The Arf GTPase module in vesicle formation: coordination of coat recruitment and membrane bending with ArfGAP activity

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Arf GTPases regulate vesicular transport within the Golgi-endosomal system and at the plasma membrane. During carrier formation Arf-GTP interacts with the membrane and with clathrin coat and adaptor proteins. Membrane tubulation and bud formation are accompanied by recruitment of Arf GTPase activating proteins (ArfGAPs), some of which can associate with coat proteins including COPI and the AP-1 complex. In the case of COPI vesicles ArfGAP activity is regulated by coat protein binding as well as membrane shape. Moreover, GTP hydrolysis on Arf may be linked to COPI-mediated cargo protein sorting. Much less is known in the case of clathrin/ AP-1/ Arf 1, clathrin/ GGAs/ Arf1, or clathrin/ AP-2/ Arf6 coats and their candidate ArfGAPs. Our preliminary studies indicate that Arts 1 and 6 display potent GTP-dependent membrane deforming activity. Moreover, we have identified the Arf6-specific GAP GIT1 as a novel direct binding partner for the AP-2 associated endocytic sorting adaptor stonin2 in brain. We propose to build on these observations to reconstitute distinct Arf1 and Arf6 functional modules comprising Arf, clathrin coat proteins, and ArfGAPs on model membranes in vitro and to examine these in living cells, to explore the interactions between ArfGAPs and clathrin coat and adaptor proteins, and to functionally analyze the Arfö/ GIT1/ stonin2 module in clathrin-mediated vesicle cycling at synapses.