protein targeting: insertion into the er membrane
TRANSCRIPT
Protein targeting:
insertion into the ER membrane
promoter coding region
messenger RNA
messenger RNA
Nascent protein
Gene transcription
by RNA-polymerase
Gene translation
by ribosomes
Protein folding
and transport
(Genomic DNA strand)
Endoplasmic reticulum
Essential chapter in Molecular Biology of the Cell,
Alberts et al., 4rth or 5th edition
Chapter 12
Intracellular compartments and protein sorting
Page 695-749
Protein synthesis, soluble or
membrane spanning?
Two synthesis routes for nuclear encoded genes:
1) Cytosol (soluble ribosomes)
2) Endoplasmic reticulum surface (membrane
bound ribosomes)
Both synthesis routes can lead to soluble as well
as membrane proteins
How are membrane proteins synthesized?
Antibody coding regions contain N-terminal
signal peptides
After translocation,
the signal peptides are cleaved
“Mature” poly-peptides
“Nascent” polypeptides
Assembly to tetramers
Soluble protein after translocation
Lumen
or extracellular
matrix
Cytosol
But some antibodies are membrane spanning
Membrane
bound IgM:
Relevant to our
Immune system
ER-, Golgi-,
vesicle- lumen,
or extracellular
matrix
Cytosol
Membrane proteins
can be classified into different groups
Phospholipid bilayer
ER lumen (oxidising)
Topologically like the
outside of the cell
Cytosol (reducing)
N-terminus (the beginning of the protein)
C-terminus (the end of the protein)
Type 1
Membrane protein
Membrane proteins
can be classified into different groups
Phospholipid bilayer
ER lumen (oxidising)
Topologically like the
outside of the cell
Cytosol (reducing)
C-terminus
N-terminus
Type 2
Membrane protein
Some proteins span the membrane many times
Phospholipid bilayerMultiple membrane spanning
protein
Some proteins span the membrane many times
A range of topologies is possible
NC
NC
N
C
C
N
The various transmembrane domains can be predicted
from the primary structure deduced from the coding region
The difference between membrane spanning proteins and membrane proteins
Phospholipid bilayer
Membrane spanning proteins
ER lumen (oxidising)
Topologically like the
outside of the cell
Cytosol (reducing)
Peripheral
membrane protein
Peripheral
Membrane
protein
How are antibodies inserted into membrane?
The heavy chain is
membrane spanning
The light chain is soluble,
but is membrane associated
via its interaction with the
heavy chain.
ER-, Golgi-,
vesicle- lumen,
or extracellular
matrix
Cytosol
Transmembrane domains are not cleaved and
remain part of the mature peptide
After translocation,
the signal peptides are cleaved
“Mature” poly-peptides
“Nascent” polypeptides
Assembly to tetramers
tethered to the membrane
TM domain
Cytosolic tail
Translocation of type I membrane spanning
proteins starts the same way as for soluble proteins
Portion coding for the
transmembrane domain
Cleavage of the signal peptide
Translocation of type I membrane spanning
continues like this until the transmembrane domain
enters the translocation pore
Portion coding for the
transmembrane domain
When the transmembrane domain reaches the
translocation pore, it stops translocation, hence the
name “stop-transfer” for the TM domain
Portion coding for the
transmembrane domain
Protein synthesis completes, the transmembrane
domain remains associated with the translocation
pore and the cytosolic tail remains in the cytosol
Lateral diffusion from the translocation pore into
the membrane and further folding
Phospholipid bilayer
ER lumen (oxidising)
Topologically like the
outside of the cell
Cytosol (reducing)
N-terminus (the beginning of the protein)
C-terminus (the end of the protein)
Type 1
Membrane protein
SP TM
Coding region of a type I membrane protein
cytosoliclumenal
Phospholipid bilayer
ER lumen (oxidising)
Topologically like the
outside of the cell
Cytosol (reducing)
Smaller N-terminal domain
Bigger C-terminal domain
Type 1
Membrane protein
SP TM
Coding region of another type I membrane protein
cytosoliclumenal
Membrane proteins
can be classified into different groups
Phospholipid bilayer
ER lumen (oxidising)
Topologically like the
outside of the cell
Cytosol (reducing)
N-terminus
C-terminus
Type 2
C-terminus
N-terminus
Type 1
Type II membrane proteins have no signal peptide
Phospholipid bilayer
ER lumen (oxidising)
Topologically like the
outside of the cell
Cytosol (reducing)
C-terminus
N-terminus
Type 2
Membrane protein:
(No signal peptide!)
TM
Type II membrane spanning proteins are often
post-translationally translocated
Portion coding for the
transmembrane domain
Less clearly
established
N-terminus
Type II membrane spanning proteins are often
post-translationally translocated
Less clearly
established
C-terminus
N-terminusSRP-dependent or independent
(competition assays in vitro)
Some type II membrane proteins are called
tail-anchored”
Phospholipid bilayer
ER lumen (oxidising)
Topologically like the
outside of the cell
Cytosol (reducing)
C-terminus
N-terminus
Extreme case of Type 2
Tail anchored
(No signal peptide!)
TM
Tail anchored molecules are always
post-translationally translocated
Novel SRP-independent route,
first published in 2007, translocation pore unknown
Transmembrane domain-
encoding
The protein leaves the
ribosome before SRP
could possibly bind
Post-translational modification
Folding and assembly into homo or hetero multimers
Proteolytic cleavage of portions
Glycosylation
Myristoylation,
Prenylation,
Phosphorylation
CN C C
O
G
HH
O
Protein anchored to membrane by
Fatty acid chain linked to N-terminal
Glycine (G: the side chain is H)
Amide
linkage
Post-translational modification
CN C C
O
H
HH
O
Protein anchored to membrane by
Fatty acid chain linked to N-terminal
glycine
Other examples of membrane anchoring include
GPI anchoring, prenylation (farnesyl or geranylgeranyl group)
or palmitylation (linking to palmitic acid) and involve other
amino acids (i.e. cysteine) and often the protein C-terminus.
Amide
linkage
NC
Myristic
acid
Membrane linkage via C-terminal prenylation
C
CH2
S
CH2
C O CH3
H O
N
CCysteine
Methylated
C-terminus
Where do we find membranes?
NE
RG
Lytic vacuole
Storage vacuole
Secretion
Mitochondria
Chloroplasts
Peroxisomes
Signals direct proteins to
Organelles
• With the exception of a few plastid and
mitochondrial proteins the proteins
destined for organelles are made in the
cytosol.
• To be incorporated into the right
organelle they need a signal within the
protein and a receptor which specifically
recognises the signal
Three mechanisms of transport
• Gated transporte.g. between nucleus and cytoplasm, mediated by
nuclear pore complexes
• Transmembrane transporte.g. across the membrane of the ER, mitochondria,
plastid or peroxisome
• Vesicular transporte.g. between organelles of the endomembrane system
(the secretory pathway)
Two main-stream protein targeting groups
Continued synthesis
on rough ER
Translocation
at the ER
Further transport via
vesicle budding and fusion
ER export via vesicles to
reach the Golgi apparatus
Golgi export in vesicles
destined to various places
Cytosolic
Nuclear
mitochondria
chloroplasts
peroxisomes
Nuclear export of mRNA
Translation of mRNA in the cytosol
1) Synthesis in cytosol or 2) SRP arrest
Transport to the nucleus
Proteins are not synthesized
in the nucleus
To act in the nucleus, they
have to be imported from the
cytosol
Examples:
Polymerases
Transcription factors
Ribosomal proteins
Typically 5 mm
in diameter
Possible Evolutionary Origin of the Nuclear
Membrane
Note the ER and nuclear membrane are continuous
More than 50 different proteins
called nucleoporins make up the
nuclear pore
Water filled pore
Scanning EM of the nucleus side of the nuclear
envelope
The nuclear pore complex from
the cytoplasmic side.
The membrane has been removed
with detergent.
Section of
nuclear
membrane,
showing that
the inner and
outer envelopes
are continuous.
•Molecules < 5,000 MW are freely permeable (e.g ATP, cofactors, small metabolites)
•Molecules of 17,000 MW equilibrate slowly (e.g. small proteins)
•Molecules of 60,000 (e.g.larger proteins) require active transport
9 nm
This equates to a channel size of 9 nm.
However much larger proteins and ribosome subunits have to move between
nucleus and cytoplasm. How is this achieved?
Nuclear targeting signals
• Usually a stretch of basic (i.e. amino acids
that are positively charged at cellular pH)
• The exact sequence and the location in
the protein are not usually important
T- antigen is a viral protein that works in the
Nucleus.
It is identified here by a fluorescent antibody.
When the second lysine (basic amino acid) is
changed to a threonine, the protein can
no longer accumulate in the nucleus. This shows
the lysine is NECESSARY for the function of
the signal.
If the sequence PPKKKRKV is attached
to a cytosolic protein it can confer nuclear
localisation. It is therefore SUFFICIENT to
function as a NLS.
Nuclear import and export is receptor-mediated
Mitochondria
The “powerhouse” of the cell
Outer membrane
Inner membrane: highly folded, 5-fold the surface of the outer
membrane, contains respiratory chain molecules,
ATP synthetase, and transport proteins.
Inter membrane space: similar to cytoplasm and
contains enzymes that use ATP
Matrix: Mitochondrial genome,
Protein synthesis machinery
Variety of enzymes
0.5 –1 mm
In diameter
(like bacteria)
4 different places for proteins to go
within the mitochondria
matrix Outer membrane Inner membrane Inter membrane
space
Example: Transport into the matrix
•Mitochondrial presequences do not have a
specific primary amino acid sequence
•They are enriched in BASIC (red)
HYDROPHOBIC (yellow) and POLAR usually
hydroxylated amino acids (blue).
•When folded into an alpha helix the basic and
polar residues lie predominantly on one face
of the helix and the hydrophobic residues on the
other.
•This feature is recognised by the presequence
receptor proteins
Features of mitochondrial presequences
TOM and TIM complexes
interact to form a ‘contact site’
at which proteins transported
into the matrix can cross both
membranes
Recognise
the presequence
Transport into the Matrix
Targeting to chloroplasts or plastids
Figure 1-44 from Biochemistry and Molecular Biology of Plants Buchanan et al.,
Chloroplasts are just one member of a family
of organelles called plastids
Development of chloroplasts from etioplasts
Is light dependent
Increasing time in light
Genes encoding photosynthetic proteins and enzymes are expressed,
the proteins translated and imported into the organelle.
The colourless chlorophyll precursor protochlorophyllide is converted to
Chlorophyll.
The prolamellar body (PB) contains very high amounts of lipids that assemble
with the newly synthesised membrane proteins to form the thylakoids
Fig 4.4 Biochemistry and Molecular Biology of Plants Buchanan et al.,
Protein import has been
studied mainly in chloroplasts
because it is easy to get
enough material
Import is post translational
Signals are often composites
and transport can occur in
multiple steps
Transport to the thylakoid space, several steps:
Outer membrane
Inner membrane
Chloroplast
Inter membrane space
Stroma
Thylakoid space
Chloroplast
transit peptide
Thylakoid
signal sequence
Mature
peptide
Chloroplast targeting signals
•No conserved primary amino acid sequence
•Forms a random coil in aqueous solution
Stromal transit peptide when fused to a passenger protein
directs transport to the stroma.
When deleted protein stays in cytoplasm
Therefore necessary and sufficient
Experimental evidence for a role in transport:
fusion and deletion
Protein import machinery of chloroplasts
TOC
TIC
cytosol
Outer envelope
Inner envelope
Stroma
Chaperones
Chaperones
TOC = Translocon of the Outer Chloroplast envelope
TIC =Translocon of the Inner Chloroplast envelope
Mechanism of import I
Ribosome-nascent
chain
Chloroplast protein
Bound to cytosolic
chaperone
TOC
ATP
GTP
Protein handed
Over to TOC machinery
Two of the proteins of the TOC complex are GTPases whose receptor activity
Is regulated by GTP binding and hydrolysis a bit like SRP and the SRP receptor
in ER targeting.
Mechanism of Import II
TOC
TIC
cytosol
Outer envelope
Inner envelope
Stroma
Chaperones
ATP
ADP
Chaperones
ATP
ADP
Stromal
peptidase
ATP hydrolysis by chaperones in the intermebrane space and stroma traps/pulls
the protein across the membrane.
Entry into the thylakoid space is very similar to translocation into the endoplasmic reticulum
or secretion across the plasma membrane in bacteria
Chloroplast
transit peptide
Thylakoid
signal sequence
Mature
peptide
In the thylakoid
In the stroma
In the cytosol
bacterium
‘Complex’ plastids in protists
• Some photosynthetic eukaryotes (e.g. dinoflagellates,
euglena) arose as a result of a secondary
endosymbiosis when a photosynthetic eukaryote (which
already had a chloroplast) was engulfed by another
eukaryotic cell.
• As a result their plastids are surrounded by three or
more membranes and are called complex plastids
• Proteins targeted to the complex plastids are first
targeted to the ER and then possibly via vesicles to the
plastid.
Peroxysomes have a main function in
• Breaking down fats
In mammals peroxisomes and mitochondria
co-operate to break down fatty acids.
Peroxisome enzyme systems shorten long
chain fatty acids, which are then exported
to mitochondria to finish the job.
Peroxisomes also deal with branched
Fatty acids like phytanic acid that are important
components of the diet
In plants and yeasts peroxisomes are the ONLY place in the cell where fatty acids are
degraded
Targeting to peroxisomes
• Unlike mitochondria and chloroplasts
peroxisomes have just a single membrane
and no DNA
• Like mitochondria and chloroplasts they
import proteins post translationally
• Unlike mitochondria and chloroplasts they
import proteins that are FOLDED
Peroxisomal proteins are targeted in two ways
PTS1: Soluble proteins use C-terminal SKL signals and
variants (i.e. SRL)
PTS2: membrane proteins are possibly targeted from the ER,
but this is currently under debate
SKL
H2N-(R/K)X6(Q/H)(L/A)
The signals are recognised by specific receptorsMuch less is known about the translocation mechanism