Dynamics of endomembrane docking and fusion

描述(由申请人提供)：内膜系统中的对接和融合事件由Rab GTP酶、SNARE蛋白及其辅因子调节和执行。关于这些蛋白质及其相互作用，人们已经了解了很多。然而，导致融合的分子事件对于任何细胞内运输步骤都没有详细的了解。解决单个事件而不是事件群体的总体行为的能力促进了生物学许多领域的重大进展。我们认为，如果我们能够在单个对接和融合事件的水平上追踪和扰动拴系、对接和融合的亚反应，将极大地促进膜对接和融合的机制剖析。在这项建议中，我们将对单一事件监测问题的直接攻击与生化和遗传学研究相结合，这些研究将同时表征关键分子，并为单一事件的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，包括它的大小足以方便光学显微镜，萌芽酵母的遗传和基因组工具包，以及一个在实验灵活性和现有知识库方面无与伦比的无细胞融合系统。这些实验将建立在威廉·威克纳博士团队的PI博士后工作期间开发的方法上，并在很大程度上扩展。微制造技术和微光定量荧光显微镜将与无细胞空泡融合系统相结合，以监测和扰乱单个酵母空泡在系绳、停靠和融合时的情况。我们将集中精力研究Ypt7p，液泡Rab，其Vps-C/Hop效应复合体，以及Vam7p，一种可溶的陷阱。这些蛋白质在整个系链融合序列中具有关键功能，许多功能和物理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。Rab Ypt7p上的GTP水解和交换是否影响系绳的力或可逆性，或对接接头组件的动力学(目标1)？Snare Vam7p在对接和融合中执行各种功能时会触摸到什么(目标2)？VPS-C复合体是如何组织的，该复合体的亚基是否经历了结构重排，以响应Ypt7p结合或其他对接或融合事件(目标3)？Ypt7p是否“知道”何时发生系绳、对接或融合(目标4)？

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)?