Secretion in mammalian cells - 1

Question 1

What are CRT (calreticulin) and CNX (calnexin) and what do mouse knockouts in both show (regarding phenotype)

Answer 1

CRT = ER resident protein which in knockouts caused cardiac problems
CNX = calnexin, chaperone protein which knockouts resulted in problems in myelination

Question 2

What is Bip ?

Answer 2

Bip = binding immunoglobulin protein. Essential in all cells. Part of the UPR.

Question 3

ER FOLDING DISEASES - examples?

Answer 3

Cystic fibrosis - CFTR protein not trafficked. Gets stuck in the ER. Protein often functional if trafficked to the membrane even if mutated
Hypothyroidism - mutant thyroglobulin protein, which controls homeostatic mechanisms. Causes proliferation of the ER and apoptosis.
Emphysema - no secretion of anti-antitrypsin which is needed for cilia functioning .

Question 4

ALS - why is it a trafficking disease? What characterises the disease ?

Answer 4

Amyotrophic lateral sclerosis - various mutations involved eg IRE1 and PERK.
Mainly caused by overactivation of ER stress pathway.

Question 5

COPII coated vesicles - componants and when were they identified?

Answer 5

SAR1 - small GTPase - INITIATES BUDDING
SEC23/24 - adaptor protein which links membrane and cargo - ENGAGES CARGO
SEC13/31 - forms the coat in a structural lattice - PROMOTES FISSION AND COAT POLYMERISATION

First discovered in Yeast secretory pathway mutants

These structures are DISTINCT FROM THE ER

Question 6

How are COPII coated vesicles formed?

Answer 6

1) GTPase is activated by SEC12 protein in the ER membrane
2) Recruits adaptor proteins
3) Coat assembles
4) Bud forms off from the ER –> pinches off –> vesicles form

Question 7

COPII and CRANIOFACIAL DISEASE – what characterises the disease and how can formation of vesicles in cells be tested?

Answer 7

Abnormal trafficking in homozygous mutants causing a DISBANDED ER STRUCTURE

Use of collagen (SECRETED PRODUCT) in mutant cells to measure secretion

Question 8

F382L mutation and COPII vesicle formation – what were the findings?

Answer 8

• Cell membranes were permeabilized and the components purified
• Introduced components into suspension
• Allowed vesicles to form

What shown that in F328L (SEC23A) mutants, budding could still occur with Sar1a but NOT Sar1b (unless the concentration was increased)

Question 9

Sar1b — how is this effected by F328L mutation?

What does this suggest about Sar1a

Answer 9

GTP hydrolysis has been impacted by Sar1b mutations —- budding only still occurs when the concentration of Sar1b is increased enough

Budding can still occur in presence of Sar1a with this mutant – suggests Sar1a’s activity on GTP hydrolysis is independent of F328L mutation