MVU10 - INTRACELLULAR TRAFFICKING 1 Flashcards
how does the Er sustain protein qualitry control?
ER chaperones
Timer: ER N-linked glycosylation
misfolded protein degradation -> exported out of the ER
unfolded protein response
in which direction do COPII vesicles transport?
transport from ER to Golgi (anterograde)
in what direction do COPI vesicles transport?
from golgi back to ER (retrograde)
in which direction do clathrin coated vesicles (CCV) transport?
from golgi and PM to endosomes
what are the stages of vesicle formation?
- initiation
- coat formation
cytosolic adaptor proteins interact with initiator
adaptors collect cargo or cargo receptors
protein framework is formed on top of adaptors to shape the coat - fission: bus is pinched off to separate vesicle from membrane
- uncoating
coat is removed to allow vesicle targeting and fusion
what are the different layers of recognition?
receptor recognises cargo
adaptor recognises receptor
coat recognises adaptor
what are Ras GTPases?
monomeric GTPase switches
GTP bound state provides binding site for various effectors
what GTPases are involved in COP vesicle initiation?
Sar1 (COPII) and Arf (COPI)
what is Rab responsible for?
vesicle targeting
what is the GTPase cycle?
GAPs stimulate GTP hydrolysis (render the molecule inactive)
GEFs cause release of GDP and binding of GTP (make it active)
explain the COPII vesicle initiation
COPII vesicles form at specific ER exit sites
proteins with exit signals are collected (cargo receptors)
misfolded proteins are kept away (calnexin)
transmembrane GEF at exit site induces GTP binding by Sar1
Sar1-GTP exposes amphipathic helix and partially inserts into membrane to initiate vesicle formation
what are the two forms of Sar1
Sar1-GDP
inactive
soluble
amphipathic helix hidden inside the protein
Sar1-GTP
active
membrane bound
helix inside the membrane
explain COPII coat formation
adaptor proteins (Sec23 and Sec24) bind activated Sar1 and TM cargo proteins, or cargo receptors for lumenal proteins
Coat proteins (Sec13 and Sec31) bind adaptors and shape the membrane into vesicle
completed coat pinches the vesicle off from membrane
energy for shaping and pinching off the vesicle is only from protein interactions, not from GTP hydrolysis
explain COPII uncoating
coat (sec13/sec31) forms cage like structure around vesicle
adaptor sec23/24 acts as GAP that allows Sar1 to hydrolyse GTP
acts as timer
Sar1-GDP releases from vesicle membrane
adaptors separate from Sar1 and coat separates from adaptors
uncoating is necessary for vesicle function and golgi
what is bulk flow, and the issue that arises with it?
proteins in the ER are transported to the GA and PM by default, even without an exit signal
proteins that are supposed to be at the ER are taken out by accident
what are ER exit signals?
TM proteins have them on cytosolic side
di-phenylalanine at C terminus (FF)
ASP-X-GLU (DxE) within a sequence
what recognises ER exit signals?
sec23/24 adaptors
what are lumenal proteins recognised by?
various cargo receptors, which are TM proetins with exit signal FF facing cytosol
what are signals on lumenal proteins that return them to the ER? and what recognises that?
KDEL-COO- at C terminus
recognised by a TM KDEL receptor which itself has a KKxx motif at the cytosolic C terminus
what are signals on TM proteins that return them to the ER? and what recognises that?
KKxx-COO- at cytosolic N terminus
NH3+-MxxRR at cytosolic N terminus
motifs are recognised by COP-I coat adaptors
explain the formation of COp-I coated vesicles?
Arf1-GTP initiator inserts amphipathic helix into membrane
adaptors and coats are unrelated to COPII but function similarly
adaptors (beta/delta, gamma/zeta subunits collect cargo)
COPI coat (alpha/beta subunits) assemble on adaptors, shape and pinch off vesicle from membrane
adaptors are GAPs for Arf1, to dissociate coat
how are CCV and PI initatied?
PI phosphates in PM and golgi initiate vesicle formation
PI on cytosolic face of membrane can be phosphorylated at different hydroxyl positions by PI kinases
what are the different phosphorylation patterns on PI and their effect?
PI(4,2)P2: PM clathrin adaptors, dynamin
PI(4)P: golgi clathrin adaptors
other phosphorylation states are used for other things
what do CCV adaptors do?
adaptor proteins (AP-1, AP-2 and others) bind to PI phosphates and cargo in membrane
many different signals for selection of cargo
Arf GTPase assists some adaptors but does not initiate CCVs
clathrin coat binds adaptors
how is the clathrin cage formed?
clathrin triskeletons (oligomers, 3 heavy and 3 light chains)
assemble on adaptors to shape the membrane and form coated pits
clathrin forms cage around vesicle
what are the differences between the clathrin and the COPI/COPII coats?
clathrin coats cannot pinch off vesicle by itself, needs dynamin
clathrin is larger than COPI/COPII
clathrin doesnt need RasGTP
how does CCV fission work?
dynamin GTPase piches off CCVs (not a member of Ras family)
dynamin monomers assemble in GTP bound state into oligomeric rings at base of bud
GTP hydrolysis causes the ring to become smalle rand pinch off the vesicle
dynamin rings disassemble in a GDP bound state
how does clathrin uncoating work?
Pi phosphatases modify PI(4,5)P2 to weaken adaptor binding
auxilin: DNAJ with clathrin binding domain and J domain
binds to the clathrin cage, activates HSC70
HSC70 binds clathrin and makes conformational change, disassembles coat into triskelions
clathrin is released from HSC70 and recycled to membrane
what are the two mechanisms that ensure that vesicles transport their contents to the correct acceptor membranes?
Rab GTPase proteins provide specificity of vesicle targeting and attachment to acceptor membrane
SNARE fusion proteins provide specificity during fusion of vesicles with acceptor membrane
