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?

真核细胞是由它们的膜系统划分的。细胞功能的这种空间分离防止了不同代谢途径之间的不希望的干扰，并且需要在细胞器之间调节蛋白质、脂质和溶质的运输。含有货物分子的小囊泡从每个供体隔室中冒出，穿过细胞质，特异性地与目标细胞器的膜对接并融合。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融合的问题。这个建议的重点是膜融合的第一步：“系住”。对其他膜融合系统的分析表明，系带复合物具有很大的结构和功能变异性。对于酵母液泡融合，我将确定捆扎复合物啤酒花如何在融合前启动膜之间的第一次接触。为此，我打算：(1)确定基本系带单元的化学计量组成。(2)采用交联技术来绘制直接分子相互作用的图谱。(3)建立一种新的栓系测定方法，以量化栓系动力学和其他因素（如磷酸肌苷和SNAREs）在这一过程中的作用。(4)量化系缚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)通过体外和体内实验将这些相互作用与整体融合过程联系起来。

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.