sfsu.edu eal 0612-01 Human Physiol. Facebook Chegg Tutors l Online T. 0350-01 Ce

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sfsu.edu eal 0612-01 Human Physiol. Facebook Chegg Tutors l Online T. 0350-01 Cell Biology S https://ilearn.sfsu.edu/a... How could you try to pr Favorites 5. 5a. Imagine that the ARF1 GTPase protein was mutated so that it could not hydrolyze GTP into GDP, regardless of its binding partners. Would you expect COPI-coated vesicles to form normally? How might subsequent or downstream steps in transport mediated by COPI-coated vesicles be affected if ARF1 cannot hydrolyze GTP into GDP? 5b. What if ARF1 protein was mutated so that it was locked in a GDP-bound state, regardless of its binding partners. How would you expect this mutation to affect COPI- coat formation?

Explanation / Answer

5. a)In mammalian and yeast cells, COPI-coat formation is triggered by the conversion of Arf1 in its GDP-form to a GTP-form, a process which can be inhibited by brefeldin A , leading to the release of membrane bound coatomer . The primary effect of brefeldin A on plant cells is rapid displacement of coatomer into the cytosol, and is similar to that in other eukaryotes. The subsequent manifestation of this toxin on the endomembrane system is, however, very different . It is interesting that the events lying between the BFA-induced loss of COPI coats from the Golgi on the one hand, and fusion of Golgi membranes with the ER on the other hand, are strikingly different between mammalian and plant cells . These differences most likely reflect the different structural organization and functional requirement  of the Golgi apparatus in two systems . In this way the COPI-coated vesicles are affected.

5.b)Uncoating of COPI vesicles is mediated by an ArfGTPase activating activity, for which three Golgi-specific enzymes are described, ArfGAPs1-3. This activity stimulates the hydrolysis of GTP in Arf1, yielding Arf-GDP that no longer binds to membranes and thus dissociates from the vesicle, initiating coat disassembly. Interestingly, an ArfGAP activity is needed also in the formation of COPI-coated vesicles, to allow uptake of cargo. Various reports have appeared in the past on the molecular mechanism that underlie vesicle formation, mainly based on in vitro reconstitution experiments. Varying functions have been reported for roles that ArfGAP proteins play in COPI vesicle biogenesis. Specifically, ArfGAP1 on the one hand was described as an enzyme to catalyze uncoating of COPI coated vesicles, and on the other hand as a stoichiometric structural coat component . In order to investigate COPI vesicle formation in a reconstituted system, it was found that it allows vesicle formation and purification in the presence of GTP, rather than its poorly hydrolyzable analogs, in the numbers needed for quantitative analyses. Therefore it allows a close examination of the roles that ArfGAP1 plays in COPI vesicle biogenesis and significantly addresses some of the discrepancies in the field. In this way the mutation affects COPI-coat formation.

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