Protein translocation across

membranes (3)
• Transport into the
endoplasmic reticulum (ER)

• Methods to study protein

translocation across
membranes
 Translocation

Translocation means “a change of location”.

In the context of protein trafficking, translocation
refers to a protein crossing a membrane.
 Nascent

Nascent means “coming into being” (nascent from

Latin nascens, “being born”). A nascent chain
is therefore a newly synthesized polypeptide chain
 Glycosylation

Glycosylation refers to the addition of sugars to

a protein or lipid
 Protein transport across membranes
•Receptor to recognise
signal

signal sequence •Channel through which

to guide protein

Protein •Receptors and channels

are made of proteins, with
one notable exception: SRP

lipid bilayer
One third of all genes encode proteins that must be
targeted and translocated into the ER
 3. Protein import into the
endoplasmic reticulum (ER)

An example of an
ER signal sequence
(this is prelysozyme)
+H N-MRSLLILVLCFLPLAALG K--
3
signal peptidase
cleavage
A typical ER signal sequence has one or more positively
charged amino acids followed by a stretch of 6-12
hydrophobic amino acids
 ER protein import
mRNA
(cotranslational)
signal Translocon (Sec61 complex)
sequence
•Receptors: SRP and
SRP receptor
•Channel: Sec61 complex

SRP receptor binds SRP

SRP and ribosome
(signal recognition
particle)

ER
membrane
 Import into the ER

GTP hydrolysis powers assembly of the nascent

chain/ translocon complex and release of the
SRP/ SRP receptor
Molecular Biology of the Cell
SRP: 6 proteins + 1 small RNA molecule

Nature Structural & Molecular Biology 11, 1049 - 1053 (2004)

Translocation of a soluble protein
into the ER lumen
 Translocation of a membrane protein
into the ER membrane

Molecular Biology of the Cell

 The insertion of multi-spanning
membrane proteins into the ER
membrane

Molecular Biology of the Cell

 N-linked
glycosylation
in the ER

N=asparagine
(single aa code)

N-X-S
or N-X-T
Figure 12-51 Molecular Biology of the Cell (© Garland Science 2008)
Oligosaccharyl transferase is the
enzyme that transfers sugars from
the lipid-linked oligosaccharide
to the asparagine side chain of the
protein
 ER translocation can be co- or post-
translational, and SRP-dependent or
-independent

Naama Aviram, and Maya Schuldiner J Cell Sci

2017;130:4079-4085
© 2017. Published by The Company of Biologists Ltd
 SRP “SRP pathway”
receptor
Sec61
Co-translational

N-terminal signal
sequence, cleaved or
uncleaved

GTP-dependent

SRP-dependent

Oligosaccharyl Signal sequence or TM

transferase
N C
 “SEC62/63 pathway”
Sec61 SRP-independent

N-terminal signal
sequence

ATP-dependent

Co- or post-translational

SEC complex and BiP

(an ATPase)

Signal sequence
Oligosaccharyl
transferase N C
“TRC40 pathway”
SRP-independent

C-terminal membrane
domain/ GPI
(glycosylphosphatidyl
inositol) anchor

Post-translational

ATP dependent

GET pathway in yeast

TM or GPI
N C
 Inside the ER, polypeptides are glycosylated and
helped to fold by “chaperone” proteins

Chaperones help proteins to fold correctly,

and also mark incorrectly folded proteins for
degradation

The ER is also the site of disulphide bond formation

(S-S). Disulphide bonds (between cysteine residues)
help stablise the tertiary and quarternary structure
of proteins.
 ERAD

Molecular Biology of the Cell

 Misfolded proteins are transported back out of the
ER (a process involving accessory exit
factors) and degraded in the cytosol
“retrotranslocation” or “dislocation”

The process of degrading misfolded proteins is called

ER-associated degradation or ERAD
 ER import

• Via translocon complex (usually Sec61)

• Requires signal sequences

• Can be co- or post-translational

• Proteins must be unfolded to pass

through translocon

• Can be ATP- or GTP-dependent

 Methods to study protein translocation
across membranes
1. Transfection approach. Does a particular amino acid
sequence act as a signal?

Gene (cDNA) Signal sequence to test

encoding
cytosolic protein eg.
GFP
 Methods to study protein translocation
across membranes
2. Biochemical approach. Does a protein co-purify
with a particular organelle?

ER
Protein of interest
 Methods to study protein translocation
across membranes
3. Genetic approach.

Yeast as a model organism

•widely used in protein trafficking studies

•yeast screens have identified, for example, the Sec61

ER translocon
See page 703 in MBOC (Alberts, 5th Edition)
or page 632 in MCB (Lodish, 7th Edition)
Protein trafficking
from the ER (1)
General mechanisms of protein trafficking
and transport
 The ER is the starting place for
proteins destined for many
other locations
•Once translocated into or through
the ER membrane, proteins
are glycosylated and folded. If
they pass ER quality control, they
can be transported to the Golgi
complex

•Some proteins are maintained in

the ER “resident proteins” eg. BiP.

Molecular Biology of the Cell

 Trafficking routes from the ER
Proteins can be “resident to a compartment” or “en route
to another compartment

Molecular Biology of the Cell

 •Biosynthetic/ secretory pathway
•Endocytic pathway
•Retrograde/ recycling pathways

Molecular Biology of the Cell

 Signals direct proteins to the
correct place

These signals can be

Amino acid sequences: signal sequences

or signal patches

Protein modifications: glycosylation,

ubiquitination,
lipid modifications
 How is a protein transported from
one membrane-bound compartment
to another?

1. Vesicular transport
vesicle
budding vesicle
fusion
donor target
vesicle movement
compartment compartment
(cytoskeletal elements)

eg. ER to Golgi transport

 GFP-Rab5 Dynein heavy chain Overexpression of
(early endosomes) depletion p50, a subunit of
dynactin

Organelles are not static!

Flores-Rodriguez, Neftali et al. “Roles of dynein and dynactin in early endosome dynamics
revealed using automated tracking and global analysis.” PloS one vol. 6,9 (2011): e24479.
White et al.
Journal of
Cell Biology
1999, 147:
743-759
Not every step requires
vesicles:
some steps are better
described as “direct fusion”
eg. late endosome to
lysosome fusion
 How is a protein transported from
one membrane-bound compartment
to another?

2. Direct fusion

fusion
compartment compartment hybrid
1 2 organelle

eg. late endosome/ lysosome fusion

Bright et al. Current Biology 2005, 15: 360-365