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

Question 1

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 ___________.

Answer 1

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

Question 2

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

Answer 2

GlcNac glycosyltransferase

Question 3

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

Answer 3

GlcNac

Question 4

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

Answer 4

1. “recipient”

2. Wild-type

Question 5

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

Answer 5

1. ATP

2. radioactively labeled GlcNac

Question 6

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

Answer 6

Radioactively labeled GlcNac.

Question 7

How is Golgi transport monitored?

Answer 7

By the accumulation of radioactively labeled GlcNac.

Question 8

How is the accumulation of radioactively labeled GlcNac measured?

Answer 8

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

Question 9

EM analysis of the transport reaction yielded what?

Answer 9

Small number of coated and uncoated vesicles.

Question 10

EM analysis of blocked transport reaction is accomplished by what?

Answer 10

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

Question 11

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

Answer 11

coated vesicles accumulate

Question 12

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

Answer 12

Uncoated vesicles accumulate.

Question 13

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

Answer 13

Uncoated vesicles accumulate.

Question 14

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

Answer 14

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

Question 15

What are the different vesicle coats?

Answer 15

1. COP1
2. COP2
3. Clathrin
4. Retromer

Question 16

What is the vesicle coat COP1 used for?

Answer 16

Golgi, constitutive secretion.

Question 17

What is the vesicle coat COP2 used for?

Answer 17

ER to golgi.

Question 18

What is the clathrin vesicle coat used for?

Answer 18

Endocytosis, TGN to endosome

Question 19

What is the retromer vesicle coat used for?

Answer 19

Endosome to TGN

Question 20

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

Answer 20

1. triskelions

2. Spontaneously

Question 21

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

Answer 21

1. 3 alpha

2. 3 beta

Question 22

_____ _____ link clathrin to specific _____ receptors.

Answer 22

1. Adaptor proteins

2. cargo

Question 23

Adaptor proteins interact with ______.

Answer 23

Clathrin

Question 24

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

Answer 24

1. phosphoinositides

2. membranes

Question 25

Adaptors also recognize ________ receptors.

Answer 25

transport

Question 26

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

Answer 26

1. Arf1 | 2. Adaptor

Question 27

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

Answer 27

1. kinases 2. phosphatases 3. organelles

Question 28

Distinct PIPs can interact with specific ____-______ proteins.

Answer 28

PIP-binding

Question 29

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

Answer 29

adaptor proteins

Question 30

COP 2 coated vesicles are necessary for what?

Answer 30

Necessary for ER to Golgi transport.

Question 31

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

Answer 31

Sec13/31

Question 32

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

Answer 32

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

Question 33

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

Answer 33

1. Sar1 2. GTPase 3. sec23/24 4. membrane

Question 34

The Arf1 coat recruitment GTPase recruits?

Answer 34

Clathrin and COP1 assembly.

Question 35

The Sar1 coat recruitment GTPase recruits?

Answer 35

COP II assembly.

Question 36

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

Answer 36

1. N-terminal | 2. membranes

Question 37

The myristil tail is only exposed when _____ bound.

Answer 37

GTP

Question 38

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

Answer 38

1. GEFs | 2. coat

Question 39

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

Answer 39

Arf1-GTP

Question 40

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

Answer 40

Sar1-GEF

Question 41

Sar1-GTP interacts with the ______ _______.

Answer 41

lipid bilayer

Question 42

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

Answer 42

sec23

Question 43

sec23 recruits what?

Answer 43

sec24 and cargo/cargo receptor.

Question 44

sec23/24 recruit what?

Answer 44

coat proteins sec13/31

Question 45

Coated vesicles are derived from what?

Answer 45

coated domains of membrane.

Question 46

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

Answer 46

1. proteins | 2. vesicle

Question 47

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

Answer 47

1. coat | 2. cargo