Topic 2.

1
Endomembrane system
Overview of the endomembrane system
– organelles, compartments & vesicle transport

Biosynthetic, secretory, and endocytic pathways

Suggested Textbook Readings: Morris Ch. 5: p33

Lodish Ch. 14: p267 – 270
 Endomembrane System
! Dynamic, coordinated network
of all the cell’s organelles and
related structures (except plasma
membrane
peroxisomes, mitochondria &
chloroplasts)
Golgi endosomes
– endoplasmic reticulum (ER)
– Golgi
– Endosomes
– lysosomes/vacuoles lysosome
(vacuole)
– secretory granules
– plasma membrane endoplasmic reticulum

! Large amounts of material (e.g.,

lipids, proteins, etc) are mRNA/protein
nucleus
exchanged (trafficked) between
each organelle/structure via
small, membrane-bound
mitochondrion
transport vesicles chloroplast
 Endomembrane system

! The organelles of the endomembrane system are

structurally and functionally distinct from one another
– contain a particular set of proteins
– perform a unique set of activities
– provides compartmentalisation and functional diversity
– conserved in eukaryotes
– dynamic structures
 Vesicle Transport
! Trafficking through the endomembrane
system via transport vesicles
Four general steps of vesicle transport:
! Step 1: ‘cargo’-containing vesicle buds
off the donor membrane compartment
– vesicle coat proteins select which donor
membrane & lumenal ‘cargo’ proteins
can enter (or not enter) the nascent
transport vesicle AND regulate vesicle
formation and budding
 Vesicle Transport
! Step 2: nascent vesicle is transported
through the cytosol to the recipient
membrane compartment
– vesicle receptor proteins regulate the
intracellular trafficking of the vesicle to the
proper recipient membrane
– involves molecular motors and cytoskeleton
‘highways’ i.e., motor proteins direct vesicle
movement within the cell by linking to the
vesicle surface and to the cytoskeleton
element
 Vesicle Transport
! Step 3: vesicle ‘fuses’ with the proper
recipient membrane compartment
! receptors proteins also regulate vesicle-
recipient membrane fusion
! vesicle (donor) membrane & lumenal
cargo proteins are incorporated into the
recipient compartment
 Vesicle Transport

! Step 4: entire process of budding and

fusion is repeated and can occur in the
reverse direction
! other receptors proteins regulate the
recycling of any proteins that ‘escaped’
to the recipient compartment back to
the donor membrane compartment
 Distinct Trafficking Pathways
Several distinct trafficking pathways
exist within the endomembrane plasma
system; all rely on transport vesicles membrane

Biosynthetic pathway
Golgi
– materials are transported endosomes
from the ER to the Golgi, to
endosomes, and then to
either lysosomes (referred to lysosome
as vacuoles in plants and (vacuole)

fungi/yeasts) or, in some endoplasmic reticulum

instances, to the plasma
membrane (PM)
mRNA/protein
nucleus

mitochondrion
chloroplast
 Distinct Trafficking Pathways
Secretory pathway (two types) extracellular space

! Constitutive secretion plasma

membrane
– ER-derived materials are secretory
vesicle
continually transported from
the Golgi to the PM and/or Golgi endosomes

‘released’ (via exocytosis)

outside of the cell (i.e.,
extracellular space) lysosome
– Secretory transport vesicle (vacuole)
membrane components are endoplasmic reticulum
incorporated into the PM, and
vesicle lumenal ‘cargo’ is
released into the extracellular mRNA/protein
nucleus
space

mitochondrion
chloroplast
 Distinct Trafficking Pathways
extracellular space
Secretory pathway (two types)
secretory plasma
! Regulated secretion granule membrane

! Occurs only in specialized cells

Golgi endosomes
! ER-derived materials from the
Golgi are stored in secretory
granules
lysosome
! In response to a cellular signal, (vacuole)
secretory granules fuse with the
PM and release (via exocytosis) endoplasmic reticulum
their lumenal ‘cargo’ into the
extracellular space mRNA/protein
nucleus
! e.g., regulated release (secretion)
of neurotransmitters by nerve
cells into the synaptic cleft mitochondrion
chloroplast
 Distinct Trafficking Pathways
extracellular space
Secretory pathway (two types)
secretory plasma
granule membrane

Golgi endosomes

lysosome
(vacuole)

endoplasmic reticulum

mRNA/protein
nucleus

mitochondrion
chloroplast
 Distinct Trafficking Pathways
Endocytic pathway extracellular space

plasma
• Operates in the opposite direction membrane
of the secretory pathway (i.e.,
materials move into the cell)
Golgi endosomes
• Materials from the PM (e.g.,
receptor proteins destined for
degradation) and/or extracellular
space are incorporated into the transport lysosome
vesicles (vacuole)
cell (via endocytosis) and then
transported to endosomes and to endoplasmic reticulum
lysosomes (vacuoles)
mRNA/protein
nucleus

mitochondrion
chloroplast
 Endomembrane System
extracellular space

• Dynamic, coordinated network of secretory plasma

organelles and related structures granule membrane
secretory
• Materials (e.g., lipids, proteins, etc) vesicle
move within the endomembrane
Golgi endosomes
system via the biosynthetic,
secretory and endocytic pathways
via transport vesicles
• Endoplasmic reticulum (ER) is the lysosome
starting point for the secretory & (vacuole)
biosynthetic pathway
– site of protein (& lipid) synthesis, endoplasmic reticulum
protein folding, and processing
mRNA/protein
nucleus
Topic 2.2
Endoplasmic reticulum
Structure of the ER
Targeting of Soluble Proteins to the ER

Suggested Textbook Readings: Morris Ch. 5: p36 - 337

Lodish Ch. 13: p221 – 229
 Endoplasmic reticulum

Highly complex network of membrane-enclosed, rod-like

tubules and sheet-like cisternae (i.e., flattened sacs)
– organelle with largest surface area
 Endoplasmic reticulum: Structure

• Lumen - aqueous space inside of ER tubules and cisternae

• tubules and cisternae shapes are mediated by reticulons
– unique ER integral membrane proteins that possess a ‘hair-pin’ (V-shaped)
secondary structure and regulate ER membrane curvature (‘bending’)

cisterna

tubule

Illustration of an ER tubule and cisterna Illustration of an ER tubule with membrane-

bound reticulons (left) – each of which possess
[Park & Blackstone, 2010]
http://www.ncbi.nlm.nih.gov/pubmed/20559323
a ‘V-shaped’ secondary structure (right)
[Hu et al. 2011]
http://www.ncbi.nlm.nih.gov/pubmed/22153070
 Endoplasmic reticulum: Structure
ER is a highly dynamic network – ER tubules and cisternae are in constant
flux (e.g., undergo constant bending, fusion, fission, etc)
 Endoplasmic reticulum: Structure

• ER consists of multiple
subdomains
– distinct regions of the ER
network that possess
unique morphologies
and/or functions

• Rough ER and smooth

ER are two ‘classic’
examples of ER
subdomains
– RER: mostly cisterane with
bound ribosomes, involved
in protein and membrane
phospholipid synthesis
 Endoplasmic reticulum: Structure

• ER consists of multiple
subdomains
– distinct regions of the ER
network that possess
unique morphologies
and/or functions

• Rough ER and smooth

ER are two ‘classic’
examples of ER
subdomains
– SER: mostly curved tubules
lacking ribosomes, involved
in Ca2+ storage and
hormone synthesis
 Endoplasmic reticulum
>20 other ER subdomains - each subdomain possesses a unique complement of
proteins and membrane lipids that mediate its distinct function(s)

More examples of ER subdomains

• Outer nuclear membrane:
- continuous with the RER, contains
NUPs and attached ribosomes
• Mitochondria and Plasma
membrane-Associated Membranes
(MAM & PAM):
- regions of ER that make direct
contact with mitochondria or PM;
involved in membrane lipid exchange
• ER Exit Sites (ERES):
– regions where transport vesicles
bud off from the ER en route to the
Golgi [Lynes & Simmen, 2011]
http://www.ncbi.nlm.nih.gov/pubmed/21756943
 Protein synthesis

One of two main sites for protein

plasma
synthesis (translation) in the cell: membrane
1. ‘free’ ribosomes in the cytosol
• Fate of the nascent protein in the
cytosol…. remains in the cytosol (e.g., Golgi endosomes
glycolytic enzyme)
OR
• targets (post-translationally) to the
lysosome
proper intracellular destination (e.g., (vacuole)
mitochondria, chloroplasts, nucleus,
etc) endoplasmic reticulum Cytosol

mRNA/protein
nucleus

mitochondrion
chloroplast
 Protein Synthesis

Rough endoplasmic reticulum (RER)

plasma
2. ER ‘membrane-bound’ ribosomes membrane
• Fate of the nascent soluble or
membrane protein in the RER:
– remains in the RER or localizes Golgi endosomes
(moves laterally in the ER
membrane) to another ER
subdomain
OR lysosome
– targets (via transport vesicles) (vacuole)
from the ER to another post-ER
compartment in the endoplasmic reticulum
endomembrane system (i.e., Golgi
apparatus, endosomes,
mRNA/protein
lysosomes, PM, etc.) nucleus

mitochondrion
chloroplast
Co-translational translocation of a soluble protein into
the RER lumen
STEPS
1 and 2

• In the cytosol, translation of an mRNA begins on a ‘free’ ribosome

• N-terminus of the nascent, growing polypeptide eventually emerges from the
ribosome
• N-terminus contains a signal sequence – stretch of 8-15 hydrophobic amino acids
that serve as an ER targeting signal
• Exposed signal sequence is recognized by the signal recognition particle (SRP)
– SRP = ribonucleoparticle consisting of 6 proteins and 1 small RNA
• SRP then binds to the ribosome and stops protein translation
Co-translational translocation of a soluble protein into
the RER lumen
* Both SRP &
SRP receptor
STEP 3 * are G proteins

• SRP targets the entire complex (i.e., ribosome, ‘stalled’ nascent

polypeptide, mRNA) to the surface of the ER (i.e., RER)
• SRP binds to the SRP receptor
– SRP receptor = hetero-dimeric ER integral membrane protein complex
• Cytosolic-facing domains of SRP receptor serve as the ‘docking site’ for
the incoming SRP
Co-translational translocation of a soluble protein into
the RER lumen
STEP 4

• SRP is ‘released’ from the SRP receptor and simultaneously the ribosome
binds to the cytosolic side of the translocon (Sec61)
– SRP returns to the cytosol for another round of protein import
• ‘Release’ step relies on GTP hydrolysis which cause a conformational change
in the SRP and SRP receptor
– SRP receptor is also released for another round of import
Co-translational translocation of a soluble protein into
the RER lumen
STEP 4 cont’d

• Binding of the ribosome to the translocon (Sec61) results in the continuation

of protein translation
• Signal sequence interacts with the interior of the translocon
• Results in a conformational change in the translocon subunits, leading to the
opening (widening) of the pore ring and displacement of the ‘plug’
 Co-translational translocation of a soluble protein into
the RER lumen
STEPS
5 and 6

BIP

• Growing polypeptide moves through the translocon (i.e., translocated across the ER membrane)
• as the signal sequence enters the lumen (or just before) it is cleaved by the signal peptidase
– ER integral membrane protein (protease) located next to the translocon – catalytic domain of signal peptidase
faces the ER lumen
• As the nascent (cleaved) protein enters the lumen, it is glycosylated (addition of sugars to the polypeptide) and
begins to be properly folded by reticuloplasmins
– reticuloplasmins = chaperones that operate in the ER
– bind to nascent proteins and mediate their proper folding and oligomeric assembly (prevent protein
aggregation)
– e.g., BiP (Binding immunoglobulin Protein), calnexin, calreticulin
Co-translational translocation of a soluble protein into
the RER lumen
STEPS
7 and 8

• following the termination of protein translation and translocation,

ribosome is ‘released’ from the translocon
• ribosome returns to the cytosol for another round of protein import
• ‘release’ of the ribosome leads to closing of the translocon pore ring &
return of the ‘plug’
Co-translational translocation of a soluble protein into
the RER lumen

Ribosome
mRNA
ER Targeting Signal
Signal Recognition Particle
(SRP)
SRP Receptor
Translocon (Sec61)
Signal Peptidase (protease)
Topic 3.3
Endoplasmic reticulum
Co-translational import of integral membrane
proteins
Chapter 8 pg 273-279

Membrane biosynthesis at the ER – maintaining protein & lipid

topology throughout the endomembrane system

ER protein glycosylation and folding

Chapter 8: pg 285-289
 Maintenance of Membrane Asymmetry

Two mechanisms
• Lipid composition
• Modification & orientation of integral
membrane proteins (IMPs)
– Lumenal domain(s) of IMPs
• Remains in the lumen of endomembrane
compartments; glycosylated
• Forms extracellular domain on exoplasmic
(extracellular) face of PM
– Cytoplasmic domain is always in cytoplasm

Fig. 8.14
 Co-translational insertion of an integral membrane
protein into the RER

• Most membrane proteins are also synthesized on membrane-bound

ribosomes at the ER (RER)
– including resident membrane proteins of the ER and all other post-ER
compartments of the endomembrane system (Golgi, endosomes, lysosomes,
pm, transport vesicles, etc.)
• Translocated during translation into the ER membrane in similar manner
to the import of a soluble protein into the ER lumen
– i.e., N-terminal signal sequence is recognized by SRP, SRP delivers the ‘stalled’
polypeptide-ribosome-mRNA complex ……
– ….except, there are a few important mechanistic differences resulting
in mature (membrane) protein being inserted (anchored) into the ER
membrane
Types of Membrane Anchored Proteins
Types of Membrane Anchored Proteins

The translocon
is a polar
molecule with a
concentration of
positively charged
amino acids on its
--- lumenal side and
negatively
charged amino
acids on its
+++ cytoplasmic side
 Co-translational insertion of a Type 1 Integral
Membrane Protein

STEPS 1, 2, and 3
• N terminus of the nascent polypeptide enters the translocon
• Signal sequence is cleaved (in the same manner as soluble ER proteins)
• Growing protein’s first or only transmembrane domain (TMD) enters the
interior of the translocon
– TMD = typically an α-helical, hydrophobic stretch of ~16-25 amino acids
• TMD serves as a stop-transfer sequence
– interaction of the hydrophobic TMD with hydrophobic pore ring stops any further
translocation of the nascent protein through the translocon
 Co-translational insertion of a Type 1 Integral
Membrane Protein

STEPS 4, 5 and 6

• Interaction of TMD with hydrophobic pore ring blocks translocation and

signals the translocon to ‘open’ laterally
• TMD segment of the protein is ‘released’ laterally into the membrane
lipid bilayer
• The ribosome is released from the mRNA and translocon
 Co-translational insertion of a Type 2 Integral
Membrane Protein

The translocon
is a polar
molecule with a
concentration of
positively charged
amino acids on its
--- luminal side and
negatively
charged amino
acids on its
+++ cytoplasmic side
STEP 1

• Type 2 IMPs do not contain a signal sequence. They begin translation on a free
ribosome until the “signal anchor sequence (SAS)” emerges
• The SAS is bound by the SRP in an analogous manner as the signal sequence.
Translation is paused and the nascent polypeptide is tranferred to the translocon
• Upstream (towards the N-terminus) of the SAS is a series of positively charged
residues that are repelled by the positively charged residues on the luminal side of
the translocon
• The SAS inverts, sending the N-terminus out into the cytoplasm
 Co-translational insertion of a Type 2 Integral
Membrane Protein

The translocon
is a polar
molecule with a
concentration of
positively charged
amino acids on its
--- luminal side and
negatively
charged amino
acids on its
+++ cytoplasmic side
STEPS
2 and 3

• Translation continues until the C-terminus emerges from the ribosome and is
deposited into the lumen of the ER.
• The ribosome disassembles and moves off of the translocon
 Co-translational insertion of a Type 3 Integral
Membrane Protein

The translocon
is a polar
molecule with a
concentration of
positively charged
amino acids on its
--- luminal side and
negatively
charged amino
acids on its
+++ cytoplasmic side

• Type 3 IMPs possess a signal anchor sequence (like Type 2 IMPs), however, they
have positively charged residues downstream of the SAS (towards the C-
terminus)
• These positively charged residues are attracted to the negatively charged amino
acids on the cytoplasmic side of the translocon so the alpha-helix does not
undergo reorientation
• Translation continues until the C-terminus emerges from the ribosome
 Insertion of Tail-Anchored Proteins

• Tail-anchored proteins are

embedded in the ER membrane via
a different mechanism than the
SRP, SRP receptor, andtranslocon
• They are entirely translated on free
ribosomes
• Their hydrophobic alpha-helix is
always on the C-terminus of the
protein
• Upon emergence from the
ribosome, the alpha-helix is
recognized and bound by Get3-ATP

GET = Guided Entry of Tail-Anchored Proteins

 Insertion of Tail-Anchored Proteins

• Upon completion of translation on

the free ribosome, Get3-ATP (bound
to the tail-anchored protein) binds
with the Get1/Get2 heterodimer
located on the ER membrane
• Get1/Get2 hydrolyze the ATP of
Get3, which facilitates the transfer
of the alpha-helix to a membrane
spanning channel of the dimer
• The alpha-helix is then released into
the membrane
• Empty Get1/Get2 exchanges the
ADP for ATP in Get3, releasing it
• Tail-anchored proteins may move to
other organelles via pathways that
will be discussed later…
GET = Guided Entry of Tail-Anchored Proteins
 Membrane biosynthesis at the ER
• Membranes do not form de novo
– all membranes arise from pre-existing
membranes
plasma
• Most membrane proteins and lipids are membrane
synthesized at the ER
Exceptions:
– glycolipids synthesized in the Golgi Golgi endosomes
– unique chloroplast and mitochondrial
proteins & lipids
• Nascent ER membrane proteins & lipids
can traffic to other membranes in the lysosome
(vacuole)
cell
– e.g. move to other ER subdomains (via endoplasmic reticulum
lateral diffusion through the bilayer) OR
to other ‘downstream’ organelles of the
endomembrane system (via transport mRNA/protein
vesicles) nucleus

• Results in each organelle possessing a

unique complement of membrane
proteins & lipids mitochondrion
chloroplast
 Membrane biosynthesis in the ER
• Nascent ER membrane proteins and lipids are distributed
and/or orientated in the lipid bilayer in an asymmetric
manner
– Integral membrane proteins
• different regions of the protein face the cytosol and/or
ER lumen
– Peripheral membrane proteins
• located on either the cytosolic or lumenal side of the
ER membrane
– Membrane phospholipids
• distributed unequally between the cytosolic & lumenal
leaflets of the bilayer
• Protein and lipid asymmetry is established at the ER and
maintained throughout the rest of the endomembrane
system Karp Fig 8.14
– e.g. ER lumenal protein or region(s) of an ER membrane-
spanning protein facing the lumen are located in the lumens
of all other endomembrane compartments in which it
resides or, at the PM, on the outside of the cell (i.e.,
extracellular space)
Processing of newly-synthesized proteins in the ER

• ER = ideal processing site for nascent proteins

– first compartment in the endomembrane system (i.e., biosynthetic & secretory
pathways)

• Final steps in co-translational translocation pathway involve ‘processing’ of

the nascent protein in the ER lumen
1. Signal sequence cleavage: removal of the N-terminal signal sequence
2. Initial stages of glycosylation: addition of unique carbohydrate side chains to
specific amino acids of the nascent protein – glycoproteins (required for proper
folding, protein-protein binding, etc.)
3. Protein folding and assembly: nascent protein is folded into the proper 3D
conformation by molecular chaperones (reticuloplasmins)
4. Quality control: misfolded or improperly assembled proteins are recognized and
degraded
 Glycosylation of Proteins in the ER
• Glycosylation
– Addition of oligosaccharide chains to proteins = form glycoproteins
• Oligosaccharide = short chains of sugars monomers linked to form an oligomer
– Order of monomers in oligosaccharide important
• Varies from one glycoprotein to the next
• Impacts function of the glycoprotein
• Functions of oligosaccharides on glycoproteins
– Assists in binding with other macromolecules
– Assists in protein folding
– Important in intracellular trafficking – targets protein to specific
subcellular destination
 Protein Glycosylation in the ER
• Most proteins (soluble and membrane) synthesized in the ER are glycoproteins
– proteins linked to one or more sugar chains (oligosaccharides) attached to specific
amino acids within the nascent polypeptide
• Most common type of glycosylation is N-linked glycosylation
– addition of specific short chains of sugar monomers (linked together in a specific order
to form an oligosaccharide) to the terminal amino group of an asparagine (N)
• N-linked glycosylation consists of two stages:
– i) core glycosylation
– ii) core modification

Note: for some glycoproteins that are transported to other post-ER

compartments, core modification continues in the Golgi [see later]
 Protein glycosylation: Core Glycosylation
First stage of N-linked glycosylation - a complex 13-step process
• Various ER membrane-bound glycosyltransferases synthesize the core oligosaccharide
– ‘core’ = branched oligosaccharide chain consisting of 14 sugar residues, including a 3-glucose-long
terminal branch
• Each enzyme adds (one at a time) a specific sugar to a specific position on the growing core
oligosaccharide
 Protein glycosylation: Core Glycosylation
• Final step involves a glycosyltransferase that links the core oligosaccharide to a specific N
residue(s) on a soluble or integral membrane protein
• core oligosaccharide only transferred to luminal-facing N residue of the sequence -N-x-S/T-

ER Retention
or…
 Protein glycosylation: Core Modification
• After transfer to the nascent protein (1), the 14-sugar core oligosaccharide(s) is gradually trimmed
• First two (of the three) terminal glucose units are removed by glucosidase I & II (2 and 3); both are ER
lumenal (soluble) enzymes
• During N-linked glycosylation and modification, the nascent glycoprotein is also properly folded -
mediated by reticuloplasmins (molecular chaperones = BiP, calreticulin and calnexin)
• During folding, glycoprotein is also subjected to ER quality control (3a) to ensures that the protein
possesses the correct sugar attachments and is properly folded (i.e., no mutations and/or errors made
during protein synthesis)
• Mature protein is released (4) and is either retained in the ER (if it functions in the ER) or is sent to the
Golgi

ER Retention
or…
 ER protein quality control
• Nascent glycoprotein (with one remaining terminal glucose residue) binds to calnexin
– membrane-bound reticuloplasmin that mediates the glycoprotein’s final folding steps
• Glucosidase II then removes (‘trims’) the last (terminal) glucose unit from the core
oligosaccharide
• protein is released from calnexin
• If the protein (soluble or membrane-bound) is properly folded:
 ER protein quality control

• If the protein released from calnexin is

misfolded….
• Recognized by the GT monitoring enzyme
– ER lumenal (soluble) glucosyltransferase (GT) that
recognizes hydrophobic residues that are usually
‘masked’ by attached sugars in a correctly folded
protein
• GT monitoring enzyme adds back a single glucose
residue to the terminal end of the trimmed ‘core’
• Misfolded protein binds (again) to calnexin
– mediates (again) the final steps in proper protein
folding
• Entire process continues (recycles) until the protein
•
is properly folded
OR… Karp Figure 8.17

UGGT = UDP–glucose glycoprotein:glucosyltransferase

Vessey MCB*2050 F16 T3 S48
 ER protein quality control

• Misfolded (abnormal) proteins in the ER lumen (or

membrane) are eventually destroyed via the ER-
Associated Degradation (ERAD) pathway
– e.g. most CF patients – mutant transporter protein
(CFTR) is degraded by ERAD and therefore not
targeted to the PM
• Misfolded protein is translocated back out of the
ER into the cytosol via the translocon
– mechanism responsible for ER lumen-to-cytosol =
retro-translocation; not well understood

Karp Figure 8.17

Vessey MCB*2050 F16 T3 S49

Topic 3.5

ER protein quality control – ER-associated degradation

(ERAD) and the unfolded protein response (UPR) pathways
Chapter 8: pg 285-289

Vesicle trafficking: ER è Golgi; Golgi è ER

ER-to-Golgi vesicle trafficking
ER exit sites (ERES)
COPII-coated vesicle assembly at the ERES
Vesicle targeting and fusion at the Golgi
Golgi-to-ER ‘retrograde’ vesicle trafficking
Suggested Textbook Readings:
Chapter 8: pg 289, 295–298 & 300-303

Vessey MCB*2050 F16 T3 S50

 ER-Associated Degradation (ERAD)
protein destined
• In the cytosol oligosaccharide chains are for degradation
removed and the misfolded protein is poly- Ub poly-Ub
ubiquitinated - misfolded protein is linked to
a chain of repeating (poly) ubiquitin units
– Ubiquitin (Ub) - small (76 aa-long) protein
involved in diverse cellular functions
– mono-Ub – serves as a ‘signal’ for membrane
protein import into endosomal vesicles [see
later]
– Poly-Ub – serves as a ‘signal’ for ER protein
degradation and most other cellular proteins
destined for normal turnover via degradation
by the proteasome
– Proteasome = complex ‘barrel-shaped’, multi-
Karp Figure 12.64c
subunit protein-degrading machine located in
the cytosol (& nucleus)
• Ub-protein binds to the ‘lid’ of the proteasome, Ub chain is removed (and
recycled), the protein is ‘threaded’ into the proteasome, where it is degraded (via
proteolysis), and amino products are reused for new protein synthesis
Vessey MCB*2050 F16 T3 S51
 Proteasome structure

Karp Figure 12.64a/b

Vessey MCB*2050 F16 T3 S52
 Unfolded Protein Response (UPR) pathways
ER protein quality control
• Under certain conditions, misfolded proteins
accumulate in the ER to very high levels (too high for
the ERAD pathway)
– e.g., in various diseases (CF, Alzheimers, etc) misfolded
proteins accumulate in the ER and form toxic
aggregates
• Results in ‘ER stress’ that signals the Unfolded
Protein Response (UPR) pathways
• Mediated by various protein sensors – ER integral
membrane proteins
– e.g. PERK (Protein kinase RNA-like ER Kinase)
ATF6 (Activating Transcription Factor 6)
• Possess ER lumenal-facing ‘stress-sensing domains’
that bind to molecular chaperones in the ER lumen
(e.g. BiP)
• In non-stress conditions, PERK and ATF6 sensors are
inactive due to their binding to BiP Karp Figure 8.18

Vessey MCB*2050 F16 T3 S53

 Unfolded Protein Response (UPR) pathways
In ER-stress conditions, UPR pathways are activated
e.g. PERK-mediated UPR pathway
• BiP is released from PERK in order to aid in the
folding of accumulating (misfolded) proteins
• PERK sensors dimerize and become ‘active’
• Cytosolic-facing kinase domains of ‘activated’ PERK
dimer phosphorylate and inhibit the protein
translation factor eIF2α
– cytosolic protein required for the initiation of protein
synthesis – participates in ribosome-mRNA binding
• protein synthesis in the cell (including at the RER)
decreases
• available molecular chaperones can focus on pre-
existing (misfolded) proteins in the ER
• ER stress is alleviated OR (if not) cell death occurs

Karp Figure 8.18

Vessey MCB*2050 F16 T3 S54

 Unfolded Protein Response (UPR) pathways
E.g. ATF6-mediated UPR pathway
• in ER-stress conditions, BiP is released from ATF6
– BiP needed in the folding of accumulating (misfolded)
proteins
• ‘active’ ATF6 moves from the ER to the Golgi (via
transport vesicles)
• At the Golgi, the cytosolic-facing, transcription factor
domain of ATF6 is cleaved off (by a Golgi associated
protease) and targets to the nucleus
• ATF6 transcription factor domain up-regulates a
number of genes encoding:
– ER molecular chaperones – assist in proper protein
folding (e.g. BiP)
– ER export components – assist in moving (via transport
vesicles) properly folded proteins out of the ER
– ERAD components – assist in degrading misfolded
proteins
• ER stress is alleviated OR (if not) cell death occurs
Karp Figure 8.18

Vessey MCB*2050 F16 T3 S55

 Cystic Fibrosis and ER Stress

It results from mutations in the CFTR gene (cystic fibrosis transmembrane conductance
regulator). Different mutations cause different problems with the encoded protein, including
One major symptom of CF the processing of the translated protein.
is blocked airways.

Vessey MCB*2050 F16 T3 S56