Mechanisms of Membrane Fusion

项目总结/摘要 膜融合对于细胞生长、激素分泌、神经传递， 和病原体的细胞入侵。膜融合机制是保守的， 酵母对人类我们已经开发了酵母液泡融合作为模型系统， 鉴定用于膜融合的基因，用纯化的膜融合蛋白建立体外融合测定， 空泡，并纯化每种相关的蛋白质和脂质，用于重建成 蛋白脂质体忠实地重建融合的各个方面。这些研究 揭示了新的元素，最近：1。令人眼花缭乱的蛋白质和脂质， 配合有序的脂双层应变和重排，产生融合而不裂解。 2.被称为"SNARE"的膜结合蛋白质之间的复合物组装 并不是自发的它们的组装实际上是由一种 一种称为HOPS的大型六聚体复合物，它识别每个个体， 捕捉并将它们组装成活性中间体，准备快速融合。3. SNARE的伴侣，称为NSF/Sec18和aSNAP/Sec17， 据信，在融合后仅起分解SNARE复合物的作用， 核聚变4.脂质在融合中起着至关重要的积极作用。每一个机械的 我们将追求的洞察力;我们的目标，在未来5年是了解 HOPS催化SNARE组装的中间体，它们的结构， 分子伴侣Sec17/Sec18，以及这些蛋白质如何触发脂质重排， 核聚变理解这一途径的重要性是强调的核心作用， HOPS在致病病毒和细菌入侵人体细胞中的作用。

Project Summary/Abstract Membrane fusion is essential for cell growth, hormone secretion, neurotransmission, and cell invasion by pathogens. Membrane fusion mechanisms are conserved from yeast to humans. We have developed yeast vacuole fusion as a model system, identifying genes for membrane fusion, establishing an in vitro fusion assay with purified vacuoles, and purifying each relevant protein and lipid for reconstitution into proteoliposomes which faithfully reconstitute each aspect of fusion. These studies have revealed novel elements, most recently: 1. A dazzling array of proteins and lipids which cooperate for orderly lipid bilayer strain and rearrangement, giving fusion without lysis. 2. The assembly of complexes among membrane-bound proteins termed "SNAREs" isn't spontaneous, as heretofore believed. Their assembly is actually catalyzed by a large hexameric complex termed HOPS, which recognizes each of the individual SNAREs and assembles them in active intermediates, poised for rapid fusion. 3. Chaperones to the SNAREs, termed NSF/Sec18 and aSNAP/Sec17, which had been believed to only function to disassemble SNARE complexes after fusion, also promote fusion. 4. Lipids have a vital and active role in fusion. Each of these mechanistic insights will be pursued; our goals for the next 5 years are to understand the intermediates of HOPS-catalyzed SNARE assembly, their structures, the roles of chaperones Sec17/Sec18, and how these proteins trigger the lipid rearrangements of fusion. The importance of understanding this pathway is underscored by the central role of HOPS in the invasion of human cells by pathogenic viruses and bacteria.