Dynamics of endomembrane docking and fusion

内膜对接和融合的动力学

• 批准号: 7078232
• 负责人: Alexey Jarrell Merz
• 金额: $ 28.87万
• 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/7078232
• 关键词: Saccharomyces cerevisiae; fluorescence microscopy; fungal genetics; fungal proteins; guanosinetriphosphatases; intracellular membranes; intracellular transport; membrane activity; membrane fusion; membrane proteins; vesicle /vacuole

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）：膜系统中的对接和融合事件由Rab GTPases、SNARE蛋白及其辅助因子调控和执行。关于这些蛋白质及其相互作用，我们已经了解了很多。然而，导致融合的分子事件并没有详细了解任何细胞内运输步骤。解决单个事件的能力，而不是事件群体的总体行为，已经在生物学的许多领域取得了重大进展。我们认为，如果我们能够在单个对接和融合事件的水平上跟踪和干扰系缚、对接和融合的子反应，将大大促进膜对接和融合的机械解剖。在这个建议中，我们将对监测单一事件的问题的直接攻击与生化和遗传学研究结合起来，这些研究将同时表征关键分子并产生用于单一事件工作的新试剂和探针。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，包括它的大小，它足够大，便于光学显微镜，出芽酵母的遗传和基因组工具包，以及在实验灵活性和现有知识基础方面无与伦比的无细胞融合系统。这些实验将建立在威廉·威克纳博士的团队在PI博士后工作期间开发的方法的基础上，并在很大程度上扩展。微加工技术和微光定量荧光显微镜；结合无细胞液泡融合系统，监测和干扰单个酵母液泡，因为它们系住，对接和融合。我们将把重点放在液泡Rab的Ypt7p、它的Vps-C/HOPS效应复合物和Vam7p（一种可溶性SNARE）上。这些蛋白质在从系索到融合的整个过程中起着至关重要的作用，许多功能和物理上的相互作用将它们联系在一起。我们要解决的生物学问题很简单。GTP在Rab Ypt7p上的水解和交换是否会影响系绳的力或可逆性，或对接结装配的动力学（Aim 1）？当SNARE Vam7p在对接和融合中执行各种功能时，它会接触什么（目标2）？Vps-C复合物是如何组织的，该复合物的亚基是否会在ypt7p结合或其他对接或融合事件中进行结构重排（Aim 3）？Ypt7p是否“知道”何时发生了捆绑、对接或融合（目标4）？