Structural Analysis of Membrane Tethering and Fusion Proteins

项目概要/摘要 由运输囊泡建立的运输模式对于蛋白质定位是至关重要的， 修饰和在真核细胞内的功能。由这些囊泡运输的货物通过 囊泡与靶细胞器的膜融合，或在胞吐作用的情况下，与血浆融合 膜的膜融合是由连接囊泡和靶点的SNARE复合物完成的 膜。这些复合物的形成需要四种不同的SNARE蛋白，锚定在两个 不同的膜，经历耦合折叠和组装反应，在此期间，陷阱图案拉链 形成平行的四螺旋束。这个复杂的过程在体外是低效的，并且肯定是均匀的。 在体内更具挑战性，它必须与各种非同源和非同源途径的形成竞争， 陷阱情结。我们假设细胞中的SNARE复合物组装反应是由 “拓扑感知”分子伴侣，称为多亚基系链复合物（MTCs）。我们还建议 催化四螺旋束形成的关键任务福尔斯落在Sec 1/Munc 18（SM）蛋白上， 与MTCs一起，有时作为MTCs的整体亚基。因此，这一总体目标 建议是实现对MTC和SM功能的改进的结构和机制理解， 特别是当它们彼此相关时，在膜融合SNARE复合物的组装中。目标1是 专注于SM蛋白，目的是表征它们在SNARE复合物中的精确催化作用 组装件.主要通过使用X射线晶体学和互补单分子光学 利用光镊实验，我们将确定SM-束缚SNARE的结构和热力学稳定性 组装中间体。在目标2和3中，我们扩大了重点，将多边贸易中心包括在内。在目标2中，我们将研究 最简单的已知MTC，酵母Dsl 1复合物，及其与SNARE和SM蛋白Sly 1的相互作用。 与Dsl 1复合物和Sly 1复合的捕获SNARE组装中间体的Cryo-EM研究 旨在揭示Dsl 1复合体和Sly 1如何协作。在目标3中，我们将把注意力转向 同型融合和液泡蛋白分选（HOPS）复合物，一种研究充分的MTC， 晚期内体和溶酶体/空泡。重要的是，HOPS含有SM蛋白作为整合亚基， 使其成为研究MTC-SM协作的理想系统。为了阐明HOPS如何组织 SNARE的组装，我们将扩大我们正在进行的冷冻电镜研究的HOPS，包括结合SNARE， 陷阱组装中间体。总的来说，这项研究计划有可能彻底改变我们的 对伴侣SNARE复合物组装的机械理解，对 阐明了不同的生物过程及其在感染和疾病期间的颠覆。虽然拟议的 这项工作的基础性大于应用性，它将为操纵贩运和其他非法活动奠定基础。 需要膜融合的过程，具有潜在的未来应用于治疗干预。

PROJECT SUMMARY / ABSTRACT The traffic patterns established by transport vesicles are of fundamental importance for protein localization, modification, and function within eukaryotic cells. Cargo transported by these vesicles is delivered through the fusion of the vesicle with the membrane of a target organelle or, in the case of exocytosis, the plasma membrane. Membrane fusion is executed by SNARE complexes that bridge the vesicle and target membranes. The formation of these complexes requires that four different SNARE proteins, anchored in two different membranes, undergo a coupled folding and assembly reaction during which the SNARE motifs zipper up into a parallel four-helix bundle. This complicated process is inefficient in vitro, and is certain to be even more challenging in vivo, where it must compete with the formation of various non-cognate and off-pathway SNARE complexes. We hypothesize that SNARE complex assembly reactions in the cell are orchestrated by `topologically aware' chaperones called multisubunit tethering complexes (MTCs). We furthermore propose that the key task of catalyzing four-helix bundle formation falls to the Sec1/Munc18 (SM) proteins, working together with—and sometimes as integral subunits of—the MTCs. Therefore, the overarching goal of this proposal is to achieve an improved structural and mechanistic understanding of MTC and SM function, especially as they relate to one another, in the assembly of membrane fusogenic SNARE complexes. Aim 1 is focused on SM proteins with the goal of characterizing their precise catalytic role in SNARE complex assembly. Principally through the use of X-ray crystallography and complementary single-molecule optical tweezers experiments, we will determine the structures and thermodynamic stabilities of SM-bound SNARE assembly intermediates. In Aims 2 and 3, we broaden our focus to include MTCs. In Aim 2, we will investigate the simplest known MTC, the yeast Dsl1 complex, and its interactions with SNAREs and the SM protein Sly1. Cryo-EM studies of arrested SNARE assembly intermediates in complex with both the Dsl1 complex and Sly1 are designed to reveal how the Dsl1 complex and Sly1 collaborate. In Aim 3, we will turn our attention to the homotypic fusion and vacuole protein sorting (HOPS) complex, a well-studied MTC that is required for fusion at late endosomes and lysosomes/vacuoles. Importantly, HOPS contains an SM protein as an integral subunit, making it an ideal system for studying MTC–SM collaboration. In order to elucidate how HOPS organizes SNAREs for assembly, we will expand our ongoing cryo-EM studies of HOPS to include bound SNAREs and SNARE assembly intermediates. Overall, this research program has the potential to revolutionize our mechanistic understanding of chaperoned SNARE complex assembly, with potentially profound implications for elucidating diverse biological processes and their subversion during infection and disease. While the proposed work is more fundamental than applied, it will lay a foundation for efforts to manipulate trafficking and other processes entailing membrane fusion, with potential future applications to therapeutic intervention.