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Understanding the mechanism of membrane fusion: How does the vacuolar tethering complex HOPS initiate the first contact between membranes? And by which means does it integrate SNARE, Rab, and lipidic functions in membrane fusion?

了解膜融合的机制：液泡束缚复合体 HOPS 如何启动膜之间的第一次接触？

基本信息

批准号：
196590410
负责人：
Dr. Michael Zick
金额：
--
依托单位：
Department of Biochemistry and Cell Biology
依托单位国家：
德国
项目类别：
Research Fellowships
财政年份：
2011
资助国家：
德国
起止时间：
2010-12-31 至 2012-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/196590410?language=en
关键词：
Understanding mechanism membrane fusion How

项目摘要

Eukaryotic cells are compartmentalized by their endomembrane system. This spatial separation of cellular functions prevents undesired interference between different metabolic pathways, and necessitates the regulated transport of proteins, lipids, and solutes between organelles. Small vesicles containing cargo molecules bud from each donor compartment, travel across the cytosol, and specifically dock and fuse with the membrane of the target organelle. Yeast vacuole fusion is a perfectly established model to study this process of membrane fusion. As the core components of this fusion machinery have been discovered, it is now possible to address a series of questions on how they mechanistically achieve the merging of two distinct membranes. This proposal focuses on the initial step of membrane fusion: "tethering". The analysis of other membrane fusion systems revealed a great structural and functional variability of the tethering complexes. For yeast vacuole fusion, I will determine how the tethering complex HOPS initiates the first contacts between membranes prior to fusion. To this end, I intend to:(1) Determine the stoichiometric composition of the fundamental tethering unit.(2) Employ cross-linking techniques to map the direct molecular interactions that underlie tethering.(3) Establish a novel tethering assay to quantify the dynamics of tethering and the role(s) of other factors such as phosphoinositides and SNAREs in this process.(4) Quantify the kinetic constants of the tethering interactions to determine whether there is synergy amongst the individual components.(5) Relate these interactions to the overall fusion process through both in-vitro and in-vivo assays.

真核细胞由内膜系统分隔。细胞功能的这种空间分离防止了不同代谢途径之间的不期望的干扰，并且需要调节蛋白质、脂质和溶质在细胞器之间的转运。含有货物分子的小囊泡从每个供体隔室出芽，穿过细胞质，并特异性地与靶细胞器的膜对接和融合。酵母液泡融合是研究膜融合过程的理想模型。随着这种融合机制的核心组件被发现，现在有可能解决一系列关于它们如何机械地实现两种不同膜的合并的问题。该提案集中在膜融合的初始步骤：“拴系”。其他膜融合系统的分析揭示了一个很大的结构和功能的变化的拴系复合物。对于酵母液泡融合，我将确定如何拴系复合物HOPS启动融合前膜之间的第一次接触。为此，我打算：（1）确定基本拴系单元的化学计量组成。(2)采用交联技术来绘制构成拴系基础的直接分子相互作用。(3)建立一种新的拴系试验，以量化拴系的动力学以及其他因素（如磷酸肌醇和SNARE）在此过程中的作用。(4)量化拴系相互作用的动力学常数，以确定各个组分之间是否存在协同作用。(5)通过体外和体内试验将这些相互作用与整体融合过程联系起来。