REGULATION OF ADP-RIBOSYLATION FACTOR

这项工作的主要目的是阐明生化机制。 调节GTP结合蛋白ARF，并在这样做的同时，进一步促进我们的 了解ARF在膜运输中的作用。 我们发现ARF的氨基末端α螺旋与共价结合 肉豆蔻酸盐形成对GTP和磷脂敏感的开关，这 结构域是与ARF GAP和Gs相互作用所必需的。我们目前正在 测试这是蛋白质的效应域的假设。 ARF与靶蛋白的相互作用依赖于磷脂。我们 发现ARF既与脂质具有非特异性相互作用，又与 洗涤剂和与磷脂酰肌醇4，5- 二磷酸(PIP2)。这些磷脂相互作用可能是 解体了。ARF的氨基末端对于非特异性 与磷脂有关，但不涉及特定的 与PIP2的相互作用。利用突变分析，我们有了初步的 在PIP2结合中涉及ARF的几个特定残基的数据。我们是 进一步检验ARF是PIP2结合蛋白的假设 PIP2作为ARF相互作用的辅助因子的相关假设 靶蛋白。 已经发现ARF-ARF间隙相互作用需要PIP2和PA， 我们在后者的纯化方面已经取得了一些进展 蛋白。ARF GTP酶激活蛋白与笼状蛋白组装共纯化 蛋白质复合体2。我们目前正在检验AP-2是 ARF间隙。这些数据支持这样一种假设，即 ARF对GTP的水解调节外壳蛋白在细胞表面的组装 运输囊泡。

The main objective of this work is to elucidate the biochemical mechanisms regulating the GTP-binding protein ARF and, in so doing, to further our understanding of the role of ARF in membrane traffic. We found that the amino terminal alpha helix of ARF and covalently bound myristate form a GTP- and phospholipid-sensitive switch and that this domain is necessary for interaction with ARF GAP and Gs. We are currently testing the hypothesis that this is an effector domain of the protein. ARF's interaction with target proteins is dependent on phospholipids. We found that ARF has both nonspecific interactions with lipids and detergents and a specific interaction with phosphatidylinositol 4,5- bisphosphate (PIP2). These phospholipid interactions could be dissociated. The amino terminus of ARF was necessary for the nonspecific association with phospholipids but was not involved in specific interactions with PIP2. Using mutational analysis we have preliminary data implicating several specific residues of ARF in PIP2 binding. We are further testing the hypothesis that ARF is a PIP2 binding protein and the related hypothesis that PIP2 acts as a cofactor for ARF interaction with target proteins. Having found that PIP2 and PA were required for ARF - ARF GAP interaction, we have been able to make some progress in the purification of the latter protein. ARF GTPase activating protein copurified with clathrin assembly protein complex 2. We are currently testing the hypothesis that AP-2 is ARF GAP. These data support the hypothesis that the binding and hydrolysis of GTP by ARF regulates the assembly of coat proteins on transport vesicles.