MVU10 - INTRACELLULAR TRAFFICKING 1

Question 1

how does the Er sustain protein qualitry control?

Answer 1

ER chaperones
Timer: ER N-linked glycosylation
misfolded protein degradation -> exported out of the ER
unfolded protein response

Question 2

in which direction do COPII vesicles transport?

Answer 2

transport from ER to Golgi (anterograde)

Question 3

in what direction do COPI vesicles transport?

Answer 3

from golgi back to ER (retrograde)

Question 4

in which direction do clathrin coated vesicles (CCV) transport?

Answer 4

from golgi and PM to endosomes

Question 5

what are the stages of vesicle formation?

Answer 5

1. initiation
2. 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
3. fission: bus is pinched off to separate vesicle from membrane
4. uncoating
   coat is removed to allow vesicle targeting and fusion

Question 6

what are the different layers of recognition?

Answer 6

receptor recognises cargo
adaptor recognises receptor
coat recognises adaptor

Question 7

what are Ras GTPases?

Answer 7

monomeric GTPase switches
GTP bound state provides binding site for various effectors

Question 8

what GTPases are involved in COP vesicle initiation?

Answer 8

Sar1 (COPII) and Arf (COPI)

Question 9

what is Rab responsible for?

Answer 9

vesicle targeting

Question 10

what is the GTPase cycle?

Answer 10

GAPs stimulate GTP hydrolysis (render the molecule inactive)
GEFs cause release of GDP and binding of GTP (make it active)

Question 11

explain the COPII vesicle initiation

Answer 11

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

Question 12

what are the two forms of Sar1

Answer 12

Sar1-GDP
inactive
soluble
amphipathic helix hidden inside the protein
Sar1-GTP
active
membrane bound
helix inside the membrane

Question 13

explain COPII coat formation

Answer 13

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

Question 14

explain COPII uncoating

Answer 14

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

Question 15

what is bulk flow, and the issue that arises with it?

Answer 15

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

Question 16

what are ER exit signals?

Answer 16

TM proteins have them on cytosolic side
di-phenylalanine at C terminus (FF)
ASP-X-GLU (DxE) within a sequence

Question 17

what recognises ER exit signals?

Answer 17

sec23/24 adaptors

Question 18

what are lumenal proteins recognised by?

Answer 18

various cargo receptors, which are TM proetins with exit signal FF facing cytosol

Question 19

what are signals on lumenal proteins that return them to the ER? and what recognises that?

Answer 19

KDEL-COO- at C terminus
recognised by a TM KDEL receptor which itself has a KKxx motif at the cytosolic C terminus

Question 20

what are signals on TM proteins that return them to the ER? and what recognises that?

Answer 20

KKxx-COO- at cytosolic N terminus
NH3+-MxxRR at cytosolic N terminus
motifs are recognised by COP-I coat adaptors

Question 21

explain the formation of COp-I coated vesicles?

Answer 21

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

Question 22

how are CCV and PI initatied?

Answer 22

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

Question 23

what are the different phosphorylation patterns on PI and their effect?

Answer 23

PI(4,2)P2: PM clathrin adaptors, dynamin
PI(4)P: golgi clathrin adaptors
other phosphorylation states are used for other things

Question 24

what do CCV adaptors do?

Answer 24

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

Question 25

how is the clathrin cage formed?

Answer 25

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

Question 26

what are the differences between the clathrin and the COPI/COPII coats?

Answer 26

clathrin coats cannot pinch off vesicle by itself, needs dynamin
clathrin is larger than COPI/COPII
clathrin doesnt need RasGTP

Question 27

how does CCV fission work?

Answer 27

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

Question 28

how does clathrin uncoating work?

Answer 28

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

Question 29

what are the two mechanisms that ensure that vesicles transport their contents to the correct acceptor membranes?

Answer 29

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