Structural Analysis of Golgi Trafficking Proteins

描述(申请人提供)：运输囊泡和其他膜载体建立的运输模式对于真核细胞内蛋白质的定位、修饰和功能是至关重要的。囊泡的对接和融合除了需要可溶的NSF附着蛋白受体(SNARs)外，还需要多亚单位系留复合体(MTCs)。这一建议集中在三个MTC上，从酵母到哺乳动物都是保守的：DSL1复合体，保守的寡聚体高尔基体(COG)复合体，以及同型融合和空泡蛋白分类(HOPS)复合体。DSL1复合体在COPI囊泡从高尔基体到内质网(ER)的运输中发挥作用，这是顺行运输机制和内质网驻留蛋白回收所必需的途径。COG复合体在高尔基体逆行运输中起作用。因此，COG对于正常的高尔基体结构和功能是必不可少的，COG缺陷会导致先天性糖基化障碍。最后，内小体和溶酶体/液泡晚期的膜融合依赖于啤酒花复合体。人类啤酒花的所有六个亚基都是最近发现的马尔堡和埃博拉病毒进入所需的七种宿主蛋白之一。我们推测，MTCs通过与RABS、SNARES、Sec1/Munc18蛋白、囊泡外壳蛋白和磷脂的相互作用，发挥协调运输囊泡的对接和融合的功能。要从机制上更深入地理解MTC的功能，关键取决于阐明它们的结构，并确定它们如何与 贩卖人口的机器。为此，我们提出了三个具体目标。在目标1中，我们将使用X射线结晶学和单粒子电子显微镜(EM)来表征DSL1复合体与其他转运因子之间的功能相互作用。此外，我们将利用 通过设计突变体，用于蛋白质组学和合成遗传筛选，以寻找更多与DSL1相互作用的伙伴，从而对我们的DSL1复合体的完整结构进行研究。在目标2中，我们将使用单粒子EM来完成我们将八个不同的亚基映射到COG复合体的整体结构中，并辅之以对COG亚组件中相互作用元素的X射线结晶学研究。此外，我们将确定COG-SNARE复杂结构，以阐明COG引导如何诱捕组装。最后，在目标3中，我们提出了一个全新的项目，即啤酒花复合体的结构研究及其与陷阱的相互作用。我们将确定关键的啤酒花亚基和亚组件的结构，然后作为体内和体外功能研究的蓝图。此外，与前两个目标一样，w将使用x射线结晶学来研究具有陷阱的络合物。由于啤酒花复合体与DSL1和COG复合体无关，这项工作应该揭示MTC之间的类别差异和共同原则，从而加深我们对细胞内转运机制的这些有趣组成部分的机械理解。

DESCRIPTION (provided by applicant): The traffic patterns established by transport vesicles and other membrane carriers are of fundamental importance for protein localization, modification, and function within eukaryotic cells. Vesicle docking and fusion requires, in addition to soluble NSF attachment protein receptors (SNAREs), multisubunit tethering complexes (MTCs). This proposal focuses on three MTCs, conserved from yeast to mammals: the Dsl1 complex, the conserved oligomeric Golgi (COG) complex, and the homotypic fusion and vacuole protein sorting (HOPS) complex. The Dsl1 complex functions in COPI vesicle transport from the Golgi apparatus to the endoplasmic reticulum (ER), a pathway essential for the recycling of the anterograde transport machinery and the retrieval of ER-resident proteins. The COG complex functions in retrograde transport within the Golgi. As a result, COG is essential for normal Golgi structure and function, and defects in COG give rise to congenital disorders of glycosylation. Finally, membrane fusion at late endosomes and lysosomes/vacuoles depends on the HOPS complex. All six subunits of human HOPS are among the seven host proteins recently discovered to be required for Marburg and Ebola virus entry. We hypothesize that MTCs, through interactions with Rabs, SNAREs, Sec1/Munc18 proteins, vesicle coat proteins, and phospholipids, function to orchestrate the docking and fusion of transport vesicles. Achieving a deeper mechanistic understanding of MTC function depends critically on elucidating their structures and determining how they interact with other elements of the trafficking machinery. To this end, we propose three specific aims. In Aim 1, we will characterize functional interactions between the Dsl1 complex and other trafficking factors using x-ray crystallography and single particle electron microscopy (EM). In addition, we will capitalize on our complete structure of the Dsl1 complex by designing mutants to use in proteomic and synthetic genetic screens for additional Dsl1-interacting partners. In Aim 2, we will use single-particle EM to complete our mapping of the eight different subunits into the overall structure of the COG complex, complemented by x-ray crystallographic studies of interacting elements within COG sub- assemblies. Furthermore, we will determine COG-SNARE complex structures in order to elucidate how COG guides SNARE assembly. Finally, in Aim 3 we propose an entirely new project, structural studies of the HOPS complex and its interaction with SNAREs. We will determine structures of key HOPS subunits and sub- assemblies, which can then serve as blueprints for in vivo and in vitro functional studies. In addition, as in the first two Aims, w will use x-ray crystallography to study complexes with SNAREs. Because the HOPS complex is unrelated to the Dsl1 and COG complexes, this work should reveal both class-specific differences and common principles among MTCs, thereby deepening our mechanistic understanding of these fascinating components of the intracellular trafficking machinery.