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ANAT458 > Endocytosis and trafficking (lect7-8) > Flashcards

Endocytosis and trafficking (lect7-8) Flashcards

1
Q

where in the endocytosis pathway are sorting decisions made?

A

PM, sorting endosome, late endosome

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2
Q

what is pinocytosis?

A

when the cell “drinks” the ECM randomly, without signal needed

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3
Q

what type of endocytosis is rafts vs non-rafts

A

clathrin-mediated is associated with non-rafts;
caveolae is associated with rafts

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4
Q

what are the main characters of clathrin-mediated endocytosis?

A

cargo receptors and molecule, clathrin, adaptin, dynamin, PIP2, Rab5, SNAREs

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5
Q

name cargo proteins of clathrin-mediated endocytosis and where they are most found

A
  • present in coated pits = LDLR, transferrin
  • recruited to coated pits = EGFR, PDGF receptor
  • excluded from coated pits = ion channels
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6
Q

what is the clathrin coat triskelion made of?

A

heavy chain (192 kDa) + light chain (36 kDa) organized in trimers via their c-terminus

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7
Q

what does the C vs N terminus of the clathrin triskelion each do?

A

c-terminus holds the triskelion together;
n-terminus mediates protein-protein interactions

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8
Q

what is the most important adaptor protein for clathrin?

A

AP2: found in coated pits and selects proteins that concentrate in coated pits

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9
Q

what are the subunits that make AP2? (and others) and what do they each bind?

A
  • alpha and beta2: large subunits that each bind one PIP2
  • u2: binds YXXO and PIP2
  • o2
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10
Q

what AP2 subunit maintains the configuration open and how?

A

u2 subunit by being phosphorylated by AAK-1

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11
Q

apart from phosphorylation, what favors AP2 to be in open configuration?

A

binding to PIP2

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12
Q

what is the actual name for PIP2?

A

Phosphatidylinositol 4,5 bisphosphate

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13
Q

where is PiP2 found?

A

in the intracellular leaflet

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14
Q

in what case is PIP2 an important signaling molecule?

A

when it is a substrate of phospholipase C

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15
Q

what kind of protein is dynamin? function?

A

a small GTPase that hydrolyzes GTP to constrict the neck of the forming clathrin-coated vesicle

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16
Q

what is the proposed sequence of events of clathrin coat assembly?

A

1) Binding of AP2 to PIP2:
2) AAK1 phosphorylates u2 subunit: u2 set in “open” configuration
3) Binding of AP2 to an internalization sequence: stabilizes the “open” state
4) Clathrin coat assembly

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17
Q

what causes the invagination of clathrin-coated pit? (2 things)

A
  • actin microfilament polymerization which bind to the coat via Epsin protein
  • Epsin binds PIP2 and promotes curvature by inserting an amphipathic alpha-helix in the cytoplasmic leaflet
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18
Q

what causes the budding of clathrin coat vesicle?

A
  • amphiphysin and endophilin associate with a curved-lipid surface and couple with the growing actin to help narrowing the neck
  • Dynamin activates & hydrolyzes GTP
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19
Q

what are the steps of uncoating of clathrin-coated vesicles

A

1) Auxilin protein: associates with the hub region of the triskelion
and recruits Hsc70
2) Hsc70 hydrolyzes ATP and associates with the N-terminus of the coat

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20
Q

what is Hsc70?

A

a chaperone protein

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21
Q

what is synaptojanin?

A

an enzyme bound to the membrane that converts PIP2 to PIP

22
Q

what is most likely the way how clathrin coat disassembles? explain

A

via phosphoinositides remodeling:
- synaptojanin enzyme converts PI-4,5-P2 to PI-4-P -> no more PIP2, AP2 can’t bind the subunit anymore and closes

23
Q

what are the 2 other hypothesizes (apart from phosphoinositides remodeling) of how clathrin vesicles uncoat?

A
  • H+ pump (ATP dependent) acidifies the budded vesicle once sealed
    or
  • drastic fall in Ca2+ due to a Ca2+ channel that exports Ca2+ out of the vesicle
24
Q

what is EEA1?

A

early endosome antigen 1: binds activated Rab5 on early endosome and PI3P also on early endosomes and endocytic vesicles

25
Q

how does the now uncoated clathrin vesicle fused with the early endosome?

A
  1. EEA1 tethers the 2 membranes via Rab5 and PI3P
  2. Rab5 then causes EEA1 to collapse and that pulls the two membranes closer together
  3. membrane docking via SNAREs
26
Q

remember what is V vs T snare?

A

v-SNARE = vesicle
t-SNARE = target (in case of clathrin vesicles, it’s the early endosome)

27
Q

name functions of Rab5

A
  • fusion of uncoated vesicle with endosome via EEA1 and PI3P
  • promotes binding of vesicle to microtubules
  • Rab5GTP activates SNAREs to promote docking and fusion
  • Rab5GTP in sorting endosomes: recruits its own deactivators and activate Rab7GTP -> mature to late endosome
28
Q

how do snares fuse membranes?

A
  1. V and t SNAREs fuse together via their n-terminal
  2. the bilayers fuse
  3. fusion pore appears
  4. SNARE’s four helix bundle rearranges to a more stable conformation, which release energy required for membrane fusion
29
Q

what is pinocytosis and what is it used for?

A

bulk-phase uptake: retrieve synaptic vesicles under intense stimulation when clathrin machinery is saturated

30
Q

what is faster, clathrin-mediated endocytosis of pinocytosis?

A

clathrin-mediated (15s)

31
Q

give an example of a marker for clathrin-coated pits

A

transferrin

32
Q

how can you test if your protein is internalized by clathrin or by bulk phase?

A

by measuring the colocalization of your protein with transferrin (clathrin pit marker) vs with a protein that floats around in ECM

33
Q

caveolin has a high concentration of what domains?

A

raft domains

34
Q

describe caveolin

A

caveolin coat protein with an unusual hairpin-shape membrane interaction domain (TM domain that doesn’t fully go through membrane) and is palmitoylated on the c-terminus

35
Q

what kind of molecules are often found in caveolin?

A

signaling molecules (high conc of then in caveolae)

36
Q

in what type of cells is endocytosis abundant?

A

in mechsnically stressed cells such a muscle, fibroblasts, endothelial, adipocytes

37
Q

what can caveolae form when the tension of the membrane decreases? in what case are they formed?

A

rosette: clusters of caveolae can form when cytoskeleton is disrupted, when cell adhesion is lost

38
Q

what proteins are involved in caveolae endocytosis?

A
  • filamin = docking protein
  • mDia1 = actin polymerization
  • Dynamin = seals neck
39
Q

more precisely, what is mDia1?

A

an actin nucleation factor that acts as a Rho GTPase effector protein. It accelerates actin elongation by rotating around the long-pitch helix of F-actin
(BASICALLY polarizes actin filaments to drive the caveolin vesicle to the early endosome)

40
Q

what happens in the sorting endosome?

A

ph drops to 6, causing cargos to detach from their receptors

41
Q

why does the pH dropping cause cargo to detach from receptor?

A

the H+ competes with the hydrophilic interactions

42
Q

what complex recognizes membrane proteins in sorting endosomes that are destined to the late endosome?

A

ESCRT

43
Q

how is the sorting of material in sorting endosomes based on geometry?

A
  • middle of endosome = goes to late endosome/lysosome
  • thin tubules extending from endosome = goes back to PM
44
Q

how is the surface/volume ratio in tubules of sorting endosomes vs middle?

A

high in the tubules, low in the middle

45
Q

what is ERC?

A

endocytic recycling compartments that brings proteins back to the membrane

46
Q

gives an example of a membrane protein that is recycled via ERC

A

Glut4: insulin triggers its recruitments to the PM in adipocytes and muscle cells

47
Q

what is ERC tightly bound to?

A

cytoskeleton

48
Q

what type of vesicles transport between trans/cis golgi and ER?

A
  • COPII = anterograde transport from ER to cis-golgi
  • COPI = retrograde transport from trans-golgi to cis-golgi to ER
49
Q

how are COPI and COPII vesicles formes and disassembled?

A

formed via small GTPases
disassembled via GTP hydrolysis

50
Q

how does COP II cage form?

A

like clathrin coat: sequentially
- Sar1GTP recruits Sec23/24 adapter proteins
- Sec13/31 are recruited (coat)

51
Q

how does COP I form?

A

coat is preassembled and recruited via GTP-Arf1

51
Q

what happens to COPI/II vesicles once they arrive at their destination?

A
  • GTP hydrolysis disassembles the coat
  • v-/t-SNARE fuse the membranez

ANAT458 (10 decks)

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