Dynamics of endomembrane docking and fusion

内膜对接和融合的动力学

• 批准号: 7192517
• 负责人: Alexey Jarrell Merz
• 金额: $ 28.09万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7192517
• 关键词: Dynamics; endomembrane; docking; fusion

DESCRIPTION (provided by applicant): Docking and fusion events in the endomembrane system are regulated and executed by Rab GTPases, SNARE proteins, and their cofactors. A great deal has been learned about these proteins and their interactions. However, the molecular events leading to fusion are not understood in detail for any intracellular transport step. The ability to resolve individual events rather than the aggregate behavior of event populations has potentiated major advances in many areas of biology. We believe that the mechanistic dissection of membrane docking and fusion would be greatly facilitated if we could trace and perturb the subreactions of tethering, docking, and fusion at the level of individual docking and fusion events. In this proposal we combine direct attacks on the problem of monitoring single events with biochemical and genetic studies that will simultaneously characterize critical molecules and yield new reagents and probes for the single-event work. Our experimental system, the yeast vacuole, offers numerous experimental advantages for these studies including its size, which is large enough to facilitate light microscopy, the genetic and genomic toolkit of budding yeast, and a cell-free fusion system that is unsurpassed in its experimental flexibility and existing knowledge base. These experiments will build on - and substantially extend - approaches developed during the PI's postdoctoral work in Dr. William Wickner's group. Microfabrication techniques and low-light, quantitative fluorescence microscopy will be; combined with the cell-free vacuole fusion system to monitor and perturb individual yeast vacuoles as they tether, dock, and fuse. We will focus our efforts on Ypt7p, the vacuole Rab, its Vps-C/HOPS effector complex, and Vam7p, a soluble SNARE. These proteins have critical functions over the entire span of the tethering-to fusion sequence, and many functional and physical interactions link them. The biological questions that we address are straightforward. Does GTP hydrolysis and exchange on the Rab Ypt7p influence the force or reversibility of tethering, or the dynamics of docking junction assembly (Aim 1)? What does the SNARE Vam7p touch as it executes its various functions in docking and fusion (Aim 2)? How is the Vps-C complex organized, and do subunits of this complex undergo structural rearrangements in response to Ypt7p-binding or other events of docking or fusion (Aim 3)? Does Ypt7p "know" when tethering, docking, or fusion have occurred (Aim 4)?

描述（由申请人提供）：内膜系统中的对接和融合事件由RAB GTPase，SNARE蛋白及其辅助因子调节和执行。关于这些蛋白质及其相互作用，已经了解了很多。然而，对于任何细胞内转运步骤，尚未详细了解导致融合的分子事件。解决个别事件而不是事件人群的总体行为的能力增强了许多生物学领域的主要进步。我们认为，如果我们能够在单个对接和融合事件的水平上追踪和扰动膜对接和融合的机械解剖会大大促进。在此提案中，我们将监测单个事件问题的直接攻击与生化和遗传研究结合在一起，这些研究将同时表征关键分子并为单个事项工作产生新的试剂和探针。我们的实验系统，酵母液泡，为这些研究提供了许多实验优势，包括其大小，足够大，可以促进光学显微镜，出现酵母的遗传和基因组工具包以及无细胞的融合系统，在其实验灵活性和现有知识库中无与伦比。这些实验将基于PI在William Wickner博士小组的博士后工作中开发的 - 并实质上扩展了方法。微分化技术和低光定量荧光显微镜将是；与无细胞的液泡融合系统结合使用，以监测和扰动单个酵母液泡的束缚，码头和保险丝。我们将精力集中在YPT7P，Vacuole RAB，其VPS-C/HOPS效应子络合物和可溶性圈套的VAM7P上。这些蛋白质在整个链球融合序列的整个跨度中具有关键功能，许多功能和物理相互作用将它们连接起来。我们解决的生物学问题很简单。 GTP水解和在RAB YPT7P上的交换会影响束缚的力或可逆性，还是对接连接组件的动力学（AIM 1）？ SNARE VAM7P在对接和融合中执行各种功能时触摸什么（AIM 2）？ VPS-C复合物如何组织，并且该复合物的亚基会响应于YPT7P结合或对接或融合的其他事件（AIM 3）而经历结构重排？ YPT7P在束缚，停靠或融合时是否“知道”（AIM 4）？