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MCB > vesicular traffic and secretion > Flashcards

vesicular traffic and secretion Flashcards

1
Q

how is the transport of cargo proteins in the secretory pathway governed?

A

by a series of budding and fusion reactions from the donor compartment to the target compartment

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2
Q

what two types of techniques were used to investigate vesicular transport?

A
  • genetic approaches

- biochemical approaches

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3
Q

what did schekman do in his genetic experiments regarding vesicular transport?

A

generated yeast mutants with defects in secretion using ionising radiation

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4
Q

what biochemical techniques did Rothman use to measure transport of proteins?

A

cell free transport assays - this allows for fractionalisation of the cytosol and precise characterisation of protein complexes

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5
Q

how were yeast budding experiments used for investigation of proteins involved in secretion?

A
  • temperature sensitive mutants were created
  • these can be transferred from lower to higher temperatures and will accumulate vesicles in the part of the secretory pathway that their mutation blocks
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6
Q

why are yeasts a good model organism for the study of vesicular transport?

A

they are single celled but do secrete some enzymes

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7
Q

what is the defective function of class A mutants?

A
  • transport into the ER

- these accumulate in the cytosol

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8
Q

what is the defective function of class B mutants?

A
  • budding of vesicles from the rough ER

- vesicles accumulate in the ER

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9
Q

what is the defective function of class C mutants?

A
  • budding of vesicles from rough ER

- accumulation in ER to Golgi transport vesicles

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10
Q

what do yeast budding assays tell us about vesicular transport?

A

studies confirm that a secreted protein is synthesised and processed, it moves from the cytosol to the rough ER -> transport vesicles -> Golgi cisternae -> secretory vesicles -> protein is exocytosed

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11
Q

what did Rothman measure in cell free assays?

A

incorporation of radio labelled N-acetylglucosamine into the oligosaccharide on proteins in vesicles in cells with defective or wild type NAG transferase function

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12
Q

what is the function of protein coats on vesicles?

A

to facilitate curvature of the membrane and selection of cargo through distortion of the membrane

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13
Q

how is budding initiated?

A

recruitment of small GTP-binding proteins to a patch on the donor membrane, complexes of coat proteins in the cytosol then bind to the cytosolic domain

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14
Q

outline the steps of protein coat formation

A

1- four active GTP binding proteins associate with the forming bud
2- The GTP binding proteins recruit the components of the protein coat
3- the protein coat components are able to selectively bind to cargo proteins via membrane receptors
4- these receptors can bind to soluble cargo proteins to selectively incorporate these into vesicle formation

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15
Q

what type of transport do COPII coated vesicles mediate?

A

anteretrograde transport

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16
Q

which type of transport do COPI coated vesicles mediate?

A

retrograde transport

17
Q

what is anteretrograde transport?

A

when newly synthesised proteins are transported from the ER to cis golgi

18
Q

what does Brefeldin A do?

A

blocks anteretrograde transport

19
Q

how can brefeldin A be used experimentally?

A

provides evidence for retrograde transport - if there was no retrograde transport, when brefeldin A is added to culture, the protein would remain localised to the Golgi

20
Q

what is Arf2?

A

a monomeric GTPase that controls COPI and clathrin coat assembly at Golgi membranes

21
Q

what is Sar1?

A

a monomeric GTPase that mediates COPII coat assembly at the ER membrane

22
Q

what is a Rab protein?

A

a family of monomeric GTPases that are associated with one or more membrane enclosed organelles in the secretory or endocytic pathways

23
Q

what is the purpose of Rab GDI?

A
  • dissociation inhibitor

- keeps Rab soluble and inactive

24
Q

which sorting signal is found on ER membrane resident proteins?

A

KKXX - found at C terminus, binds directly to COPI coats

25
Q

which sorting signal is found on soluble ER resident proteins?

A

KDEL - binds to KDEL receptor

26
Q

what is the purpose of sorting signals?

A

ensure ER resident proteins are returned to the ER via COPI coated vesicles

27
Q

how do we know that sorting signals aren’t the only mechanism for ER retention?

A

mutants with KDEL deletions still secrete proteins, but this is slow

28
Q

how does the SNARE complex mediate vesicle fusion?

A
  • transport vesicles have a v-SNARE called VAMP
  • VAMP carries Rab, which binds to the target effector when in GTP bound form
  • in the target membrane, the SNARE is called syntaxin and SNAP-25
  • tails of the t-SNARE and v-SNARE associate to form a coiled structure
29
Q

what is the function of the SNARE complex?

A

holds the vesicle close to the target membrane and assists fusion

MCB (16 decks)

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