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HELENP CBG: Cell Architecture, Communication and Regulation > CBG Lecture 36: Vesicular Traffic > Flashcards

CBG Lecture 36: Vesicular Traffic Flashcards

1
Q

what are the main experiments that led to the concept of trafficking

A

pulse chain:radioactivity
GFP - encoded by cDNA then attach GFP tag to viral glycoprotein VSVG - use glycoprotein as this will undergo postranslational modification - in Golgi

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2
Q

why do proteins move

A

prevent depletion of organelle-resident proteins

prevent depletion of lipids

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3
Q

what is KDEL sequence

A

Golgi to ER signalling sequence

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4
Q

where must proteins destined for the signalling pathway go

A

to the RER

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5
Q

via what do proteins enter the RER

A

via SRP signal peptide recognition partice

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6
Q

where does synthesis and modification of proteins occur

A

RER

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7
Q

where does synthesis of lipids occur

A

SER

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8
Q

where does calcium storage occur

A

sarcoplasmic reticulum in muscle cells

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9
Q

how does vesicular transport betwen compartments occur

A

budding and fusion

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10
Q

what are the different types of protein vesicle coats

A

clathrin
COPI
COPII

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11
Q

what do protein coats do

A

organise vesicular traffic and give shape to vesicles and concentrate membrane proteins in a specialised patch

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12
Q

what is a vesicle coat

A

a small membrane bounded organelle with a cage of proteins (coat) on its cytosolic surface.
formed by piclking off of a coated region of membrane - coated pit

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13
Q

where do clathrin coats mediate transport to

A

golgi

PM

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14
Q

where do COPI coats mediate transport to

A

ER and Golgi cisternae

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15
Q

what are the major components of clathrin coated vesicles

A

clathrins and adaptins

each clathrin unit has 3 large heavy and 3 light pp chains that form a triskelion

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16
Q

where is triskelion found

what is it made up of

A

triskelion found in clathrin coated vesicles
made up of 3 large heavy and 3 light pp chains
alpha zigzag and beta propellor- 36 triskelions make up a hexagonal clathrin cage

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17
Q

what can isolated triskelions do

A

self assemble sponatenously

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18
Q

what are adaptin proteins

A

requierd to bind the clathrin coat to membrane and to trap transmembrane receptors that capture soluble cargo molecules

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19
Q

outline the structure of clathrin

A

alpha zigzag
beta propeller makes the triskelion
36 triskelions make up a hexagonal clathrin cage

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20
Q

how many triskelions make up a hexagonal clathrin cage

A

36

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21
Q

discuss assembly and disassembly of clathrin coat

A

assembly of coat thought to induce curvature into membrane, which leads to formation uniformly sized coated buds

  1. adaptins bind both clathrin triskelions adn membrane bound cargo receptors, thereby mediating the selective recruitment of both membrane and cargo molecules into the vesicle
  2. ARF GTPase initiastes assembly
  3. dynamin pinches off vesicle
  4. ARF switching and Hsc70 ATP hydrolysis triggers uncoating of vlathrin
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22
Q

which enzymes initiate assembly of clathrin coated vesicles

A

ARF GTPase

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23
Q

which enzymes trigger uncoating of clathrin coated vesicles

A

ARF switching

and Hsc70 ATP hydrolysis

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24
Q

what are the four adaptor proteins present in clathrin coated vesicles

A

AP1 AP3 AP2 GGA

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25
Q

which adaptor proteins are involved in movement from TGN trans golgi network to endosome

A

AP1 or GGA

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26
Q

which adaptor proteins are involved in movement from TGN to PM

A

AP2

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27
Q

which adaptor proteins are involved in movement from TGN to lysosomes

28
Q

which adaptor proteins are involved in movement from PM to endosomes

29
Q

what movement IS MEDIATED By AP1 or GGA

A

TGN to endosome

30
Q

what movement is mediated by AP2

31
Q

what movement is mediated by AP1

A

TGN to lysosome

32
Q

what movement is mediated by AP2

A

PM to endosome

33
Q

discuss retrograde trafficking via COPI

A

from Golgi back to ER
coat formed from 7pps
1.ARF-GDP complex binds receptor on Golgi membrane
2.ARF activation by GEF, which phosphorylates GDP to GTP so complex gives ARF-GTP
3.ARF mediated recruiment of preassembled COP1 coat
4.Cargo sorting motif recognition by COPI

34
Q

what are ARF proteins

A

coat recruitment proteins-used in COPI and clathrin coatin assembly @ Golgi membranes
Sar1 is used instead for COP2

35
Q

what are common themes in how vesicles bud

A
  1. GTPase initiates assembly
  2. GTP hydrolysis triggers disassembly
  3. Selection of cargo by accessory proteins
  4. Coat that accommodates diverse cargo
36
Q

how many pps make up a COP1 coat (coatamer)

37
Q

what is Arf1 similar to from COP2 system

A

Arf1 from COP1 is similar to Sar1 from COP2 sytem

38
Q

in COP1 retrograde trafficking, what is GEF similar to

A

GEF in COPI is similar to Sec12 from COP2

39
Q

hwere are coat recruitment GTPases usually found

A

in high concns in the cytosol, in an inactive GDP bound state

40
Q

discuss budding of vesicles from the ER via COPII

A

1, inactive soluble Sar1-GDP binds GEF(Sec12) in Er membrane, so Sar1 releases GDP and binds GTP

  1. GTP triggered conformatoinal change in Sar1 exposes its hydrophobic tail, which inserts into the ER membrane
  2. membrane bound, active Sar1-GTP recruits COP2 subunits to the membrane
  3. causes membrane to bud - then pinches off into vesicle
41
Q

what are the 3 components of COP2 trafficking

A

inner coat - Sec23/24
Outer Coat - Sec13/31
GTP-binding protein Sar1

42
Q

what are the roles of COP2 trafficking

A

cargo sorting
shapes membrane
associates with GTPase switch so that assembly is reversilble

43
Q

when is vesicle formation of COP2 triggered

A

when Sec12(GEF) exchanges Sar-1GDP for GTP

44
Q

main steps for COP2

A

budding -> GTP hydrolysis by GEF - > uncoating

vesicle formation is triggered when Sec12 (GEF) exchanges Sar-1 GDP for GTP

45
Q

what does Sar1(GTP) binding trigger

A

triggers Sec23/24 assembly

46
Q

what is Sec24 responsible for

A

cargo recognition (DXE)

47
Q

what does Sec23 do

48
Q

how many Sec13/31 heterodimers form an cuboctohedron

A

24 - flexible enough to transport large cargos, rigid enough to impact curvature on the membrane

49
Q

why does vesicle fusion need to be highly selective

A

specific cos very diverse membranes

50
Q

what is recognition of vesicles controlled by

A

SNAREs

Rabs

51
Q

what are SNAREs

A 
soluble NSF (N-ethyl-maleimide-sensitive fusion protein) Attachment Protein Receptor
centrol role in providing specificity and catalysing fusion
52
Q

what are Rabs

A

proteins that work with other proteins to regulate initial docking and tethering of vesicle to target membrane

53
Q

what proteins help docking to Target membrane

54
Q

what proteins ensure specificity and catalysis of vesicles to membranes

55
Q

how many different SNAREs in mammalian cells - why?

A

30

diversity confers specificity

56
Q

what two categories of snare are there

A

v-SNARE: 3 helix bundle in transport vesicles made of synaptobrevin - comes from Vesicular
t-SNARE: comes from Target membrane, synTaxin

57
Q

give an example of t-SNARE

A

Snap25 - 2 alpha helixes

58
Q

what is synaptobrevin aka

59
Q

outline trans snare complexes

A

laways consist of 4 tightly intertwined multiple chaines

60
Q

what haappens when v-SNARE interacts with t-SNARE

A

the helical domains of one wrap around the helical domains of another to form TRANS SNARE complexes - lock the 2 membranes together by exerting an inward force

61
Q

what are SM proteins

A

they direct fusion: Sec1/Munc18 - like proteins

shaped like clasps

62
Q

what are SM proteins bound with trans-SNARE called

A

SNAREpins complexes

63
Q

give an example of a SM protein

A

Rab GTPase

64
Q

outline vesicle fusion process

A

a tight SNARE pairing forces lipid bilayers into close apposition so that water molecules are expelled from the interface
lipids of the two interacting leaflets of the bilayers then flow between the membranes to form a connecting stalk
lipids of the other two leaflets then contact eachother forming a new bilayer which wides the fusion zone causing a hemifusion
rupture of the new bilayer completes the fusion reaction

65
Q

what is a core complex made of

A

snap25&syntaxin (t-SNARE) and synaptobrevin (v-SNARE)

66
Q

what are the requirements for vesicle fusion

A

SNAREs alone can fuse vesicles just not v fast

not physiological condns cant mix and match

67
Q

how can SNAREs be recycled

A

requires energy - ATP hydrolysis

NSF and alpha SNAP proteins essential

HELENP CBG: Cell Architecture, Communication and Regulation (5 decks)

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