Lecture 6/7- Golgi BOdy

Question 1

ERAD pathway

Answer 1

Role to contribute to maintenance of ER and cell homeostasis

1) n-linked glycostlation (glycan with 3 glucose)
1. 5)2 glucose removed- calnexin (chaperone)
2) incorrect folding sensed by glucosydase transferase

Question 2

Invtro golgi assay

Answer 2

VSVG as a protein of intereset
normal
golgi with defective N-acetlyglucosamine transferase (N-glycan modifying enzyme)
Immunoprecipitated Radiolabelled VSVG will only appear in mutant golgi if transportation occurs

Question 3

What did it reveal

Answer 3

GTP analgoue- ARF

NEM(alkylating agent) vesicle formation- Revealed NSF

Question 4

ARF role in COP1 vesicle formation

Answer 4

Discovered microscopy of isolated uncoated vesicles
GEF make ARF-GDP to ARF-ATP (membrane bound due release of myrosilated group)
recuites coatomer
ARF-COP recruit GTPase activating protein required for uncoating
coat non linear organisation causes curvature and budding

Question 5

COP11 vesicle formation

Answer 5

same but with different proteins
sar1(ARF)
sec12(GEF)
coat proteins Sec23,24,13,31(coantomer

Question 6

What did incubation with alkylating agent show and how were specifics found

Answer 6

reconstitution using fractioned cytsol, after it was estored then used inividual proteins
Revealed NSF (NEM sensitive factor) and SNAPS
SNARES identified by TIRF when looking for binding partners of SNAPS and NSF

Question 7

Roles of these proteins

Answer 7

SNARE- 4 bundle- 1V, 2T, 1 suntaxin like T SNARE
added a layer of specificity of targetting
removes water, membrane fusion

NSF (ATPase) and SNAPS segregates bundles