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Translocation through the endoplasmic reticulum membrane

Institute of BiochemistryBenoît Kornmann

Benoît Kornmann Institute of Biochemistry ETH Zürich18.09.13

Mittwoch, 18. September 2013

Protein sorting

Endo

mem

bran

e sy

stem



Permeable to proteins but not to ions

IgG tetramer (16 nm)

Fully hydrated Ca2+ ion (0.6 nm)



The signal hypothesis

Blobel, G. & Sabatini, D. D. 1971 in Biomembranes Vol. 2 (ed. Manson, L. A.) 193–195



The Endoplasmic reticulum

Sheets and tubules

Rough and smooth Sheets ~ Rough Tubules ~ Smooth

Tubules

Sheets

Nuclear envelope



Professional secretory cells

Plasma cell (activated B lymphocyte) secrete ~500 IgG molecules per second. More than their own dry weight everyday!



Rough endoplasmic reticulum

Ribosomes associated to ER membrane Co-translational translocation



Principal players in protein translocation

Ribosome Signal-recognition particle

(SRP)

SRP-receptor (SR) on ER membrane

Aqueous channel (translocon)



Challenges in SRP-mediated targeting

SRP must recognize nascent signal peptides and bind them with high affinity and selectivity

Once released, the nascent polypeptide must engage with the translocon

Finally SRP and SR must dissociate for being recycled

SRP must release peptide upon binding to SRP-receptor (SR)

Therefore energy is needed for completion of the cycle



Signal sequences

target proteins for secretion and membrane insertion (PM proteins, secreted proteins and proteins of secretory organelles)

Are located at the N-terminus of pre-protein

are typically cleaved off by signal peptidase

typical length: 15-25 amino acid residues

Bear no sequence homology but characteristic 3-partite structure

n-region: hydrophilic, basic

h-region: hydrophobic, 7-15 amino acid residues

c-region: 2-9 polar, small amino acid residues (consensus site for cleavage by signal peptidase)

Signal sequences end-up inserted in the ER membrane



SRP is conserved across all three domains of Life



Eukaryotic SRP pauses translation through its ALU domain

The SRP Alu domain competitively inhibits elongation factor binding by covering the same site on the ribosome

(eEF2 promotes the translocation step of amino-acyl-tRNA from A to P site during protein synthesis)



Signal recognition particle

(N-terminal)

Interaction with SR

and ribosome

GTPase activity/interaction with ribosome

(methionine-rich)

Interaction with

signal peptide

From Sulfolobus solfataricus (Archea)



Signal recognition in the M-domain

Signal peptide

N-Domain

G-Domain

M-Domain

T. Hainzl, et al., Nature structural & molecular biology. 18, 389-91 (March 2011).



Binding to the SRP receptor: the N- and G-domains

Two subunits: alpha and beta (SRα and SRβ) SRα resembles SRP54

N-domain

G-domainN-domain

G-domain

M-domain

A-domain

N

N-domain

N

CN

C

N

N C

C

SRP54 (Mammalian)

Ffh (E. Coli)

SRα (Mammalian)

FtsY (E. Coli)

SRP

SR



The SRP-SR complex Quasi two-fold symmetrical heterodimer

Extensive contacts between G-domains

Major rearrangements in N-domain between monomer and complex

Ffh

Light: Monomer

Dark: Dimer:SRP:SR



Reciprocal stimulation of GTPase activity

SRP and SR reciprocally stimulate each other’s GTPase activity -

after GTP hydrolysis the complex dissociates.



Reciprocal stimulation of GTPase activity

SRP and SR reciprocally stimulate each other’s GTPase activity -

after GTP hydrolysis the complex dissociates.

The two GTPase sites form a composite active site with the nucleotides packed in a head-to-tail manner

Symmetrical hydrogen bonds between the 3’OH ribose of one nucleotide and the γ-phosphate of the other

GTP-hydrolysis severs these connections and leads to complex dissociation

a.w. attacking water



Last step of the SRP reaction: the SRP-RNC binds to the translocon

Binding of SRP to SR exposes a translocon binding site close to the peptide exit channel on the ribosome



SRP cycle

SRP M-domain binds to signal peptide

SRP-SR interaction liberates a translocon-binding domain on the ribosome

GTP hydrolysis causes SRP-SR complex disassembly

This cause rearrangement in N- and G-domains allowing interaction with SRP receptor



Next questions:

How does signal binding promote SRP-SR complex formation? How does binding in M-

domain rearrange NG-domains?

How does formation of SRP-SR complex cause peptide release? How does a change in NG

domain cause a conformational change in M-domain?

The answer probably lies in the RNA moiety of the SRP

Linker is ordered and

elongated

One RNA base is flipped toward GTPase

Ataide et al., Science. 331, 881-886 (February 2011).



RNA may participate in GTPase reaction

Flipped base

Ataide et al., Science. 331, 881-886 (February 2011).



Animation of SRP targeting

Ribosome-bound SRP scan nascent chains for emerging signal peptides.

Upon signal sequence binding, conformational changes are transmitted to the GTPase core, allowing SR binding

SR binding displace Srp54/Ffh from Ribosomal protein L23

L3 is now free to bind to translocon

SRP54/SR complex is free to interact with flipped base on SRP RNA

GTP hydrolysis dissociate the complex



Translocation

The ribosome translocon complex



c

The translocon

Bacteria: SecY SecE SecG

Eukaryotes: Sec61α Sec61β Sec61γ Archea

Blue: Sec61α Red: Sec 61β Green: Sec61γ

Sec61 from Methanococcus Jannaschi (Archea)



Helix 2a serves as a plug in the closed state (a)

Six hydrophobic residues work as a seal in the open state (b and c)

These two features likely maintain a membrane barrier during membrane protein synthesis

The pore size of 5-8 Å would not allow passage of folded domains

Imp0rtant features of the Sec61 channel



Membrane integration requires sideway opening of the translocon

The transmembrane helix needs to exit the channel through a side opening (seam)



Lateral opening of the translocon

c

Methanococcus Jannaschi Pyrococcus Furiosus

P. F. Egea, R. M. Stroud, PNAS. 107, 17182-7 (October 2010).B. Van den Berg et al., Nature. 427, 36-44 (January 2004).



Topology of membrane proteins

Membrane topology is established co-translationally in the ER and can't be changed afterwards

How does the ribosome know that it has to stop transferring through translocon when a TM domain happens?



Topology of membrane proteins: Type I



Topology of membrane proteins: Type II



Topology of membrane proteins: Type III (or Type Ia)



Topogenesis of membrane proteins in the ER



What determines the orientation of TMHs?

Observations: Charged residues flanking the hydrophobic core of the signal:

Positive-inside rule - the more positively charged segment stays in the cytosol

Hydrophobicity of the signal: a. N-terminal signals initially insert in the Nexo/Ccyt orientation and then invert based

on their charge distribution

b. The more hydrophobic the signal, the harder to invert due to higher affinity for the translocon

Other possible causes Protein folding (internal signals)

Folding of hydrophilic sequences N-terminal to a signal sterically hinders N-terminal translocation

...but the detailed molecular mechanisms are unknown



Signal sequence cleavage

Blobel, G. & Dobberstein, B. J. Cell Biol. 67, 835–851 (1975).

Achieved by signal sequence peptidase Co-translational



Co- and post-translational targeting

No additional energy source on the cytosolic side

ATP hydrolysis by BiP (HSP70) in ER lumen

ATPase activity of SecA pumps protein through the pore of translocon



Further reading

S. F. Ataide et al., Science. 331, 881-6 (February 2011). T. Hainzl, et al Nat struct mol biol. 18, 389-91 (March 2011). P. F. Egea, R. M. Stroud, PNAS. 107, 17182-7 (October 2010). Halic, M. et al. (2004) Nature, 427, 808-814 Egea, P. F. et al. (2004) Nature, 427, 215-221 Shan S. et al. (2004) PloS Biology, 2, 1572-1581 Rosendal, K. R. et al. (2003) PNAS, 100, 14701-14706 van den Berg, B. et al. (2003) Nature, 427, 36-44 Mitra et al. (2005) Nature, 438, 318-324

Reviews Osborne, Rapoport, van den Berg Annu. Rev. Cell Dev. Biol.2005. 21:529–

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institute of biochemistry benoît kornmann - eth zn.ethz.ch/~nielssi/download/5....

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