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

囊泡形成中的 Arf GTPase 模块：外套招募和膜弯曲与 ArfGAP 活性的协调

• 批准号: 46239794
• 负责人: Professor Volker Haucke, Ph.D.
• 金额: --
• 依托单位: Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2007
• 资助国家: 德国
• 起止时间: 2006-12-31 至 2010-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/46239794?language=en
• 关键词: Arf; GTPase; module; vesicle; formation

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.

ARF GTP酶调节高尔基体-内体系统和质膜上的囊泡运输。在载体形成过程中，Arf-GTP与膜相互作用，并与包膜蛋白、外壳蛋白和接头蛋白相互作用。膜管形成和芽形成伴随着Arf GTPase激活蛋白(ArfGAPs)的募集，其中一些与包括COPI和AP-1复合体在内的外壳蛋白有关。在COPI囊泡的情况下，ArfGAP的活性受外壳蛋白结合和膜形状的调节。此外，GTP在Arf上的水解可能与COPI介导的货物蛋白分选有关。对于clathrin/ap-1/arf 1、clathrin/ggas/arf1或clathrin/ap-2/arf6涂层及其候选ArfGAP，所知的要少得多。我们的初步研究表明，ART1和ART6显示出强烈的依赖GTP的膜变形活性。此外，我们还发现Arf6特异的GAP GIT1是脑内AP-2相关的内吞分选接头Stonin2的新的直接结合伙伴。我们建议在这些观察的基础上，在体外重建模型膜上不同的Arf1和Arf6功能模块，包括Arf、笼蛋白外壳蛋白和ArfGAP，并在活细胞中检测这些功能模块，探索ArfGAP与笼蛋白外壳和接头蛋白之间的相互作用，并从功能上分析分子筛介导的突触处小泡循环中的Arfö/GIT1/Stonin2模块。