): Studies of the small GTPase ADP-ribosylation Factor 1 (ARF1) have contributed much to our mechanistic understanding of intracellular protein sorting. Activation of ARF1 by its binding of GTP recruits the coat complex COPI from the cytosol to Golgi membranes to initiate the formation of COPI-coated vesicles (CVs). The activation of ARF1, by the hydrolysis of its bound GTP to GDP, is regulated by a GTPase-activating protein (GAP), and this event is thought to regulate the uncoating of COPI-CVs. In preliminary studies, we have reconstituted budding of COPI-CVs using purified components and show that, rather than regulating uncoating of COPI-CVs, GAP regulates their formation. This finding suggests the possibility that the currently elucidated mechanisms for the biogenesis of vesicles coated by COPII and clathrin might be relevant to a further understanding of mechanisms that regulate budding of COPI-CVs from Golgi membranes and their subsequent uncoating that is required to complete their transport itinerary. First, based on mechanisms elucidated for COPII-CVs, we will test whether GAP interacts with the COPI coat, and how this potential interaction might regulate budding of COPI-CVs. Second, a potential role for GAP in sorting cargo proteins into COPl-CVs will be tested, and a potential cargo receptor for GAP will be identified. Third, as GAP promotes budding of COPI-CVs, what might regulate their uncoating becomes an open question. Taking potential clues from clathrin-CVs, we will test whether a member of the heat shock protein (hsc7O) participates in uncoating of COPI-CVs, and also take a complementary approach to systemically characterize the uncoating activity. Fourth, we will test whether vesicle budding promoted by GAP might involve mechanisms that modify the phospholipid composition of the Golgi membrane. Results of these proposed studies may suggest fundamentally similar mechanisms of transport among different intracellular transport pathways, and will be broadly applicable in the understanding and treatment of diseases, as a number of them have been shown to be due to aberrations in the transport itinerary of responsible proteins.
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