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，对于任何细胞内转运步骤，导致融合的分子事件尚未详细了解。解决单个事件而不是事件群体的聚集行为的能力增强了生物学许多领域的重大进展。我们相信，膜对接和融合的机制解剖将大大促进，如果我们可以跟踪和扰动的子反应的拴系，对接，融合在个人的对接和融合事件的水平。在这个建议中，我们结合联合收割机直接攻击的问题，监测单事件与生化和遗传学的研究，将同时表征关键分子，并产生新的试剂和探针的单事件的工作。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，这是足够大，以方便光学显微镜，芽殖酵母的遗传和基因组工具包，和无细胞融合系统，是无与伦比的实验灵活性和现有的知识基础。这些实验将建立在-并大大扩展-PI在William Wickner博士小组的博士后工作期间开发的方法。微加工技术和低光，定量荧光显微镜将与无细胞空泡融合系统相结合，以监测和扰动单个酵母空泡，因为它们系链，对接和融合。我们将把我们的努力集中在Ypt 7 p，空泡Rab，其Vps-C/HOPS效应复合物，和Vam 7 p，可溶性陷阱。这些蛋白质在整个连接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起来。我们要解决的生物学问题很简单。GTP水解和交换对Rab Ypt 7 p的影响力或可逆性拴系，或对接接头组装的动力学（目的1）？SNARE Vam 7 p在对接和融合中执行其各种功能时接触了什么（目标2）？Vps-C复合物是如何组织的，这种复合物的亚基是否会因Ypt 7 p结合或其他对接或融合事件而发生结构重排（目的3）？Ypt 7 p是否“知道”何时发生了系留、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)?