Question

Consider cargo proteins that are destined for secretion to the extracellular space. Please 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 conditions.

A.

(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) COPII coat proteins are mutated so they bind both to active Sar1 and active ARF1.

(iii) Both

B. (i) A KDEL signal sequence is added to the C-terminus of the cargo protein.
(ii) The signal recognition particle is mutated so it can’t recognize ER signal sequences.

(iii) Both

Answer 1

A. I . a specific organelle - Sar1*-GDP Cannot maintain the coat once Sar 1 - GTP is hydrolyzed by GAP and inorganic phosphate is released. The coat will disassemble. It not comes out from Endoplasmic reticulum.

ii. Transport vesicles - COPII recruitment is mediated by the active GTP-bound form of the small GTPase Sar1.

iii. a specific organelle

B. a specific organelle - ER resident protein with a 4 AA sequence: Lysine - Aspartate - Glutamate - Leucine. It's a signal sequence for ER resident SOLUBLE proteins.

ii. in the cytoplasm

iii. extracellular space