A fungal metabolite is a potent toxin that inhibits the assembly of COPI-coated

Question

A fungal metabolite is a potent toxin that inhibits the assembly of COPI-coated vesicles on the Golgi complex, and thus prevents the budding of retrograde transport vesicles between the ER and Golgi and between Golgi stacks. Theoretically, this toxin could block formation of COP coated vesicles at any point in the mechanism of their assembly (below): 5. ARF-GDP(cytosolic) + GTP-) ARF-GTP(cytosolic)+ GDP ARF-GTP(membrane) + COPI COPI-coated vesicles The following experimental observations are made in an attempt to identify the point of action in the above mechanism: a). ARF with bound GppNp (non-hydrolyzable GTP analog) causes CoPI-coated vesicles to form when added to Golgi membrane preparations. Formation of the vesicles in this experiment is not affected with the fungal toxin. b). ARF with bound GDP exchanges GDP for GTP when added to Golgi membrane preparations. Trypsin-treated Golgi membrane preparations do not stimulate GTP-for-GDP exchange. The nucleotide-exchange reaction with normal Golgi membrane preparations does not occur in the presence of the fungal toxin. c). ARF with bound GTP can be made to exchange GDP for GTP in the absence of Golgi membrane preparations by treatment with phosphatidylcholine and the detergent cholic acid. This artificial exchange is not affected by the fungal toxin. Given the above experimental observations, propose at least two different points of action for the fungal toxin.

Explanation / Answer

We are considering here a fungal toxin which has been found to inhibit the COPI coated vesicles on the golgi complex, preventing the retrograde transport of vesicles between ER to golgi and golgi stacks.

And there are some experiments tofind out the possible mechanism of action.

From 1st observation - Non-hydrolyzable GTP analog can form COPI vesicle in presence of fungal toxin with Golgi membrane preparation. It means the toxin may cut the chain of reaction at the GTP - GDP exchange point. In Non-hydrolyzable GTP analog the exchange already took place and that is irreversible, so there is no point of toxin to work. So the fungal toxin can be a cytosolic Neucleotide exchanger inhibitor.

From 2nd observation - Trypsin treated Golgi membrane works in the similar way as the fungal toxin with normal golgi membrane for GTP - GDP exchange. Usually trypsin as a proteolytic enzyme destroys the cell surface receptor proteins. Now in membrane there would be some sort of anchoring protein which facilitates to dock the GTP-ARF complex. destroying membrane structure that achoring function can be lost, so for the use of fungal toxin. That toxin possibly prevents the docking action of that achoring protein in the golgi membrane.

From 3rd observation - In the absence of Golgi membrane preparation, phosphatidyl choline with cholic acid can facilitates the GDP-GTP excahnge even in presence of fungal toxin. This also supports the hypothesis from observation 2 that fungal toxin may block the anchoring protein of the membrane from nucleotide exchange. Without golgi membrane, toxin can't bind to it and work as nucleotide exchange inhibitor.

So, We have -

1. The fungal toxin can work as a cytosolic nucleotide exchange inhibitor, which is possibly act on ARF-GDP state and not in already membrane bound GTP form.

2. The toxin may also prevents the docking action of some achoring protein in the golgi membrane to the ARF-GTP form.

Tjhanks for asking.

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