Structural Analysis of Golgi Trafficking Proteins

描述（由申请人提供）：运输囊泡和其他膜载体建立的运输模式对真核细胞内蛋白质的定位、修饰和功能至关重要。囊泡对接和融合除了需要可溶性NSF附着蛋白受体（SNAREs）外，还需要多亚单位系聚复合物（mtc）。本研究的重点是酵母到哺乳动物的三个MTCs: Dsl1复合物、保守的低聚高尔基（COG）复合物和同型融合和液泡蛋白分选（HOPS）复合物。Dsl1复合体在COPI囊泡从高尔基体运输到内质网（ER）中发挥作用，这是一个对顺行运输机制的再循环和内质网驻留蛋白的回收至关重要的途径。COG复合体在高尔基体内的逆行运输中起作用。因此，COG对于正常的高尔基体结构和功能至关重要，而COG的缺陷会引起先天性糖基化障碍。最后，内体和溶酶体/液泡后期的膜融合取决于啤酒花复合物。人类啤酒花的所有6个亚基都是最近发现的马尔堡和埃博拉病毒进入所需的7种宿主蛋白之一。我们假设mtc通过与Rabs、SNAREs、Sec1/Munc18蛋白、囊泡外壳蛋白和磷脂的相互作用，协调转运囊泡的对接和融合。实现对MTC功能的更深入的机制理解关键取决于阐明它们的结构并确定它们如何与其他元素相互作用

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.