Dynamics of Endomembrane Docking and Fusion

项目概要 膜运输是真核细胞最古老的创新之一。它的核心是 神经传递、免疫信号传导和正常发育，并在多种方面受到干扰 传染病和退行性疾病。运输囊泡与目标膜的融合是核心 许多贩运过程并采用核心且保守的机制：三个或四个 SNARE 蛋白， 分解分子伴侣 Sec17 (α-SNAP) 和 Sec18 (NSF) 以及 SM 蛋白 (Sec1/Munc18) 家庭。 SM 蛋白的功能机制尚不清楚，尽管有几种 SM 蛋白已被研究 与人类传染病、神经退行性疾病、中性粒细胞减少症和糖尿病有关。总体目标 该项目的目的是仔细研究新出现的假设，即 SM 功能的生化基础并不需要 只有 SM-SNARE 交互，而是一个优雅的物理和功能交互网络 SNARE、SM、Sec17 和 Sec18，在紧密耦合的组装和拆卸反应中运行。很多 最近关于SM蛋白和Sec17的工作是在体外进行的。该项目结合了强大的体外 具有目前可行的最先进的体内结构功能分析的生化方法 仅存在于酿酒酵母中。在目标 1 中，SM 蛋白激活和活性的机制是 体内和体外评估。在目标 2 中，Sec17 与 SNARE、Sec18 和特定 SM 的交互是 评估。在目标 3 中，应用生物物理技术来阐明 SM 组装的架构 SNARE 和 Sec17。这些研究将检验关于保守性的一般和深层假设 SNARE 介导的膜融合机制，特别强调两者之间的比较 不同的运输步骤，以及体外结果和严格定量的体内结构之间—— 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.