32 – General Mechanisms of Vesicle trafficking Flashcards
Well characterized 3 types of vesicles (Transport carriers)
COPII: transport Er to Golgi
COPI: protein retrival from Golgi
Clathrin:
-Budding from Golgi or PM
not so well charactirized:
Secretive vesicles = transport TGN to PM to extracellular space
Vesicle trafficking is…
complicated in morphology
How to know which is COPI or COPII?
Molecular regulation of vesicle formation
Design what do to for vesicle trafficking
-Coat vesicles differently to make them differently
-Remove the coat so vesicles can fuse with target
–But keep a tag so target recognize vesicle
–To achieve accurate fusion in crowded environment in cytosol
Cells use different sets of proteins to
to coat vesicles to specify their identity
Biochemically different for outer/inner layer
2 roles of coat proteins in vesicle formation
o Vesicle formation
o Cargo selection
Vesicle formation:
coat proteins polymerize on donor membrane to make (+) curvature of membrane = transport vesicle in ~80nm in diameter can assemble from membrane
COPII: Sec23-Sec24 + Sec12-Sec31 polymerize to curve ER membrane
Cargo selection:
coat protein interact with membrane cargoes/membrane cargo receptors + soluble cargoes = incorporate them into shaped vesicles
COPII: Sec24 interacts with Di-acidic sorting signal in membrane cargo
-Active cargo selection
what require Small ARF GTPases?
Initiation of vesicle formation require action of small ARF GTPase
which ARF-GTP is needed for each 3 types of vesicles?
COPII—Sar1: ARF GTP-binding protein initiates its formation from ER
COPI—ARF1: initiated formation from Golgi
Clathrin—ARF6: vesicle formation from TGN/PM
Small ART GTPase cycles between
GDP-bound & GTP-bound forms
Inactive cytosolic ARF-GDP recruited & activated by membrane associated GEF
-GEF-turn on
Active membrane bound-ARF-GTP induces vesicle formation
Once vesicle formed, active membrane associated ARF-GTP switch off by GAP
-GAP-turn off
Sar1
- trigger COPII vesicle formation & subsequent uncoating
-Hydrophobic N-terminus normally buried in ER membrane - anchored to where you want to make COPII - Sarq recruit Coat protein: Sec23/Sec24
- once Vesicle formed & bud off from ER
- Sar1 GTP become Sar1 GDP dissociate & coat protein shed off = naked vesicle
If can’t switch off Sar1
too much uncoated vesicles = cant fuse with their target=fail to transport protein from ER to Golgi
if cant turn on Sar 1:
= can’t turn on = can’t make COPII = can’t transport protein out of ER
COPII vesicle formation by COPII coat proteins + Sar1 reconstituted in vitro
Mix phospholipids tgt= mix liposomes with COPII coat proteins+Sar1
-Highly curved buds with coat proteins visible
-if with wrong ARF GTPase = no curve
what is Small Rad GTPases used for.
the tag
Cells use small Rab GTPases to maintain identity of uncoated vesicles
2 major types of Rab effectors
Microtubule motor proteins (kinesins)
Tethering factors on target membrane
how does cells use Small Rad GTPases?
- Small Rab GTPases exists as inactive Rab-GDP in cytosol
-Like small ARF GTPases - When required, inactive, cytosolic Rab-GDP targeted & attached to vesicle via lipid anchor
- Specific GEF on vesicle converts Rab-GDP to active Rad-GTP
-Vesicle is tagged - Active Rab-GTP recognized & interacts with 2 major types of Rab effectors
-Microtubule motor proteins (kinesins)
–To propel vesicle toward target membrane
-Tethering factors on target membrane
–To tether & dock the vesicle onto target membrane
—Tether: brought close to vesicles & target membrane
in human cells
> 60 Rab proteins found in human cells
-Many tag an individual type of vesicles/compartment
COPI – can come from cis OR trans Golgi
COPII – Rab1 from ER to tag COPII
Secretory vesicles - All Rab8 homologs
Need actions of different Rabs to
achieve specificity of vesicle fusion
If not=cant tether & vesicle are misrecognized
Cognate pairs of SNARE proteins mediate
fusion
v-SNARE in vesicle
t-SNARE in target membrane
=Always 3 SNARE molecules
v-SNARE & t-SNARE interact to
form a trans-SNARE complex (vesicle + target) to bring 2 membranes close enough together to fuse with each other
Action of cognate v-t-SNARE pair in vesicle fusion
- Vesicle movement & tethering & docking
-Rab effector
-v-t-SNARE - trans-SNARE complex assembly & wrap tightly
3 from target membranes + 1 from vesicle - Membrane hemi-fusion – outer layer fuse
- Full membrane fusion – vesicle fused to target membrane
- Cis-snare complex unfolded by NSF
- Disassembly of SNARE complexes
2 major types of secretion:
- In regulated secretion
- In constitutive secretion:
regulated secretion
insulin secretion/neurotransmitter secretion
Vesicle fusion only starts when cells are signaled to do so
-Trans-SNARE complex formed by doesn’t undergo tight wrap around before signal is coming
