Lecture 12

Question 1

describe secretory pathway

Answer 1

proteins sythesized in er –> golgi apparatus, may ptms —> vesicles to pm or endosome
proteins from pm can also bring cargo to inside of cell

Question 2

name 3 types of coated vesicle transport

Answer 2

cop2
cop1
clathrin coated vesicles - ccv

Question 3

describe COPII

Answer 3

vesicles transport from er to golgi
anterograde = normal direction, unidirectional
normal direction

Question 4

describe COPI

Answer 4

Vesicles transport from golgi back to ER (retrograde)
probably since cop2 made a mistake
related mechanism but diff proteins involved

Question 5

describe clathrin coated vesicles

Answer 5

transport from golgi and PM to endosomes

Question 6

what are steps common to all vesicle formation

Answer 6

initiation
coat formation
Fission
uncoating
clathrin = slightly diff but still same 4 steps
cargo must be recognized by receptor, then receptor recognized by adaptor and adaptor recognized by coat

Question 7

describe initiation

Answer 7

some event on the membrane starts the process of forming a vesicle

Question 8

describe coat formation

Answer 8

cytosolic adaptor proteins interact with initiator (recognizes initiation event in membrane)
adaptors collect transmembrane cargo, or cargo receptors (also recognize cargo and initiation events) (tm proteins - cytosolic adaptor, if soluble = need receptor to be recognized by adaptor)
coat: protein framework is formed on top of adaptors to shape the vesicle bud from the membrane (in cop1 and cop2, coat helps fission)

Question 9

describe fission

Answer 9

– bud is pinched off to separate the vesicle from membrane

Question 10

describe uncoating

Answer 10

coat is removed to allow vesicle targeting and fusion
not needed for travel so needs to be uncoated

Question 11

describe Ras GTPase family

Answer 11

initiator of cop1 and cop2
effectors, can do many things
monomeric GTPase “switches” = GTP-bound state provides binding site for various effectors (active)
when gdp bound = inactive
Sar1 and Arf: COP vesicle initiation

Question 12

what is GAP

Answer 12

GTPase Activating Proteins (GAP) – stimulate GTP hydrolysis

Question 13

what is GEF

Answer 13

Guanine Exchange Factors (GEF) – cause release of GDP and binding of GTP

Question 14

describe COPII vesicle initation

Answer 14

COP-II vesicles form at specific ER exit sites (goes to golgi)
– 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 (sar1 = soluble protein in cytosol, helix hidden in interior, but then amphipathic helix faces cytosol and allows sar1 to be inserted into membrane)

Question 15

describe COPII coat formation - adaptor

Answer 15

Adaptor proteins (Sec23 and Sec24) bind activated Sar1 and TM cargo proteins, or cargo receptors for lumenal proteins
sec23 and sec24 recognizes adaptor - bound to cargo *specificity

Question 16

why does it have to uncoat

Answer 16

membrane by itself cannot form vesicle
need to uncoat after = adaptors stimulate gtpase activity of sar 1 = make soluble molecule then disassemble = detach imitators and adaptors
sec 23 and sec 24 act as gap for sar 1

Question 17

describe cop2 uncoating

Answer 17

Coat (Sec 13/31) forms cage-like structure around vesicle
Adaptor (Sec23/24) acts as GAP that allows Sar1 to hydrolyze GTP

Question 18

how does er exit work

Answer 18

bulk flow and er exit signals

Question 19

describe bulkflow - er exit

Answer 19

proteins in the ER are transported to the Golgi and PM by default, even with no exit signals
– but many proteins are exported much more efficiently (bc certain motifs), while some others return to the ER

Question 20

describe er exit signals - er exit

Answer 20

TM proteins have exit signals on cytosolic side
– di-phenylalanine (FF) at C-terminus (tm type 1 protein)
– Asp-X-Glu (DxE) within a sequence
– recognized by Sec23/24 adaptor
*efficiently transported to golgi
Lumenal proteins are recognized by various cargo receptors, which are TM
proteins with exit signals (FF) = 2 phenylalanines facing cytosol near c terminus

Question 21

describe the forward and retrival pathways

Answer 21

forward pathway = efficiently transported into golgi
retrieval pathway = must have motif so golgi knows it has to be transported back to ER

Question 22

what is er retrieval

Answer 22

mediated by cop1
ER resident proteins are transported to Golgi by bulk flow, but have signals that return them to the ER

Question 23

describe er retrieval - lumenal proteins

Answer 23

KDEL-COO- at C-terminus
recognized by a transmembrane KDEL receptor, which itself has a KKxx motif at the cytosolic C-terminus

Question 24

describe er retrival - tm proteins

Answer 24

KKxx-COO- at cytosolic C-terminus (type 1 tm)
NH3 +-MxxRR at cytosolic N-terminus (type 2 tm)
motifs are recognized by the COP-I coat adaptors
could need both - depends on tm proteins, only recognizes if type 1 or 2

Question 25

rab vs ras

Answer 25

rab = vesicle targeting
ras = signal transduction

Question 26

describe COPII coat formation - coat

Answer 26

Coat proteins (Sec13 and Sec31) bind adaptors and shape the membrane into vesicle
recognzies adaptors and starts forming coat

Question 27

describe COPII coat formation - pinching off

Answer 27

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 28

what acts as timer for cop2 uncoating

Answer 28

gtp hydrolysis still slow - acts as timer, gdp detaches whole structure of initiator adaptor and coat protein

Question 29

describe 3 steps of cop2 uncoating

Answer 29

Detach initiator
parts fall off and recycled
Vesicles ready for transport

Question 30

describe adaptor - uncoating of cop 2

Answer 30

– GTP hydrolysis still slow – acts as timer
– Sar1-GDP releases from vesicle membrane
– adaptors separate from Sar1 and coat separates from adaptors
– uncoating is necessary for vesicle fusion at Golgi

Question 31

describe what aa sequence determines

Answer 31

folding of protein
ptms
where proteins will go

Question 32

describe cop 1 coated vesicles

Answer 32

Arf1-GTP initiator inserts amphipathic helix into membrane
Adaptors and Coats are unrelated to COP-II but function similarly

Question 33

describe cop 1 coated vesicles - adaptors

Answer 33

Adaptors (β/δ, γ/ζ subunits) collect cargo
COP-I Coat (α/β’ subunits) assembles on adaptors, shape and pinch off vesicle from membrane (recognize arf1 and receptors)
– Adaptors are GAPs for Arf1, to dissociate coat
amphipathic helix makes arf1 integral part of membrane of er

Question 34

when do proteins go from golgi to er

Answer 34

only when made mistake

Question 35

describe ccv and PI

Answer 35

no ras gtpase
PI-phosphates in PM and Golgi initiate vesicle formation (phosphatidyl inositol = sugar, OH in sugar can be phosphorylated = signalling molecule)
PI on cytosolic face of membranes can be phosphorylated at different hydroxyl positions by PI kinases
PI-phosphates provide binding sites for different proteins
Other phosphorylation states are used for signal transduction

Question 36

name binding sites for ccv

Answer 36

– PI(4,5)P2: PM clathrin adaptors, dynamin
– PI(4)P: Golgi clathrin adaptors
phosphorylated only on these positions = if any other formation = signalling diff cascade, not formation of clathrin vesicles

Question 37

describe ccv adaptors

Answer 37

Adaptor proteins (AP-1, AP-2, others) bind to PI-phosphates and cargo in membrane
many different signals for selection of cargo, including mono-ubiquitination, phosphorylation
Arf GTPase assists some adaptors, but does not initiate CCVs
Clathrin coat binds adaptors

Question 38

descrive clathrin cage

Answer 38

Clathrin triskelions (oligomers, 3 heavy and 3 light chains)
Assemble on adaptors to shape the membrane, and form “coated pits”
Clathrin forms a cage around vesicle= cannot pinch off vesicle by itself – requires Dynamin, divergent from COP-I and COP-II, larger vesicle

Question 39

describe dynamin - clathrin cage

Answer 39

extra protein to separate vesicle from compartment
monomer but make oligomer when gtp bound = ring and ring around membrane
will get smaller and smaller till pinches off
needs gtp hydrolysis
closes ring and detaches vesicle

Question 40

describe ccv fission

Answer 40

Dynamin GTPase pinches 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 a coordinated constriction of ring that pinches off the vesicle – fission
Dynamin rings disassemble in GDP-bound state

Question 41

does ccv fission need ras gtpase

Answer 41

NOO but does need gtpase

Question 42

describe clathrin uncoating

Answer 42

PI-phosphatases modify PI(4,5)P2 to weaken adaptor binding (needs help of chaperones, hsf1, recognizes protein that is not misfolded but needs to be refolded)
Auxilin (cochaperone)
Binds to assembled clathrin cage, activates HSC70 (constitutive)
HSC70 binds clathrin and induces a conformational change, that disassembles coat into triskelions (makes into monomer, releases vesicle and then can travel to next compartments)
Clathrin is released from HSC70 and recycles to membrane

Question 43

describe auxilin and its role in clathrin uncoating

Answer 43

DNAJ with clathrin-binding domain and J domain
constituitive = always on
2 domains = j binds hsp70n and cargo recognition domain = clathrin

Question 44

what can j proteins bind to

Answer 44

both constitutive and inducible hsp70

Question 45

give analogy for cell

Answer 45

cell is like nyc map
midtown = too many options