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。它是中央， 神经传递，免疫信号传导和正常发育，并在广泛的 传染病和退化性疾病。转运囊泡与靶膜的融合是 许多运输过程，并采用核心和保守的机制：三个或四个陷阱蛋白， 分解伴侣Sec 17（α-SNAP）和Sec 18（NSF），以及SM蛋白（Sec 1/Munc 18） 家人SM蛋白功能的机制尚不清楚，尽管有几种SM蛋白是已知的。 与人类感染性疾病、神经变性、中性粒细胞减少症和糖尿病相关。总目标 本项目的目的是仔细研究新出现的假说，即SM功能的生化基础不需要 只有SM-SNARE相互作用，但一个优雅的网络的物理和功能的相互作用， SNARE，SM，Sec 17和Sec 18，在紧密耦合的组装和拆卸反应中操作。大部分 最近关于SM蛋白和Sec 17的工作是在体外进行的。该项目结合了强大的体外 生物化学方法与目前可行的最先进的体内结构-功能分析 只存在于酿酒酵母中。在目的1中，SM蛋白活化和活性的机制是 在体内和体外进行评估。在目标2中，Sec 17与SNARE、Sec 18和特定SM的相互作用是 评估。在目标3中，应用生物物理技术来阐明SM组装的结构， 与Sec 17一起。这些研究将测试关于保守的 SNARE介导的膜融合的机制，特别强调两个之间的比较 不同的转运步骤，以及体外结果与严格和定量的体内结构之间的差异- 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.