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Advanced Cell Biology > Lecture 5,6 for class tomorrow starting from Golgi transport assay > Flashcards

Lecture 5,6 for class tomorrow starting from Golgi transport assay Flashcards

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1
Q

For what concerns the in-vitro assay for Golgi transport. It starts with ______ Golgi from cells infected with ____. These cells make a protein, _____-_ that is known to pass through the Golgi and be ___________.

A
  1. “donor”
  2. VSV
  3. VSV-G
  4. glycosylated
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2
Q

For what concerns the in-vitro assay for Golgi transport. These cells (the “donor” Golgi infected cells) also carry a mutation in ____ ___________.

A

GlcNac glycosyltransferase

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3
Q

For what concerns the in-vitro assay for Golgi transport. These cells (the “donor” Golgi infected cells) make lots of VSV-G that lacks ______.

A

GlcNac

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4
Q

For what concerns the in-vitro assay for Golgi transport. Also add _______ Golgi from uninfected cells that are _____ - ____ for GlcNac glycosyltransferase.

A
  1. “recipient”

2. Wild-type

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5
Q

In addition to cytosol and ___, add ________ ________ GlcNac.

A
  1. ATP

2. radioactively labeled GlcNac

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6
Q

Golgi transport can be monitored by the accumulation of _________ ________ ______.

A

Radioactively labeled GlcNac.

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7
Q

How is Golgi transport monitored?

A

By the accumulation of radioactively labeled GlcNac.

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8
Q

How is the accumulation of radioactively labeled GlcNac measured?

A

Grind up reaction contents.
IP with anti-VSV-G bound to beads.
Measure radioactivity.

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9
Q

EM analysis of the transport reaction yielded what?

A

Small number of coated and uncoated vesicles.

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10
Q

EM analysis of blocked transport reaction is accomplished by what?

A
  1. add GTP(gamma)S
  2. Leave out ATP
  3. Add NEM
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11
Q

What happens when adding GTP(gamma)S to the in-vitro assay for Golgi Transport?

A

coated vesicles accumulate

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12
Q

What happens when one leaves out ATP from the in-vitro assay for Golgi transport?

A

Uncoated vesicles accumulate.

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13
Q

What happens when NEM is added to the in-vitro assay for Golgi Transport?

A

Uncoated vesicles accumulate.

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14
Q

What is the order of the steps in vesicle transport from source to target?

A

Source -> coated vesicle – (GTPase required) –> uncoated vesicle – NEM sensitive, ATP required -> Target

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15
Q

What are the different vesicle coats?

A
  1. COP1
  2. COP2
  3. Clathrin
  4. Retromer
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16
Q

What is the vesicle coat COP1 used for?

A

Golgi, constitutive secretion.

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17
Q

What is the vesicle coat COP2 used for?

A

ER to golgi.

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18
Q

What is the clathrin vesicle coat used for?

A

Endocytosis, TGN to endosome

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19
Q

What is the retromer vesicle coat used for?

A

Endosome to TGN

20
Q

Clathrin coats are composed of a lattice-like arrangement of Clathrin ________ that can form _______.

A
  1. triskelions

2. Spontaneously

21
Q

The Clathrin coated vesicles’ coat consists of a triskelion of __ ___ and __ __ Clathrin subunits.

A
  1. 3 alpha

2. 3 beta

22
Q

_____ _____ link clathrin to specific _____ receptors.

A
  1. Adaptor proteins

2. cargo

23
Q

Adaptor proteins interact with ______.

A

Clathrin

24
Q

Most adaptors recognize specific _________ that are enriched in specific ________.

A
  1. phosphoinositides

2. membranes

25
Q

Adaptors also recognize ________ receptors.

A

transport

26
Q

The coat recruitment GTPase, _____ is a specialized ________ protein.

A
  1. Arf1

2. Adaptor

27
Q

Conversion of PI and PIPs depends on specific PIP ______ and __________. These enzymes are often localized to specific _______.

A
  1. kinases
  2. phosphatases
  3. organelles
28
Q

Distinct PIPs can interact with specific ____-______ proteins.

A

PIP-binding

29
Q

In clathrin coated vesicles, many of the _______ _______ interact with localized PIPs.

A

adaptor proteins

30
Q

COP 2 coated vesicles are necessary for what?

A

Necessary for ER to Golgi transport.

31
Q

______ forms the outer coat (and will spontaneously form a cage-like structure analogous to Clathrin).

A

Sec13/31

32
Q

_____ and ______ form the inner coat and associate with ______ and to ________.

A
  1. sec23 and sec24
  2. cargo-bound receptors
  3. sec13/31
33
Q

_____ coat recruitment ______ interacts with ______ and the _______ to stabilize coat assembly.

A
  1. Sar1
  2. GTPase
  3. sec23/24
  4. membrane
34
Q

The Arf1 coat recruitment GTPase recruits?

A

Clathrin and COP1 assembly.

35
Q

The Sar1 coat recruitment GTPase recruits?

A

COP II assembly.

36
Q

Myristoylated proteins possess an ________ hydrophobic fatty acid (myristate) that can stably insert into _______.

A
  1. N-terminal

2. membranes

37
Q

The myristil tail is only exposed when _____ bound.

A

GTP

38
Q

Integral membrane ____ localized to specific membranes regulate sites of _____ recruitment.

A
  1. GEFs

2. coat

39
Q

In clathrin coated vesicles, _____ associates with membrane and other adaptor proteins to help stabilize the coat.

A

Arf1-GTP

40
Q

The ________ is an ER-resident transmembrane protein that specifically activates Sar1 in the ER membrane.

A

Sar1-GEF

41
Q

Sar1-GTP interacts with the ______ _______.

A

lipid bilayer

42
Q

Sar1-GTP recruits the COP 2 inner coat protein ______,

A

sec23

43
Q

sec23 recruits what?

A

sec24 and cargo/cargo receptor.

44
Q

sec23/24 recruit what?

A

coat proteins sec13/31

45
Q

Coated vesicles are derived from what?

A

coated domains of membrane.

46
Q

Coats function to concentrate specific _______ for inclusion in vesicles and assist in ______ formation.

A
  1. proteins

2. vesicle

47
Q

Coat adaptor proteins, including coat-recruitment GTPases assist in _____ formation, and recruit specific ______.

A
  1. coat

2. cargo

Advanced Cell Biology (15 decks)

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