lecture 16 vesicle transport and targeting in the secretory pathway cop coated vesicles snares...
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Lecture 16
Vesicle transport and targeting in the secretory pathwayCOP coated vesiclesSNAREs
Protein sorting Secretion - Golgi to plasma membraneRetention in ERGolgi to lysosome
Transport between organelles is mediated by coated vesicles
Clathrin coated vesicles mainly involved in endocytosis (next time)
COP coated vesicles mediate ER to Golgi and back
Transport between ER and Golgi compartments occurs via “COP-coated
vesicles”…
Collection of 4-7 “coat proteins” = “COPs”…(aka “Coatomers” )COP-coated vesicles function in transport between:
ER and GolgiGolgi and ER (retrieval)
intra-GolgiTGN and plasma membrane
COP proteins
“cargo”
More COP proteins
Lipid bilayer
Sar1
COPII-coated vesicles - ER to Golgi-SarI in ER membrane
COPI coated vesicles - Golgi to ERARF (instead of Sar1) in Golgi
membraneWe will only consider Sar1
Cop coated vesicles contain many proteins
Sar1:GTPase switch
on/off
ON: binds membranerecruits COP proteins
COP proteins then recruit specific cargo
Sar1 --Similar to RAN in nuclear import
Sar1 ARF triggers vesicle formation
GAP
Large family (Ras) of proteinsMolecular “switches”
Sar1 GTPase
GTPSar1
GTPase
GDP
Pi
GDP
GTP
“on” “off”GEF
In cytoplasm, large amount in “off” form
cytoplasmic
Bound tomembrane
GTPase (GTP Binding Proteins)
Sar1 activation exposes hydrophobic tail and membrane insertion
Sar 1 in membrane recruits COP proteins
Greasy foot
The Ras “superfamily” of small GTPases…• Ras: signaling and regulating cell proliferation…
>30% of human tumors have Ras mutations…
Many (not all) Ras family members associated with membranes via covalent fatty acid tail (“greasy feet”)…
• EF-1/EF-Tu: translation…
• Ran: nuclear transport…
• Rho family (Rho, Rac, cdc42): actin assembly and organization (in a few lectures)
• Arf/Sar family of “Coat recruitment GTPases:” COP assembly and vesicle budding…
• Rab family: vesicle targeting and fusion (in a few minutes)
Aside: G-proteins and ATPases as molecular switches
Translation:
IFs (GTP), EF-1/EF-Tu (GTP)
EF-2/EF-G (GTP)
Chaperones:
HSP70 family (ATP)
HSP60 (ATP)
SRP family:
SRP54 (GTP), SRP-R(GTP)
SRP-R(GTP)
Polymer dynamics:
Actin (ATP), Tubulin (GTP)
Dynamin (GTP)
Motors:
Myosin (ATP), Dynein (ATP)
Kinesin (ATP)
Signaling:
Heterotrimeric G proteins (GTP)
Ras family (GTP)
Cells make high-affinity transient molecular complexes as trigger or switch
GTP GDP + Pi
A paradox:
High-affinity/high-specificity = stable…
Energy input is required to dissociate high-affinity complexes…
(Example: to remove Sar 1 from membrane)
Bound UnboundBA BA +
Summary of COPII-coated vesicle formation
COP subunits recruit specific cargo proteins…
Vesicle transport is a complex process
3. Targeting and docking to specific compartment…
(ATP, GTP, and cytoplasmic protein factors…)1. Formation of
coated buds…
2. Formation of coated transport vesicle…
Target compartment
Coat proteins (“COPs”)
Donor compartment
SNAREs and Rabs
The Snare hypothesis: v- and t-SNAREs target transport vesicles to the correct membrane
v-SNAREs
Budding Uncoating, targeting and docking
t-SNAREs
Specific pairing of V-SNAREs with T-SNAREs matches vesicle to target membrane compartment (>20 known snares in animals cells)
See ECB figure 15-20
Targeting and docking requires/is facilitated by specific Rab GTPase in vesicle and Rab effector in target (~30 known Rabs in animal cells)…
Cargo
Bacterial toxins target the vesicle docking and fusion machinery of
neurons
Botulism A
Botulism B
Botulism C
Tetanus
SNAP25 (t-SNARE)
VAMP (v-SNARE)
Syntaxin (t-SNARE)
VAMP (v-SNARE)
A small subunit of the toxin acts as a specific protease that cleaves and inactivates targeting proteins
Net result is to block neuronal signaling by blocking neurotransmitter release (regulated secretory pathway)
Vesicle transport is a multi-step process
3. Targeting and docking to specific compartment…
2. Formation of coated transport vesicle…
Donor compartment
1. Formation of coated buds…
(ATP, GTP, and cytoplasmic protein factors…)
Target compartment
4. Uncoating…
GTP
GDP + Pi
Coat proteins (“COPs”)
SNAREs and Rabs
GTPS and other non-hydrolyzable GTP analogs block uncoating, resulting in accumulation of docked, coated vesicles
GTP hydrolysis by Sar1 is required for uncoating
GTPS
Sar 1
Vesicle transport is a multi-step process
3. Targeting and docking to specific compartment…
2. Formation of coated transport vesicle…
Sar1
Donor compartment
1. Formation of coated buds…
(ATP, GTP, and cytoplasmic protein factors…)
GEF and Sar1
Target compartment
4. Uncoating…
GTP
GDP + Pi
Coat proteins (“COPs”)
SNAREs and Rabs
GEF in donor membrane promotes nucleotide exchange, activating Sar1 @ ER, (ARF @ Golgi) and promoting coat assembly…
GTP hydrolysis serves as “timer” delaying uncoating (GAP in target membrane?)…
GTPase “cycle” provides directionality to vesicle coating/uncoating
Vesicle transport is a multi-step process
3. Targeting and docking to specific compartment…
2. Formation of coated transport vesicle…
Coat recruitment GTPase
Donor compartment
1. Formation of coated buds…
(ATP, GTP, and cytoplasmic protein factors…)
GNRP/GEF and Coat recruitment GTPase
Target compartment
4. Uncoating…
GTP
GDP + Pi
Coat proteins (“COPs” or
“coatomer”)
SNAREs and Rabs
5. Fusion…SNARE plus other fusion
proteins
SNAREs are necessary for membrane fusion
SNAREs bring two membranes into close apposition
Lipids flow between membranes - fusion
Other proteins cooperate with SNAREs to facilitate fusion and to pry SNAREs apart
ECB 15-21
Much still to learn!!!
Lecture 16
Vesicle transport and targeting in the secretory pathwayCOP coated vesiclesSNAREs
Protein sorting/targetingSecretion - Golgi to plasma membraneRetention in ERGolgi to lysosome
How are proteins sorted to appropriate vesicles so that they are transported to proper location? What are the address label?
Two secretory pathways; constitutive and regulated
Default pathway for ER/Golgi proteins
If no address label, then secrete
Signal required to trigger secretory granule fusionExample - neurotransmitter release
However, recent data suggests there may be ER exit sequences..For now, consider secretion default
ECB 15-28
Inside lumen is equivalent to outside of cell
15.9-secretory_pathway.mov
Regulated secretion
Secretory granules containing insulin in pancreatic cells Signal for release is elevated glucose levels in blood
If secretion is default, how are resident ER proteins retained?
C, M, T Golgi
ER
Plasma membrane
OutsideCGN TGN
Constituitive secretion
Secretory granuleRegulated secretion
Ex: BiP is a member of the HSP70 family that functions in the ER…They
aren’t!
BiP escapes from ER and must be “retrieved” from the Golgi…
C-terminal KDEL in BiP sequence functions as retrieval signal…
BiP KDEL
KKXX
KDEL-R
KDEL-receptors in Golgi direct retrieval/recycling…
KKXX at C-terminus of KDEL-R binds COPI coat and targets back to ER…
Summary so far of protein targeting, revisited…
Cytoplasm
Secretion/membrane proteins
Secretory vesicles
RetrievalTransport
(constituitive secretion)
(regulated secretion)
Pro
tein
ta
rgeti
ng
Vesi
cle t
arg
eti
ng
RER
Golgi
Plasma membrane
See ECB figure 14-5
Default
Signal sequence (hydrophobic -helix)
KDEL (soluble proteins)
KKXX (membrane proteins)
Lysosomes
?Default
How are proteins targeted to the lysosome?
Lecture 16
Vesicle transport and targeting in the secretory pathwayCOP coated vesiclesSNAREs
Protein sorting Secretion - Golgi to plasma membraneRetention in ERGolgi to lysosome
How are proteins sorted to vesicles leaving TGN for lysosome?
Lysosomes degrade and recycle macromolecules…
Lysosomes in plant and animal cells contain acid hydrolases (hydrolytic enzymes) for degrading/recycling macromolecules
pH of lumen is about 5 - acidic!
How are hydrolases and other proteins targeted to lysosomes?
ECB 15-34
I-cell disease helped decipher the signal for targeting proteins to the lysosome
• Recessive mutation in single gene…
• Fibroblasts of patients contain large inclusions (I-cells)…
• Lysosomes lack normal complement of acid hydrolases…
• All lysosomal enzymes secreted (secretion is the “default” fate for proteins in the ER-Golgi pathway)…
• Lysosomal enzymes of “wild-type” (normal) cells are modified by phosphorylation of mannose on oligosaccharide (forming mannose-6-phosphate)…
• Lysosomal proteins of I-cells lack M-6-P…
• Lysosomal targeting signal resides in carbohydrate!
Mannose-6-P targets proteins from Golgi to lysosomeCis Golgi
Network (CGN)Trans Golgi
Network (TGN)
RER
M6P receptor recycling back to Golgi
Transport via clathrin-coated vesicles to…
Lysosome
M6P receptor in TGN directs transport of enzymes to lysosome via clathrin-coated vesicles
Addition of M6P to lysosomal enzymes in cis-Golgi
Patients with I-cell disease lack phosphotransferase needed for addition of M-6-P to lysosomal proteins in fibroblasts… secreted…
Lysosomal hydrolase (precursor
)
Addition of M6P
Removal of phosphate &proteolytic processing…
Maturehydrolase
M6P receptor
Clathrin coat
Uncoupling(pH 5)
Protein targeting, revisited
Next lecture: endocytosis and clathrin coats
Cytoplasm
Secretion/membrane proteins
Secretory vesicles
Lysosomes
RetrievalTransport
(constituitive secretion)
(regulated secretion)
Pro
tein
ta
rgeti
ng
Vesi
cle t
arg
eti
ng
RER
Golgi
Plasma membrane
See ECB figure 14-5
Signal sequence (hydrophobic -helix)
KDEL (soluble proteins)
KKXX (membrane proteins)
M6P
Default or
signal?
Default or
signal?