Chapter 11
Protein Trafficking into the Endoplasmic Reticulum
Learning Objectives, Part 1
Understand:
• Components of endomembrane system
• Endomembrane evolution
• Similarities between some organelle lumens and
extracellular environment
• Protein Movement through endomembrane system
• Rough vs smooth endoplasmic reticulum
• ER shape
• Continuity of ER and outer nuclear membrane and
ribosomes on outer nuclear membrane
• Overview of translation of proteins into the rough ER
Introduction to the Endomembrane
System
Anatomy, Evolution, Environment, and Movement
The Endomembrane System
• Starts at Endoplasmic
Reticulum (ER)
• Goes through Golgi
Apparatus
• Possible End Destinations
➢ Endosomes
➢ Lysosomes
➢ Peroxisome
➢ Cell Membrane
• Trafficking vesicles move
between organelles and
membranes
Endomembrane Evolution
• Likely plasma
membrane invagination
(still debated)
• Supportive evidence
➢ Nuclear envelope
double membrane
➢ Outer nuclear
membrane continuous
with ER
Environmental Similarities
• Some organelle lumens*
share chemical similarities
to extracellular matrix.
➢ “non-cytosolic
environment”
➢ E.g., endoplasmic
reticulum or Golgi body
• Very different from cytosol • Most proteins trafficked via
endomembrane system end up
*Lumen is the inside cavity of an either in the extracellular solution/
cell membrane or in endomembrane
organelle or tubular structure
compartments, such as endosomes
and lysosomes.
• Outward movement Protein Movement through
➢ RNA from nucleus to rough ER endomembrane system
➢ Protein from ER to Golgi
➢ Proteins in vesicles from Golgi to
organelles and membranes
• Some proteins (resident proteins)
need to stay in ER or Golgi so will not
progress.
• Trafficking through endomembrane
system is tightly controlled and
involves signal sequences in proteins.
• Proteins that go through
endomembrane system are translated
mostly on ER:
➢ They enter ER, and are
chemically modified and, except
for resident ER proteins, are then
packaged into vesicles and
transported to Golgi
➢ In Golgi, proteins are further
modified and then sorted to
different sites.
The Golgi “Airport” Analogy
• Protein “passengers” arrive at
and move through rough ER
(A,B).
• ER movement to Golgi like
“security check”; only properly
folded proteins can enter (C).
• Signal sequence “tickets” help
proteins to get to “gates” at
Golgi (sorting) (D).
• Resident ER and Golgi proteins
are like airport staff (E).
• Vesicle are like “airplanes”; they
ship proteins to final destination.
Entering the Rough ER
Rough vs Smooth ER, ER Signal Sequence, Signal Recognition
Particle, Soluble vs Transmembrane Proteins
Rough and Smooth ER
• ER is the largest organelle in cells and
consists of a large membrane network
• Rough ER
➢ Continuous with outer nuclear
membrane
➢ Made of flat cisternae
➢ Site of protein synthesis
➢ “Rough” because covered with
ribosomes
• Smooth ER
➢ Made of tubules
➢ Site of lipid synthesis
➢ “Smooth” because no ribosomes
• The cisternae of rough ER and tubes of
smooth ER are interconnected,
enclosing a single internal space.
ER Shape
• The two distinct ER
shapes maintained by
proteins
➢ E.g., CLIMP-63 extends
between sides of
cisternae to hold flat
shape
➢ Other proteins embedded
within the outer
phospholipid layer of the
bilayer curve the edges of
the cisternae.
The ER is continuous with the outer
nuclear membrane
• The ER is continuous with
the outer nuclear
membrane, and the outer
nuclear membrane is
continuous with the inner
nuclear membrane.
• Nuclear pore complexes
(NPCs) can prevent the
movement of proteins
between the inner and
outer nuclear membranes
Ribosomes on the outer nuclear membrane
• Because the outer
nuclear membrane is
continuous with the ER,
it is also covered in
ribosomes.
• These ribosomes
translate soluble
proteins into the
perinuclear space and
membrane proteins into
the outer nuclear
membrane. These
proteins will then move
into the rough ER
lumen and rough ER
membrane respectively.
TEM of rough ER
Overview of translation of proteins into the
rough ER
• A signal recognition particle (SRP) binds
emerging N-terminal of the protein being
translated in the cytosol.
• Ribosome-SRP complex is recruited to an
SRP receptor on rough ER membrane.
• Ribosome is handed off to a translocation
channel called a translocon.
• Soluble polypeptides are translated directly
into the rough ER lumen
• Membrane polypeptides are translated in
the same way but contain one or more
stop signals that embed protein in the
membrane.
Learning Objectives (Part 2)
Understand:
• How a protein enters the rough endoplasmic reticulum as it is being translated
• Explain the process by which a nascent protein enters the rough endoplasmic
reticulum.
➢ Understand functions of ER signal sequence, SRP, SRP receptor, and
translocon in this process
• How soluble proteins are processed as they enter the ER lumen
• How membrane-embedded proteins are processed in the rough endoplasmic
reticulum.
➢ Differences in processing of single and multi-pass membrane proteins
• Addition and function of chemical modifications that occur on proteins entering
the endoplasmic reticulum.
• Functional importance of disulfide bonds, including where they may occur
within a protein (and which amino acid forms disulfide bonds).
• What a glycoprotein is and its structure and function
• Process by which proteins are glycosylated in the rough endoplasmic
reticulum.
Entry of proteins into endomembrane system
• Proteins enter the endomembrane system as they are translated
into the rough ER by ribosomes on the cytosolic surface of the
rough ER and outer nuclear membrane:
➢ Process called co-translation translocation
Choosing the ER Pathway
• mRNA translation begins
for most proteins on a
ribosome in the cytoplasm.
➢ N-terminus of peptide
will determine whether
protein enters:
o Cytosolic pathway or
o Endomembrane
pathway ER signal sequences are
▪ If N-terminus has about15-30 amino acids long
an ER signal and are highly hydrophobic
sequence,
translation is
paused
Signal Recognition Particle (SRP)
• ER signal sequence recruits a
signal recognition complex
(SRP).
➢ SRP is complex, consisting of
6 proteins bound to RNA (A)
➢ A hydrophobic pocket at one
end of SRP binds hydrophobic
ER signal sequence (B,C)
➢ The other end of the SRP
blocks the ribosome entrance
of central chamber, preventing
entrance of tRNAs and
pausing translation
• SRP/polypeptide/ribosome
complex is then recruited by an
SRP receptor embedded in rough
ER membrane
Recruitment of SRP to the rough
• Ribosome-SRP complex binds ER and ribosome handoff
SRP receptor in rough ER
membrane.
• Both SRP and the SRP receptor
have a bound GTP, and when
they come together, both GTPs
are hydrolyzed.
• GTP hydrolysis results in transfer
of the ribosome from SRP to a
channel in the rough ER
membrane, called
a translocon, and release of SRP
from the SRP receptor.
➢ Hence, GTP hydrolysis acts
as a checkpoint to ensure
that SRP releases the
ribosome only at the rough
ER membrane, where the
SRP receptors are located.
• When ribosome binds the
Soluble Proteins
translocon, ER signal sequence
on polypeptide undergoing
translation also binds translocon,
causing the translocon central
channel to open.
• The ribosome exit tunnel lines up
with translocon channel.
• Translation resumes with signal
sequence still bound to the
translocon channel.
• As polypeptide is synthesized, it
is translocated to luminal side.
➢ Co-translational translocation
• Signal peptidase cleaves off ER
signal sequence, and
polypeptide is release to ER
lumen. Essential Cell Biology, 4th Ed.
Single pass Transmembrane Proteins
• Like soluble proteins, ER signal sequence in
single-pass transmembrane proteins is initially
bound to the translocon channel (and later cleaved
off), as the rest of the polypeptide chain is
threaded through the membrane into the rough ER
lumen.
• Membrane polypeptides have an additional signal
sequence called a stop signal that prevents the
entire polypeptide from passing across the rough
ER membrane
➢ Stop signal is rich in hydrophobic residues and
interacts with hydrophobic residues on the
inside of the channel, preventing it from
passing through into the rough ER lumen.
➢ Final configuration; N-terminal end in the rough
ER lumen; C-terminal end in the cytosol, and
transmembrane domain in the translocon
channel.
➢ When translation is complete, the
transmembrane domain is released laterally out
of the channel into the rough ER membrane, Essential Cell Biology, 4th Ed.
Multi-pass Transmembrane Proteins
• Multi-pass proteins
➢ Hydrophobic Start and stop signal
sequences work in pairs to weave
protein into membrane.
➢ In some, signal sequence is an
internal start signal that is not
cleaved--serves as start sequence
and remains embedded in
membrane.
Covalent Modifications in
the Rough ER
Disulfide Bond Formation and Glycosylation
Covalent Modifications in the ER
• Covalent modifications begin
immediately upon entering ER
• Disulfide bonds and glycosylation
only found on non-cytosolic
proteins
• Help proteins survive harsh non-
cytosolic “extracellular”
environment
➢ Proteolytic activity, fluctuations
in pH, temperature, mechanical
stress, oxidizing condition, etc.
• Disulfide bonds help provide
structural stability; can help prevent
unfolding of proteins
• Glycosylation can block proteolytic
enzymes from degrading proteins,
provide thermostability, increased
solubility, etc.
Disulfide Bond Formation
• Disulfide bonds are covalent
bonds that are formed between
the sulfur atoms from two nearby
cysteine residues either:
➢ within the same polypeptide
chain (at the level of tertiary
structure) or
➢ between different polypeptide
chains within a protein (at the
level of quaternary structure)
• These reactions are catalyzed by
the enzyme protein disulfide
isomerase (PDI) in the rough
ER lumen.
Disulfide Bond Formation
• Helps protect proteins from
oxidation
• Exposure to oxygen results in
oxidation.
➢ Oxygen takes electrons from
proteins, alters their structure
and function.
➢ Cytosol is a reducing
environment, unlike outside
the cell
• Cysteine amino acids protected
by formation of covalent bond
between sulfurs, which oxidizes
them
➢ Protects electrons (because
they are already oxidized in
disulfide bonds) and stabilizes
protein structure
Glycosylation
• Covalent addition of an
oligosaccharide generates a
glycoprotein.
➢ Most proteins that enter the
ER, both soluble and
membrane, are glycosylated
➢ Occurs on luminal side of ER
• Protective function at protein
and cellular level
➢ Glycocalyx barrier of
glycoproteins
• Cell recognition function in
multicellular organisms
Attaching the First Oligosaccharide
• 14-sugar oligosaccharide is always
first to attach:
➢ three glucose, nine mannose,
and two N-acetylglucosamine
• Starts attached to a dolichol lipid on
bilayer
• Transferred to protein by
oligosaccharyltransferase
➢ Attaches to glycosylation
signal sequence on protein:
Asn-X-Ser/Thr
➢ Attached to Nitrogen in Asn
➢ Called N-linked glycosylation
• Process one of first steps after
protein emerges into ER lumen
Learning Objectives (Part 3)
Understand:
• Exit from the ER Is controlled to Ensure Protein Quality
• Proteins go through 5 checkpoints to ensure they are properly
folded and modified
• Accumulation of unfolded and/or misfolded proteins can cause
cell death or diseases
• Proteins in the rough ER that are properly folded leave it at ER
exit sites (ERESs).
• The first checkpoint is in the ERES and ensures that all proteins
are properly folded and modified.
• Proteins that do not assemble properly, are actively retained in
the ER by chaperones
• How the 5 checkpoints function to prevent release of misfolded
proteins from ER
Exit from the ER Is Controlled to Ensure
Protein Quality
• Exit from the ER is highly
selective.
• Proteins that fail to fold
correctly, and dimeric or
multimeric proteins that do
not assemble properly, are • Accumulation of unfolded
actively retained in the ER by and/or misfolded proteins can
chaperones cause cell death or diseases
• Proteins go through 5 such as cancer, diabetes, and
checkpoints to ensure they neurodegenerative diseases
are properly folded and in humans.
modified
The 1st Checkpoint: Leaving the ER
• Proteins in the rough ER lumen
and membrane proteins in the
rough ER membrane leave the
rough ER at ER exit sites
(ERESs).
• Only proteins that are correctly
folded and modified can leave
ER via ERESs.
• The first checkpoint is in the
ERES and ensures that all
proteins are properly folded and,
if required, properly covalently
modified. Only proteins that meet
these standards will be packaged
into vesicles for departure from
Proteins are packed into vesicle at ER exit sites
• Within ERESs, soluble
and membrane proteins
are packaged into
vesicles that are
transported to the Golgi.
• Soluble proteins in the
rough ER remain
soluble in the Golgi
• Rough ER membrane
proteins remain in the
Golgi membrane.
• 1st checkpoint in ERES Quality Control:
• There are a number of
Chaperone Proteins
chaperones in ERES, including
➢ BiP and Calnexin: t bind to
misfolded proteins.
➢ BiP is a soluble protein in
ER lumen
➢ Calnexin is an integral
membrane protein in ER
membrane with N-terminal in
ER lumen
➢ BiP and Calnexin bind to
hydrophobic amino acids
that exposed in unfolded
proteins exposed
o Hold back misfolded
proteins back
o Properly folded proteins
are free to leave ERES
The 2nd Checkpoint: Getting Sent Back
• Movement from the rough ER to the
Golgi is in the forward,
or anterograde, direction.
• Improperly folded or modified
proteins sometimes escape the
rough ER chaperones by chance by
moving into a Golgi-bound vesicle.
• Such proteins are detected within
the Golgi and are returned to the
rough ER via transport vesicles
moving in the backward,
or retrograde, direction
➢ Return to the rough ER is the
second quality control checkpoint.
➢ Return of resident ER protein
involves ER retention signal, such
as KDEL, which is detected by
membrane receptors in the Golgi
• If the misfolded protein rate is The 3rd Checkpoint: UPR
high, the Unfolded Protein
Response (UPR) is triggered
➢ 3rd checkpoint
• UPR is triggered indirectly by
chaperone BiP
➢ normally binds to Ire1 and
unfolded proteins.
➢ When there are high levels
of unfolded proteins, there is
less BiP to bind to Ire1; Ire1
dimers form
➢ Ire1 induces a signaling
cascade that causes
production of more
chaperones, other quality
control proteins, and growth
of ER membrane.
The 4th Checkpoint: Dislocation
• If misfolded proteins continue to
accumulate, despite UPR: \
➢ A 4th checkpoint, called
dislocation, is implemented
o Misfolded proteins are
exported to the cytosol
through a rough ER
membrane channel
(process not well
understood).
o After dislocation, the
misfolded proteins are • Failure of one or more
tagged with ubiquitin , of these checkpoints
targeting them for can lead to various
proteasome degradation. types of diseases in
• Failure of dislocation leads to humans
apoptosis, programmed cell death:
5th and final checkpoint
Asymmetrical Membranes and Proteins
• Membranes are asymmetrical:
➢ Cytosolic and extracellular
environment (equivalent to
ER/Golgi lumen and other
compartments in endomembrane
system) are very different.
➢ Soluble proteins modified to be
compatible with extracellular
environment.
➢ Membrane proteins are
asymmetrical: are exposed to both
sides, extracellular and cytosolic:
o Orientation must be
maintained: important for cell
function; different chemistry in
different environments
o Asymmetry maintenance starts
in ER, maintained throughout
endomembrane system.