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

Arf gtpase调节高尔基-内体系统和质膜内的囊泡运输。在载体形成过程中，Arf-GTP与膜、网格蛋白外壳和衔接蛋白相互作用。膜的管化和芽的形成伴随着Arf GTPase激活蛋白（ArfGAPs）的募集，其中一些可以与包括COPI和AP-1复合物在内的外壳蛋白结合。在COPI囊泡中，ArfGAP活性受外壳蛋白结合和膜形状的调节。此外，Arf上的GTP水解可能与copi介导的货物蛋白分选有关。对于网格蛋白/ AP-1/ Arf1，网格蛋白/ GGAs/ Arf1，或网格蛋白/ AP-2/ Arf6外壳及其候选arfgap的情况知之甚少。我们的初步研究表明，art 1和art 6显示出强大的gtp依赖性膜变形活性。此外，我们已经确定了arf6特异性GAP GIT1作为脑内AP-2相关的内吞分选适配器stonin2的新的直接结合伙伴。我们建议在这些观察的基础上，重建不同的Arf1和Arf6功能模块，包括体外模型膜上的Arf、网格蛋白外壳蛋白和ArfGAPs，并在活细胞中检测这些功能模块，探索ArfGAPs与网格蛋白外壳和接头蛋白之间的相互作用，并功能分析Arfö/ GIT1/ stonin2模块在网格蛋白介导的突触囊泡循环中的作用。

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.