Question

1) how does the timing and location of Arf1-GTP hydrolysis contribute the transport of COPI coated vesicles?

2) Consider cargo proteins that are destined for secretion to the extracellular space. For each of the following pairs of mutations or conditions, predict where the cargo will end up (ex. in the cytoplasm, a specific organelle, transport vesicles, multiple places, extracellular space) for: (i) & (ii) Each individual condition; & (iii) The combination of both.

A.(i) Normal Sar1 is mutated so that it hydrolyzes GTP immediately after binding GTP.

(ii)The signal recognition particle is mutated to be unable to bind to the SRP receptor.

(iii)Both

B.(i) A mitochondrial signal sequence is added to the C-terminus of the cargo protein.

(ii)v-SNAREs on COPII coated vesicles are mutated to be unable to bind t-SNAREs

(iii)Both

C. (i) COPII coat proteins are mutated so they don’t bind to the ER membrane.

(ii)COPI coat proteins are mutated so they don’t bind to the Golgi membrane.

(iii)Both

D.(i) Normal Sar1 is mutated so it binds tightly to GDP and cannot exchange it for GTP. (This leads to Sar1*-GDP being the only form of the protein in the cell.)

(ii) A transmembrane domain is added to the cargo protein.

(iii)Both

Answer 1

1.

• ADP - ribosylation factor is necessary for COPI mediated transport from Endoplasmic reticulum and Golgi cisternae.
• COPI coated vesicles are involved in the early secretory pathway.
• The coats of COPI vesicles consist of large protein complexes that are composed of seven individual coat-protein subunits.
• The ARF proteins acts as a molecular switch.
• It binds to GDP when inactive state and when activated it binds to the GTP.
• When COPI is needed in the early secretory pathway , the ARF proteins gets activated by releasing its bound GDP and binding of GTP.
• Once activated the ARF proteins mediate COPI assembly and mediate tarnsport.

2.

A) Normal Sar1 is mutated so that it hydrolyzes GTP immediately after binding GTP

• The cargo protein will remain in the ER itself and will not get transported through COPII vesicles.
• Sar1 proteins are responsible for COPII assembly.
• When this is mutated it cannot assemble COPII vesicles.

The signal recognition particle is mutated to be unable to bind to the SRP receptor.

• SRP is responsible for targeting the proteins to ER.
• When SRP receptor is mutated SRP cannot bind to this receptor which is present in the ER and remains in the cytoplasm.

Both mutation occurs - then the protein remains in the cytoplasm because the protein cannot reach ER itself as the SRP receptor mutation persists.

B.(i) A mitochondrial signal sequence is added to the C-terminus of the cargo protein.

• Usually the mitochondrial signal sequence is added to the N-terminus , if it is added to C terminus it remains in the cytosol or in plants it is directed to peroxixomes.
• When the v-snare and T- SNARE proteins are mutated , both the membranes cannot fuse and the transport of proteins to target membrane cannot take place.
• The proteins cannot reach mitochondria or peroxixomes if the snare proteins are muated.

c)

• COPI transports proteins from Golgi to ER, if this is mutated , this transpotation cannot occur.
• COPII proteins transport from ER to golgi. If it is mutated this cannot occur.
• Both anterograde and retrograde transportation of proteins cannot occur if both are mutated.

d)

• Sar1 is responsible for COPII assembly. If sar1 is mutated it cannot move the proteins from ER to golgi.