjacent cysteine residues in the C-terminal cytoplasmic fragment of bovine rhodopsin are palmitylated. FEBS Lett 230, 1-5.
Paetzel, M., Karla, A., Strynadka, N. C. & Dalbey, R. E. (2002). Signal peptidases. Chem Rev 102, 4549-4580.
Palade, G. (1975). Intracellular aspects of the process of protein synthesis. Science 189, 347-358.
Palczewski, K., Kumasaka, T., Hori, T. & other authors (2000). Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289, 739-745.
Panzner, S., Dreier, L., Hartmann, E., Kostka, S. & Rapoport, T. A. (1995). Posttranslational protein transport in yeast reconstituted with a purified complex of Sec proteins and Kar2p. Cell 81, 561-570.
Parodi, A. J. (2000). Protein glucosylation and its role in protein folding. Annu Rev Biochem 69, 69-93.
Pool, M. R., Stumm, J., Fulga, T. A., Sinning, I. & Dobberstein, B. (2002). Distinct modes of signal recognition particle interaction with the ribosome. Science 297, 1345-1348.
Powers, T. & Walter, P. (1996). The ribosome talks back. Nature 382, 191-192.
Chapter 8 - Bibliography
166
Prinz, A., Behrens, C., Rapoport, T. A., Hartmann, E. & Kalies, K. U. (2000a). Evolutionarily conserved binding of ribosomes to the translocation channel via the large ribosomal RNA. EMBO J 19, 1900-1906.
Prinz, A., Hartmann, E. & Kalies, K. U. (2000b). Sec61p is the main ribosome receptor in the endoplasmic reticulum of Saccharomyces cerevisiae. Biol Chem 381, 1025-1029.
Raden, D., Song, W. & Gilmore, R. (2000). Role of the cytoplasmic segments of Sec61alpha in the ribosome-binding and translocation-promoting activities of the Sec61 complex. J Cell Biol 150, 53-64.
Rapoport, T. A., Jungnickel, B. & Kutay, U. (1996). Protein transport across the eukaryotic endoplasmic reticulum and bacterial inner membranes. Annu Rev Biochem 65, 271-303.
Rapoport, T. A., Matlack, K. E., Plath, K., Misselwitz, B. & Staeck, O. (1999). Posttranslational protein translocation across the membrane of the endoplasmic reticulum. Biol Chem 380, 1143-1150.
Regnacq, M., Hewitt, E., Allen, J., Rosamond, J. & Stirling, C. J. (1998). Deletion analysis of yeast Sec65p reveals a central domain that is sufficient for function in vivo. Mol Microbiol 29, 753-762.
Ridge, K. D., Lee, S. J. S. & Yao, L. L. (1995). In vivo assembly of rhodopsin from expressed polypeptide fragments. Proc Natl Acad Sci USA 92, 3204-3208.
Ridge, K. D., Lee, S. J. S. & Abdulaev, N. G. (1996). Examining rhodopsin folding and assembly through expression of polypeptide fragments. J Biol Chem 271, 7860-7867.
Römisch, K., Webb, J., Herz, J., Prehn, S., Frank, R., Vingron, M. & Dobberstein, B. (1989). Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains. Nature 340, 478-482.
Römisch, K., Webb, J., Lingelbach, K., Gausepohl, H. & Dobberstein, B. (1990). The 54-kD protein of signal recognition particle contains a methionine-rich RNA binding domain. J Cell Biol 111, 1793-1802.
Rosch, K., Naeher, D., Laird, V., Goder, V. & Spiess, M. (2000). The topogenic contribution of uncharged amino acids on signal sequence orientation in the endoplasmic reticulum. J Biol Chem 275, 14916-14922.
Chapter 8 - Bibliography
167
Rothblatt, J. A., Deshaies, R. J., Sanders, S. L., Daum, G. & Schekman, R. (1989). Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast. J Cell Biol 109, 2641-2652.
Sato, M., Hresko, R. & Mueckler, M. (1998). Testing the charge difference hypothesis for the assembly of a eucaryotic multispanning membrane protein. J Biol Chem 273, 25203-25208.
Sauri, A., Saksena, S., Salgado, J., Johnson, A. E. & Mingarro, I. (2005). Double-spanning Plant Viral Movement Protein Integration into the Endoplasmic Reticulum Membrane Is Signal Recognition Particle-dependent, Translocon-mediated, and Concerted. J Biol Chem 280, 25907-25912.
Shelness, G. S., Lin, L. & Nicchitta, C. V. (1993). Membrane topology and biogenesis of eukaryotic signal peptidase. J Biol Chem 268, 5201-5208.
Shibatani, T., David, L. L., McCormack, A. L., Frueh, K. & Skach, W. R. (2005). Proteomic analysis of mammalian oligosaccharyltransferase reveals multiple subcomplexes that contain Sec61, TRAP, and two potential new subunits. Biochemistry 44, 5982-5992.
Shieh, B. H., Stamnes, M. A., Seavello, S., Harris, G. L. & Zuker, C. S. (1989). The ninaA gene required for visual transduction in Drosophila encodes a homologue of cyclosporin A-binding protein. Nature 338, 67-70.
Simon, S. M. & Blobel, G. (1991). A protein-conducting channel in the endoplasmic reticulum. Cell 65, 371-380.
Skach, W. R. & Lingappa, V. R. (1993). Amino-terminal assembly of human P-glycoprotein at the endoplasmic reticulum is directed by cooperative actions of two internal sequences. J Biol Chem 268, 23552-23561.
Song, W., Raden, D., Mandon, E. C. & Gilmore, R. (2000). Role of Sec61alpha in the regulated transfer of the ribosome-nascent chain complex from the signal recognition particle to the translocation channel. Cell 100, 333-343.
Spiro, R. G., Zhu, Q., Bhoyroo, V. & Soling, H. D. (1996). Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi. J Biol Chem 271, 11588-11594.
Chapter 8 - Bibliography
168
Stirling, C. J. & Hewitt, E. W. (1992). The S. cerevisiae SEC65 gene encodes a component of yeast signal recognition particle with homology to human SRP19. Nature 356, 534-537.
Stirling, C. J. (1999). Protein targeting to the endoplasmic reticulum in yeast. 1997 Fleming Lecture. Microbiology 145 ( Pt 5), 991-998.
Sung, C. H., Schneider, B. G., Agarwal, N., Papermaster, D. S. & Nathans, J. (1991). Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Proc Natl Acad Sci U S A 88, 8840-8844.
Sung, C. H., Davenport, C. M. & Nathans, J. (1993). Rhodopsin mutations responsible for autosomal dominant retinitis pigmentosa. Clustering of functional classes along the polypeptide chain. J Biol Chem 268, 26645-26649.
Sung, C. H., Makino, C., Baylor, D. & Nathans, J. (1994). A rhodopsin gene mutation responsible for autosomal dominant retinitis pigmentosa results in a protein that is defective in localization to the photoreceptor outer segment. J Neurosci 14, 5818-5833.
Szczesna-Skorupa, E., Browne, N., Mead, D. & Kemper, B. (1988). Positive charges at the NH2 terminus convert the membrane-anchor signal peptide of cytochrome P-450 to a secretory signal peptide. Proc Natl Acad Sci USA 85, 738-742.
Thrift, R. N., Andrews, D. W., Walter, P. & Johnson, A. E. (1991). A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation. J Cell Biol 112, 809-821.
Valcarcel, R., Weber, U., Jackson, D. B., Benes, V., Ansorge, W., Bohmann, D. & Mlodzik, M. (1999). Sec61beta, a subunit of the protein translocation channel, is required during Drosophila development. J Cell Sci 112 ( Pt 23), 4389-4396.
Van den Berg, B., Clemons, W. M., Jr., Collinson, I., Modis, Y., Hartmann, E., Harrison, S. C. & Rapoport, T. A. (2004). X-ray structure of a protein-conducting channel. Nature 427, 36-44.
van der Sluis, E. O., Nouwen, N. & Driessen, A. J. (2002). SecY-SecY and SecY-SecG contacts revealed by site-specific crosslinking. FEBS Lett 527, 159-165.
van Geest, M. & Lolkema, J. S. (2000). Membrane topology and insertion of membrane proteins: search for topogenic signals. Microbiol Mol Biol Rev 64, 13-33.
Chapter 8 - Bibliography
169
VanValkenburgh, C., Chen, X., Mullins, C., Fang, H. & Green, N. (1999). The catalytic mechanism of endoplasmic reticulum signal peptidase appears to be distinct from most eubacterial signal peptidases. J Biol Chem 274, 11519-11525.
Voigt, S., Jungnickel, B., Hartmann, E. & Rapoport, T. A. (1996). Signal sequence-dependent function of the TRAM protein during early phases of protein transport across the endoplasmic reticulum membrane. J Cell Biol 134, 25-35.
von Heijne, G. (1985). Structural and thermodynamic aspects of the transfer of proteins into and across membranes: Academic Press, Inc.
von Heijne, G. (1989). Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues. Nature 341, 456-458.
von Heijne, G. (1998). Life and death of a signal peptide. Nature 396, 111-113.
Walter, P. & Johnson, A. E. (1994). Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu Rev Cell Biol 10, 87-119.
Wang, T. & Hebert, D. N. (2003). EDEM an ER quality control receptor. Nat Struct Biol 10, 319-321.
Wessels, H. P. & Spiess, M. (1988). Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence. Cell 55, 61-70.
Wiedmann, M., Gorlich, D., Hartmann, E., Kurzchalia, T. V. & Rapoport, T. A. (1989). Photocrosslinking demonstrates proximity of a 34 kDa membrane protein to different portions of preprolactin during translocation through the endoplasmic reticulum. FEBS Lett 257, 263-268.
Wilkinson, B. M., Critchley, A. J. & Stirling, C. J. (1996). Determination of the transmembrane topology of yeast Sec61p, an essential component of the endoplasmic reticulum translocation complex. J Biol Chem 271, 25590-25597.
Willer, M., Jermy, A. J., Steel, G. J., Garside, H. J., Carter, S. & Stirling, C. J. (2003). An in vitro assay using overexpressed yeast SRP demonstrates that cotranslational translocation is dependent upon the J-domain of Sec63p. Biochemistry 42, 7171-7177.
Chapter 8 - Bibliography
170
Wilson, C. M., Kraft, C., Duggan, C., Ismail, N., Crawshaw, S. G. & High, S. (2005). Ribophorin I associates with a subset of membrane proteins after their integration at the sec61 translocon. Journal of Biological Chemistry 280, 4195-4206.
Wittke, S., Dunnwald, M. & Johnsson, N. (2000). Sec62p, a component of the endoplasmic reticulum protein translocation machinery, contains multiple binding sites for the Sec-complex. Mol Biol Cell 11, 3859-3871.
Woolhead, C. A., McCormick, P. J. & Johnson, A. E. (2004). Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins. Cell 116, 725-736.
Yan, A. & Lennarz, W. J. (2005). Unraveling the mechanism of protein N-glycosylation. J Biol Chem 280, 3121-3124.
Yan, Q., Prestwich, G. D. & Lennarz, W. J. (1999). The Ost1p subunit of yeast oligosaccharyl transferase recognizes the peptide glycosylation site sequence, -Asn-X-Ser/Thr. J Biol Chem 274, 5021-5025.
Young, B. P., Craven, R. A., Reid, P. J., Willer, M. & Stirling, C. J. (2001). Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo. EMBO J 20, 262-271.
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APPENDICES
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(A)
Intensity of products Nascent chain Opsin band Sec61α adduct
Fraction of Sec61α adduct
Relative fraction to OP96
OP96 530932 50055 0.0943 1.00 OP109 1355772 97392 0.0718 0.76 OP130 - - - - OP150 11074 418 0.0377 0.40 OP164 722440 63663 0.0881 0.93 OP204 - - - - OP259 - - - -
(B)
Intensity of products Nascent chain Opsin band Sec61α adduct
Fraction of Sec61α adduct
Relative fraction to OPTM1PPL164
OPTM1PPL109 16493.9 824.4 0.0500 0.94 OPTM1PPL130 26036.5 898.6 0.0345 0.65 OPTM1PPL150 71729 2870.7 0.0400 0.75 OPTM1PPL164 47964.4 2553.9 0.0532 1.00 OPTM1PPL204 86374.9 3942.5 0.0456 0.86 OPTM1PPL259 - - - -
Appendix 1.1 Raw data for the quantification of products obtained from the cross-linking analysis of integration intermediates of (A) normal opsin and (B) OPTM1PPL polypeptide chain. Where appropriate, products due to uncross-linked doubly-glycosylated opsin chains and Sec61α adducts from immunoprecipitations using the α-opsin antibody were quantified using the AIDA software. The fraction of Sec61α adduct was obtained by dividing the intensity of the Sec61α adduct by the value obtained for doubly-glycosylated opsin chains. The integration intermediate with the highest fraction was set to a nominal value of 1.00 and the relative fractions of Sec61α adduct formation for the other intermediates were calculated.
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M N G T E G P N F Y V P F S N K T G V V R S P F E A P Q 28
Y Y L A E P W Q F S M L A A Y M F L L I M L G F P I N F 56
L T L Y V T V Q H K K L R T P L N Y I L L N L A V A D L 84
F M V F G G F T T T L Y T S L H G Y F V F G P T G C N L 112
E G F F A T L G G E I A L W S L V V L A I E R Y V V V C 140
K P M S N F R F G E N H A I M G V A F T W V M A L A C A 168
A P P L V G W S R Y I P E G M Q C S C G I D Y Y T P H E 196
E T N N E S F V I Y M F V V H F I I P L I V I F F C Y G 224
Q L V F T V K E A A A Q Q Q E S A T T Q K A E K E V T R 252
M V I I M V I A F L I C W L P Y A G V A F Y I F T H Q G 280
S D F G P I F M T I P A F F A K T S A V Y N P V I Y I M 308
M N K Q F R N C M V T T L C C G K N P L G D D E A S T T 336
V S K T E T S Q V A P A 348
Appendix 1.2 The amino acid sequence of bovine opsin. Cysteine residues which have been replaced with glycine residues to obtain the cysteine null opsin polypeptide chain are in blue, while residues which have been converted to cysteine residues for site-specific cross-linking are in red. Regions representing transmembrane domains are underlined in black.
M N G T E G P N F Y V P F S N K T G V V R S P F E A P Q 28
Y Y L A E P W H E E T N N E S F V I Y M F V V H F I I P 56
L I V I F F C Y G Q L V F T V K E A A A Q Q Q E S A T T 84
Q K A E K E V T R M V I I M V I A F L I C W L P Y A G V 112
A F Y I F T H Q G S D F G P I F M T I P A F F A K T S A 140
V Y N P V I Y I M M N K Q F R N C M V T T L C C G K N P 168
L G D D E A S T T V S K T E T S Q V A P A 189
Appendix 1.3 The amino acid sequence of the OPN/5-7 polypeptide chain. Residues 36 to 194 have been deleted from the sequence of opsin to form OPN/5-7. The position of the deleted region is indicated with ‘ ’. All other notations are as described in appendix 1.2.
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M N G T E G P N F Y V P F S N K T G V V R S P F E A P Q 28
Y Y L A E P W Q F S M L A A Y M F L L I M L G F P I N C 56
L T L Y V T V Q H K K L R T T P V G P N G P G N G Q V S 84
L R D L F D R A V M V S H Y I H D L S S E M F N E F D K 112
R Y A Q G K G F I T M A L N S G H T S S L P T P E D K E 140
Q A Q Q T H H E V L M S L I L G L L R S W N D P L Y H L 168
V T E V R G M K G A P D A I L S R A I E I E E E N K R L 196
L E G M E M I F G Q V I P G A K E T E P Y P V W S G L P 224
S L Q T K D E D A R Y S A F Y N L L H G L R R D S S K I 252
D T Y L K L L N C R I I Y N N N C 269
Appendix 1.4 The amino acid sequence of OPTM1PPL[cys56]. Residues representing opsin TM1 is underlined in black while residues in the preprolactin sequence are shaded in grey. The cysteine probe at position 56 is in bold.
M N G T E G P N F Y V P F S N K T G V V R S P F E A P Q 28
Y Y L A E P W Q F S M L A A Y M F L L I M L G F P I N G 56
L T L Y V T V Q H K K L R T P L N Y I L L N L A V A D L 84
F M V F G G F T T T L Y T S L H G Y F V F G P T G G N L 112
E G C F A T L G G E I A L W S L V V L A I E R Y V V V G 140
K P T P V G P N G P G N G Q V S L R D L F D R A V M V S 168
H Y I H D L S S E M F N E F D K R Y A Q G K G F I T M A 196
L N S G H T S S L P T P E D K E Q A Q Q T H H E V L M S 224
L I L G L L R S W N D P L Y H L V T E V R G M K G A P D 252
A I L S R A I E I E E E N K R L L E G M E M I F G Q V I 280
P G A K E T E P Y P V W S G L P S L Q T K D E D A R Y S 308
A F Y N L L H G L R R D S S K I D T Y L K L L N C R I I 336
Y N N N C 341
Appendix 1.5 The amino sequence of OPTM1-3PPL[cys115]. The transmembrane domains are underlined in black while residues due to preprolactin are shaded in grey. Cys115 is in bold.
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175
Appendix 1.6 Schematic representation of the secondary structure of bovine opsin (adapted from Palczewski et al., 2000). Cysteine residues which were replaced with glycines are shown in blue while residues mutated to cysteines are shown in red. Asparagine-linked glycan groups are indicated by ‘Y’ and the disulphide bridge is represented with a blue dashed line.
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