Elucidating mechanisms of intracellular transport regulated by the Coat Protein I (COPI) complex, we have found that the GTPase-activating protein (GAP) for the small GTPase ADP-Ribosylation Factor""""""""! (ARF1) acts not only as a negative regulator of ARF1, but also as its effector by being a coat component. This finding reverses the prevailing view that the GAP functions in the uncoating of COPI vesicles and should antagonize vesicle formation, but is consistent with mechanisms elucidated for the formation of COPII vesicles, where the corresponding GAP has also been shown to function as a component of the COPII coat complex. Refining the COPI vesicle reconstitution system recently, we have revealed that this process requires accessory proteins, which is similar to the formation of clathrin vesicles that have been shown to use multiple accessory proteins. Thus, as key mechanisms of COPI transport now appear to be more similar to that elucidated for the other well-characterized transport pathways than previously suspected, this observed conservation in key mechanisms also becomes the overall driving hypothesis in the current application that seeks to further elucidate mechanisms of COPI vesicle formation and uncoating. First, as we have found that GAP does not directly trigger COPI vesicle uncoating, we will identify predicted novel uncoating factor(s) by a systematic purification approach and also complement this strategy by examining for potential clues from mechanisms of uncoating that are better elucidated for clathrin vesicles. Second, as we have found recently that Brefeldin-A ADP-Ribosylated Substrate (BARS) and endophilin B play interchangeable roles as key components of the fission machinery for COPI vesicle formation, we will determine more precisely how they achieve this role, which again will likely be facilitated by potential clues from mechanisms of fission that have been better elucidated for clathrin vesicles. Third, we have identified a novel role for phospholipase D (PLD) activity in COPI vesicle formation. Thus, we will clarify this role, which will also address a current controversy regarding the precise role of this activity in COPI vesicle formation. Fourth, we will examinewhether and potentially how antiquitin, which we have shown recently to interact with ARFGAP1, plays a role in either COPI vesicle formation and/or cargo sorting. We anticipate that the completion of these studies will not only further elucidate mechanisms of COPI transport, but also advance a general understanding of vesicular transport, as key mechanisms are likely to be conserved across all transport pathways. Thus, as defects in intracellular transport pathways are being appreciated as the cause for a growing number of human diseases, our findings will likely be broadly applicable to understanding and treating these diseases.
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