Lecture 5,6 for class tomorrow starting from Golgi transport assay Flashcards
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 ___________.
- “donor”
- VSV
- VSV-G
- glycosylated
For what concerns the in-vitro assay for Golgi transport. These cells (the “donor” Golgi infected cells) also carry a mutation in ____ ___________.
GlcNac glycosyltransferase
For what concerns the in-vitro assay for Golgi transport. These cells (the “donor” Golgi infected cells) make lots of VSV-G that lacks ______.
GlcNac
For what concerns the in-vitro assay for Golgi transport. Also add _______ Golgi from uninfected cells that are _____ - ____ for GlcNac glycosyltransferase.
- “recipient”
2. Wild-type
In addition to cytosol and ___, add ________ ________ GlcNac.
- ATP
2. radioactively labeled GlcNac
Golgi transport can be monitored by the accumulation of _________ ________ ______.
Radioactively labeled GlcNac.
How is Golgi transport monitored?
By the accumulation of radioactively labeled GlcNac.
How is the accumulation of radioactively labeled GlcNac measured?
Grind up reaction contents.
IP with anti-VSV-G bound to beads.
Measure radioactivity.
EM analysis of the transport reaction yielded what?
Small number of coated and uncoated vesicles.
EM analysis of blocked transport reaction is accomplished by what?
- add GTP(gamma)S
- Leave out ATP
- Add NEM
What happens when adding GTP(gamma)S to the in-vitro assay for Golgi Transport?
coated vesicles accumulate
What happens when one leaves out ATP from the in-vitro assay for Golgi transport?
Uncoated vesicles accumulate.
What happens when NEM is added to the in-vitro assay for Golgi Transport?
Uncoated vesicles accumulate.
What is the order of the steps in vesicle transport from source to target?
Source -> coated vesicle – (GTPase required) –> uncoated vesicle – NEM sensitive, ATP required -> Target
What are the different vesicle coats?
- COP1
- COP2
- Clathrin
- Retromer
What is the vesicle coat COP1 used for?
Golgi, constitutive secretion.
What is the vesicle coat COP2 used for?
ER to golgi.
What is the clathrin vesicle coat used for?
Endocytosis, TGN to endosome
What is the retromer vesicle coat used for?
Endosome to TGN
Clathrin coats are composed of a lattice-like arrangement of Clathrin ________ that can form _______.
- triskelions
2. Spontaneously
The Clathrin coated vesicles’ coat consists of a triskelion of __ ___ and __ __ Clathrin subunits.
- 3 alpha
2. 3 beta
_____ _____ link clathrin to specific _____ receptors.
- Adaptor proteins
2. cargo
Adaptor proteins interact with ______.
Clathrin
Most adaptors recognize specific _________ that are enriched in specific ________.
- phosphoinositides
2. membranes
Adaptors also recognize ________ receptors.
transport
The coat recruitment GTPase, _____ is a specialized ________ protein.
- Arf1
2. Adaptor
Conversion of PI and PIPs depends on specific PIP ______ and __________. These enzymes are often localized to specific _______.
- kinases
- phosphatases
- organelles
Distinct PIPs can interact with specific ____-______ proteins.
PIP-binding
In clathrin coated vesicles, many of the _______ _______ interact with localized PIPs.
adaptor proteins
COP 2 coated vesicles are necessary for what?
Necessary for ER to Golgi transport.
______ forms the outer coat (and will spontaneously form a cage-like structure analogous to Clathrin).
Sec13/31
_____ and ______ form the inner coat and associate with ______ and to ________.
- sec23 and sec24
- cargo-bound receptors
- sec13/31
_____ coat recruitment ______ interacts with ______ and the _______ to stabilize coat assembly.
- Sar1
- GTPase
- sec23/24
- membrane
The Arf1 coat recruitment GTPase recruits?
Clathrin and COP1 assembly.
The Sar1 coat recruitment GTPase recruits?
COP II assembly.
Myristoylated proteins possess an ________ hydrophobic fatty acid (myristate) that can stably insert into _______.
- N-terminal
2. membranes
The myristil tail is only exposed when _____ bound.
GTP
Integral membrane ____ localized to specific membranes regulate sites of _____ recruitment.
- GEFs
2. coat
In clathrin coated vesicles, _____ associates with membrane and other adaptor proteins to help stabilize the coat.
Arf1-GTP
The ________ is an ER-resident transmembrane protein that specifically activates Sar1 in the ER membrane.
Sar1-GEF
Sar1-GTP interacts with the ______ _______.
lipid bilayer
Sar1-GTP recruits the COP 2 inner coat protein ______,
sec23
sec23 recruits what?
sec24 and cargo/cargo receptor.
sec23/24 recruit what?
coat proteins sec13/31
Coated vesicles are derived from what?
coated domains of membrane.
Coats function to concentrate specific _______ for inclusion in vesicles and assist in ______ formation.
- proteins
2. vesicle
Coat adaptor proteins, including coat-recruitment GTPases assist in _____ formation, and recruit specific ______.
- coat
2. cargo
