Cell biology Reconstitution of vesicle transport in cell - free systems was hist

Question

Cell biology Reconstitution of vesicle transport in cell - free systems was historically carried out in the presence of isolated 'donor' and ?acceptor? Golgi stacks. The donor stacks are isolated from cells expressing a viral protein and lacking a processing enzyme. The protein can be transported to the acceptor stacks (that do contain the processing enzyme) and be processed only in the presence of an energy source (such as ATP) and a cytosolic fraction. The transport vesicles that mediate this process are visualized by microscopy. Various compounds can be added to this system to help understand the mechanism of transport. Two such compounds were added in early experiments in the 1980s, both of which blocked transport of the viral protein and resulted in the accumulation of transport vesicles. However, the accumulated vesicles following treatment with one of these compounds (A) appeared to be coated, while those resulting from treatment with the other compound (B) did not. If one of these compounds is a nonhydrolyzable GTP analog and the other one is N - ethylmaleimide, which compound (A or B) do you think represents the GTP analog? Write down A or B as your answer and explain why.

Explanation / Answer

The N-Ethylmaleimide (NEM) is an alkene, which is obtained from maleic acid. This compound binds covalently to the cystine-thiol (-SH) groups in an irreversible manner. Hence, it also called Michael acceptor (NEM adds nucleophiles such as thiols) and commonly inhibit the enzymes, namely cysteine peptidases and it also blocks the vesicular transport. It causes the accumulation of uncoated vesicles; hence N-Ethylmaleimide is the compound (B). The GTPS causes the accumulation of coated vesicles, thus the compound (A) is the GTP analogue.    

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