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Mitochondria
Mitochondria
ER Targeting and Secretory Pathway
ER
Golgi
Vesicles
PM
Secretion
Lysosome
Endoplasmic Reticulum
Endoplasmic Reticulum
• Two parts:
Endoplasmic Reticulum
• Rough ER
Endoplasmic Reticulum
• What happens in the ER?
Targeting of Proteins to the ER
What proteins pass through ER?
ER residents
Other components of the
secretory pathway
Secreted proteins
Proteins going to the plasma
membrane
ER targeting is cotranslational
Ribosomes free in cytosol
Recruited to ER
Cotranslational Translocation
Targeting of Proteins to the ER
PLAYERS
In cytoplasm:
Ribosome
Peptide with signal sequence
Signal Recognition Particle (SRP)
In ER membrane:
SRP Receptor
Translocon
Signal Peptidase
BiP
Oligosaccharide Transferase
Cytoplasm
OST
BiP
ER lumen
Targeting of Proteins to the ER
• How are proteins targeted to the ER?
• N-terminal signal sequence:
Signal sequence targets proteins to ER
•In the cytosol:
•Signal Recognition Particle (SRP):
•Cytosolic ribonucleoprotein particle
•Binds GTP
•6 polypeptides bound to a 300 nucleotide RNA
molecule
•RNA acts as a scaffold
•SRP stops translation of mRNA upon binding
•Binds to ER signal sequence and large ribosome
subunit
•Directs complex to the ER membrane so it can bind
the receptor
Targeting of Proteins to the ER
•On the ER membrane:
•SRP receptor
•Alpha and beta subunits; GTP bound in ER
membrane
•Binding of GTP strengthens the interaction
between SRP and the SRP receptor
•Translocon
–Sec61α is a membrane protein with 10
membrane spanning α helicies
–Gated and tightly regulated
ER protein targeting
• Steps 1 and 2
– ER signal sequence emerges from the ribosome
– SRP binds, stops translation
ER protein targeting
• Step 3
– Complex is quickly targeted to the ER membrane and
the SRP receptor
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ER protein targeting
Step 4
Ribosome-cargo transferred to translocon
Channel opens
Signal sequence and polypeptide go into channel
Translation resumes
ER protein targeting
• Steps 5 and 6
– As the polypeptide elongates, it passes into lumen
– Signal sequence is cleaved by signal peptidase and rapidly degrades
ER protein targeting
• Steps 7 and 8
– At the end of translation, ribosome is released
– Protein is drawn into lumen
– Translocon closes
– Protein folds
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Translation drives translocation in ER lumen
Translation drives translocation
Signal sequence cleaved
Ribosome released
BiP (chaperone) - keeps protein unfolded
Oligosaccharide transferase adds sugar
Microsomes
• When cells are homogenized,
ER forms microsomes
• Contain ribosomes
Microsome allows in vitro study of membrane-bound processes
Label secretory protein
Homogenize ER to make microsomes
containing secretory protein
OR make whole cell extracts without
microsomes
Treat with protease +/- detergent
Run on gel to detect protein
Results
Cell-free
protease
detergent
Microsomes
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Secretory proteins translated in the presence of an ER are protected from protease.
This means they are produced inside the ER
Protein Insertion into the ER
• Membrane proteins for ER, Golgi and lysosomes are
sythesized in the ER
• Topogenic sequences direct membrane insertion
Integral Membrane Classes
• Differ by orientation and signal sequence
Orientation of protein in membrane important
for its function
What would happen if the Na/K ATPase pump were
misoriented in the cell?
Two types of membrane signals
Stop-Transfer
Internal alpha-helical hydrophobic
sequence of 22-25 amino acids
Signal Anchor
Internal alpha-helical hydrophobic
sequence of 22-25 amino acids with a
stretch of positively charged amino acids
Insufficient to target to translocon on its to one side
own; not a target of SRP
recognized by SRP and recruited to
Recognition of sequence causes
translocon
translational stop and transfer of
hydrophobic stretch to membrane
Signal anchor amino acids are placed into
membrane, but translation continues
Remaining translation continues in
cytosol unless another signal sequence Location of translation is dictated by the
is encountered
orientation of the anchor (+++ amino
acids)
Integral Membrane Classes
Type I--Have a cleavable ER signal sequence
Type I
• Type I proteins are targeted to the ER by SRP signal
sequence mediated path
– Signal sequence is cleaved in ER lumen
– Stop Transfer Anchor Sequence
– Sec61
– SRP and SRP receptor
– N-term:
– C-term:
Type I proteins contain N-term signal sequences and internal
stop-transfer
Type II
• Internal signal-anchor sequence (SA)
– This stretch of amino acids tells the cell two
things
• SA sequence recruits SRP for targeting to the ER
Internal Signal Anchor Sequences are targeted to the ER by SRP
Type II
• 5’ end is translated in the
cytosol
• Translation of signal anchor
sequence allows SRP to bind
• SRP receptor
• SA to translocon
• Translation finishes
Type III
• Two possible orientations for SA containing
proteins:
• The +++ charges will always be to the
cytosolic side
• In Type II, +++ on the N-terminal side of SA
• In Type III, the +++ on the C-terminal side
Internal Signal Anchor Sequences are targeted to the ER by SRP
Type III
• Steps are similar to
Type II
– N-terminus is in the
lumen
– Due to the +++ charged
amino acids on Cterminal side
Internal Signal Anchor Sequences are targeted to the ER by SRP
Type II
Type III
• laskdfalksd
Transmembrane Sequences
• Note the differences between the three types
Multipass Proteins use a combination
Multipass transmembrane proteins alternate between StopTransfer and Signal Anchor Sequences
Odd:
Even:
Multipass proteinsType Iva and IVb
• Type IVa
Multipass proteins
• Type IVb
Movie
Secretory Pathway
ER
Golgi
Secretory Vesicles
Production, processing, and sorting of material bound for cell membrane components or
secretion
Physical Apposition of Secretory Pathway Components
Secretory vesicle
Golgi
ER-to-Golgi vesicles
Rough ER
Order of travel is critical for proper
modifications
Golgi Complex
• Most proteins leave ER
within minutes
• Where do they go?
• How do they get there?
• Why?
Vesicular Transport
• Vesicles transport proteins
to different target sites
Vesicular Transport from the ER
• With proper folding correct
modifications, proteins
leave the ER
• Transport vesicles
General Steps from ER to Golgi and back again
Target
Cargo accumulation
G-protein binds to donor compartment
Coat assembly and budding
Vesicle
Coat
Coat release
Target compartment fusion
Vesicle components released
Donor
cargo
General Steps from ER to Golgi and back again
Players
G-protein that recruits Coat: Sar1 or ARF
GEF in donor compartment
Coat proteins
Sorting signal sequence in cytosolic portion
of transmembrane cargo
G-protein that targets vesicle to correct
destination: Rab
Rab effector in target compartment
Snares in vesicle and target compartment to
aid in docking and fusion
General Steps from ER to Golgi and back again
• Vesicle coat forms
• Interacts with the cytosolic
portion of membrane proteins
• Coats provide curvature needed
for budding
• Small GTP-binding protein
COPII transport
Overview
• 5 steps
General Steps from ER to Golgi and back again
• Step 1
• Sec12-GEF promotes GDP to GTP exchange in Sar1
• Causes conformation change
• This drives polymerization of COPII
General Steps from ER to Golgi and back again
• Step 2
• Sar1-GTP serves as a binding
site
• Coats provide the curvature
• Membrane cargo is recruited
by a binding site in their
cytoplasmic portions
General Steps from ER to Golgi and back again
Step 3
• Sar1 promotes GTP hydrolysis
Step 4
• Release of coat proteins
Sar1 GTP recruits coat to donor membrane
Sar1
G-protein- GDP vs. GTP induces conformational change to reveal or
hide a hydrophobic stretch of amino acids
GDP =
GTP =
Sar1 GEF (ex Sec12) - donor membrane
Rab GTP docks vesicle to target compartment
Rab
• Direct vesicles to target
membranes
• GTPase super family
Rab GTP docks vesicle to target compartment
• Step 1
• Rab binds effector
protein complex
SNARE interaction induces membrane fusion
• SNAREs promote fusion
• V-SNARE: VAMP (vesicle
assoctiated membrane
protein)
SNARE mediate fusion
• v-SNAREs are proteins in the
vesicle (VAMP)
• t-SNAREs are in the target
location
– t-SNAREs are syntaxin and
SNAP-25
– Syntaxin
– SNAP-25
– The four α-helices form a
tight interaction
SNARE interaction induces membrane fusion
• v-SNARE interacts with tSNARE and SNAP25 in target
membrane interact
• SNARE interaction is very
specific
Release of SNARES
•SNAREs must dissociate after fusion
•Need help to break apart
•Two Proteins aid in release:
•NSF (NEM sensitive factor)
• α-SNAP: soluble NSF attachment
protein
Release of SNARES
• SNAREs are
disassembled
• SNARE interactions
Differences between Anterograde and Retrograde Transport
Anterograde
Retrograde
COPII mediated
COPI mediated
ER to Golgi
Golgi to ER
Golgi to plasma membrane
Signal sequences of cargo
Ex. KKXXX for ER-resident membrane
proteins
Signal sequences of cargo
Ex. Asp-X-Glu for cargo membrane
proteins in ER
OR KDEL for ER-resident soluble
proteins (requires KDEL receptor in
Golgi membrane for cytoplasmic
sorting signal)
Retrograde transport
• COPI mediates retrograde transport
• Why is it necessary?
ER signal sequence
• KDEL sequence targets proteins to the ER
at the C-terminus
Retrograde transport
• Step 1: Identify ER
protein in Golgi
• Step 2: KDEL
sequence binds to
KDEL receptor
Retrograde transport
• GTP binding protein is ARF
– GTPase superfamily
member
Retrograde transport
• Steps 3 and 4
– Similar to COPII
mediated transport of
vesicles
Transport within the Golgi
• Cisternal maturation
• Golgi has three or four
subcompartments
Transport within the Golgi
• Little to no COPII mediated transport
• COPI mediated retrograde transport
Transport out of the Golgi
• Trans-Golgi Network (TGN) sends
proteins to three different locations
– Exocytosis
– Secretory Vesicles
– Lysosome
Transport out of the Golgi (TGN)
• Buds have a two layer coat
• Outer layer - Clathrin
• Inner layer of adapter protein (AP)
compexes
– Clathrin--triskelion
conformation
– AP target signal
• Y-X-X-Φ
Endocytosis
• Receptor mediated
endocytosis
Endocytosis
• Turnover of receptors is
very high
Studying the Secretory Pathway
Cystic Fibrosis-CFTR
Summary Movie