Dynamics of Endomembrane Docking and Fusion

项目摘要 膜运输是真核细胞最古老的创新之一。它是最重要的 神经传递、免疫信号和正常发育，并在广泛的一系列 传染病和退化性疾病。转运囊泡与靶膜的融合是 许多贩运过程和使用一个核心和保守的机制：三到四个圈套蛋白， 拆解伴侣Sec17(α-SNAP)和Sec18(Nsf)以及SM的蛋白质(Sec1/Munc18) 一家人。SM蛋白的功能机制尚不清楚，尽管有几种SM蛋白 与人类传染病、神经变性、中性粒细胞减少症和糖尿病有关。总目标 本项目的主要目的是仔细研究一种新的假设，即SM功能的生化基础不需要 只有SM-SNARE交互，而是一个优雅的物理和功能交互网络 SNARS、SMS、Sec17和Sec18，在紧密耦合的组装和拆卸反应中运行。大部分 最近关于SM蛋白和Sec17的工作已经在体外完成。该项目在体外结合了强大的 目前可行的具有最先进的体内结构-功能分析的生化方法 仅在酿酒酵母中存在。在目标1中，SM蛋白的激活和活性的机制是 在体内和体外进行评估。在目标2中，Sec17与SNARS、Sec18和特定Sms的交互是 评估过了。在目标3中，应用生物物理技术来阐明SM组装的结构 斯奈尔斯和第17节。这些研究将检验关于保守者的普遍和深入的假设 SNARE介导的膜融合的机制，特别强调比较两种 不同的转运步骤，以及在体外结果和严格的体内定量结构之间- 功能分析。

Project Summary Membrane traffic is among the most ancient innovations of eukaryotic cells. It is central to neurotransmission, immune signaling, and normal development, and disrupted in a wide array of infectious and degenerative diseases. The fusion of transport vesicles with target membranes is central to many trafficking processes and employs a core and conserved machinery: three or four SNARE proteins, the disassembly chaperones Sec17 (α-SNAP) and Sec18 (NSF), and proteins of the SM (Sec1/Munc18) family. The mechanism of SM protein function is not understood, though several SM proteins are associated with human infectious disease, neurodegeneration, neutropenia, and diabetes. The overall goal of this Project is to scrutinize the emerging hypothesis that the biochemical basis of SM function entails not only SM–SNARE interactions but an elegant network of physical and functional interactions among SNAREs, SMs, Sec17, and Sec18, operating in tightly coupled assembly and disassembly reactions. Much of the recent work on SM proteins and Sec17 has been done in vitro. This Project combines powerful in vitro biochemical approaches with state-of-the-art in vivo structure–function analyses that are currently feasible only in Saccharomyces cerevisiae. In Aim 1, the mechanisms of SM protein activation and activity are assessed in vivo and in vitro. In Aim 2, Sec17 interactions with SNAREs, Sec18, and specific SMs are assessed. In Aim 3, biophysical techniques are applied to elucidate the architecture of SM assembly with SNAREs and with Sec17. These studies will test general and deep hypotheses about the conserved mechanisms of SNARE-mediated membrane fusion, with particular emphasis on comparisons between two different transport steps, and between in vitro results and stringent and quantitative in vivo structure– function analyses.