Project 1- Reconstitution of SNAR-mediated Fusion with Native and Artificial Mem

突触囊泡的胞吐膜融合由SNARE突触融合蛋白SNAP-25和 突触泡蛋白/VAMP，它们以拉链样的方式在膜之间反式组装，从而拉动 膜结合在一起，克服了聚变的能量障碍。此外，调节蛋白 包括synaptotagmin、Munc 18和complexin与SNARE和参与SNARE的细胞相互作用。 膜，从而控制SNARE反应性和塑造钙依赖性的具体特征， 突触囊泡的胞吐作用。虽然拉链模型直观上令人满意，并得到了大量的证实， 大量的证据，蛋白质-蛋白质相互作用的精确序列和调节蛋白质相互作用的作用位点， 蛋白质沿着融合途径仍然只是部分理解和有争议的讨论。 在这里，我们使用天然分泌囊泡和用纯化蛋白质重构的脂质体的体外融合， 分离融合途径的部分反应，以了解结构，动力学和化学计量 参与蛋白质的中间状态，并确定影响动力学的参数， 每个反应。在上一个资助期，我们解决了神经元SNARE的晶体结构 复合物与其接头和跨膜结构域，并表征了突触结合蛋白的相互作用 含SNARE的脂质体。此外，我们开发了精确的测量融合的方法， 中间体，包括荧光互相关和冷冻电子显微镜，研究了融合 突触囊泡与含SNARE的脂质体，并干扰SNARE组装在部分或 完全拉链状态，从而获得融合途径的中间状态。我们现建议 利用这些成就来表征SNARE介导的融合中的中间步骤 并确定调节蛋白synaptotagmin 1、复合蛋白1和 Munc 18 -1对神经元SNARE催化的膜融合起作用。在第一个具体目标中，我们计划 阐明SNARE组装和拉链如何连接到膜对接、半融合和融合 使用天然和人工囊泡。在第二个具体目标中，我们将确定SNARE的步骤 成核和拉链作用的调节蛋白synaptotagmin，MunclS，和 复合蛋白该计划将与项目2和项目3密切合作， 采取了补充办法。我们期望获得关于 神经元胞吐作用的分子机制，这是无法通过其他方法获得的。

Exocytotic membrane fusion of synaptic vesicles is driven by the SNAREs syntaxin, SNAP-25, and synaptobrevin/VAMP, which assemble in trans between the membranes in a zipper-like fashion, thus pulling the membranes together and overcoming the energy barrier for fusion. In addition, regulatory proteins including synaptotagmin, Munc18, and complexin interact both with the SNAREs and the participating membranes, thus controlling SNARE reactivity and shaping the specific features of cacium-dependent exocytosis of synaptic vesicles. While the zippering model is intuitively satisfying and confirmed by a large body of evidence, the precise sequence of protein-protein interactions and the site of action of the regulatory proteins along the fusion pathway is still only partially understood and controversially discussed. Here we use in-vitro fusion of native secretory vesicles and liposomes reconstituted with purified proteins to isolate partial reactions of the fusion pathway, to understand the structure, dynamics and stoichiometries of the intermediate states of the participating proteins, and to determine the parameters affecting kinetics of each reaction. In the previous funding period, we solved the ci7stal structure of the neuronal SNARE complex with its linkers and transmembrane domains and characterized the interaction of synaptotagmin with SNARE-containing liposomes. Moreover, we developed refined assays for measuring fusion intermediates including fluorescence cross-correlation and cryo electron microscopy, studied the fusion of synaptic vesicles with SNARE-containing liposomes, and interfered with SNARE assembly at the partially or fully zippered state, thus gaining access to intermediate states of the fusion pathway. We now propose to take advantage of these achievements to characterize the intermediate steps in SNARE-mediated fusion and to determine the precise step at which the regulatory proteins synaptotagmin 1, complexin 1, and Munc18-1 operate on membrane fusion catalyzed by neuronal SNAREs. In the first specific aim, we plan to elucidate how SNARE assembly and zippering is connected to membrane docking, hemifusion, and fusion using native and artificial vesicles. In the second specific aim, we will determine the steps in SNARE nucleation and zippering that are acted upon by the regulatory proteins synaptotagmin, MunclS, and complexin. The program will be carried out in close collaboration with Projects 2 and 3 in which complementary approaches are pursued. We expect to obtain essential mechanistic information about the molecular mechanism of neuronal exocytosis that is not obtainable by other approaches..