Question

Question1:

Briefly answer the following questions:

a- Why do COPII coated vesicles get only formed at the ER membrane?

b- Why does the COPII coat disassemble upon vesicle detachment from the ER?

c- In the case where cells are treated with a non-hydrolysable GTP, which of these events will be observed. Highlight the event(s) you expect to observe and explain in a sentence each of your choices.

• Sar1 depletion from the cytosol
• No bud formation at the ER
• Formation of buds and COPII vesicles at the ER
• Empty COPII vesicles (with no cargo) will be formed
• Normal COPII coat dissociation after vesicle formation
• COPII abnormal fusion with plasma membrane
• Inability of COPII coated vesicles to fuse

Question2:

a- Briefly, explain how integral cargo proteins are sorted and concentrated in ER vesicles going to the Golgi?

b- Consider a wild type and mutant insulin receptors (IR) (with deleted cytoplasmic domain):

-  What is the effect of the mutation on the ability of IR to enter forming buds in the ER?

-  What is the effect of the mutation on the IR concentration in vesicles leaving the ER compare to WT?

-  What is the effect of the mutation on the Insulin concentration in vesicles leaving the ER compare to WT?

Question3:

Consider the following C-terminal sequences of some ER proteins:

Bip:                             N-…AGPPPTGEEDTAEKDEL-C

KDEL receptor:          N-…YLYITKVLKGKKLSLPA-C

a- Highlight with yellow color the sorting signal in each protein.

b- Which type of coated vesicle will specifically recruit and enrich Bip in them?

b- Explain how Bip sorting signal allows its selection and enrichment in vesicles?

Answer 1

1.

a. Coat protein complex II (COPII) is a set of highly conserved proteins that is responsible for creating small membrane vesicles that originate from the endoplasmic reticulum (ER).

This activity is restricted to the ER membrane, because Sec12, the guanine nucleotide exchange factor (GEF) that activates Sar1, is only found at the ER.

b. the COPII protein coat must be disassembled and its components released into cytosol. This uncoating is triggered by hydrolysis of the bound GTP to produce Sar1p-GDP, which has decreased affinity for the vesicle membrane. Disassociation of Sar1p-GDP from the membrane is followed by the release of the other COPII subunits.

C.

• Formation of buds and COPII vesicles at the ER.

2. a.The recruitment of cargo molecules into ER transport vesicles. By binding to the COPII coat, membrane and cargo proteins become concentrated in the transport vesicles as they leave the ER. ... The exit signals that direct proteins out of the ER for transport to the Golgi .

b. Insulin gene mutations affecting proinsulin folding in the ER

3.

b. Like other chaperones, BiP recognizes incorrectly folded proteins, as well as protein subunits that have not yet assembled into their final oligomeric complexes. To do so, it binds to exposed amino acid sequences that would normally be buried in the interior of correctly folded or assembled polypeptide chains.