Structural Analysis of Golgi Trafficking Proteins

描述（由申请人提供）：由转运囊泡和其他膜载体建立的运输模式对于真核细胞内的蛋白定位、修饰和功能至关重要。囊泡对接和融合需要，除了可溶性NSF附着蛋白受体（SNARE），多亚基拴系复合物（MTCs）。该建议侧重于三个MTCs，从酵母到哺乳动物保守：Dsl 1复合体，保守的寡聚高尔基体（COG）复合体，同型融合和液泡蛋白分选（HOPS）复合体。Dsl 1复合物在COPI囊泡从高尔基体运输到内质网（ER）中发挥作用，这是一种对顺行运输机制的再循环和ER驻留蛋白的检索至关重要的途径。COG复合物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是必需的，并且COG中的缺陷引起先天性糖基化障碍。最后，晚期内体和溶酶体/空泡处的膜融合取决于HOPS复合物。人类HOPS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质之一。我们假设，MTCs，通过与Rabs，SNARE，Sec 1/Munc 18蛋白，囊泡外壳蛋白和磷脂的相互作用，协调运输囊泡的对接和融合。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。 贩卖机器。为此，我们提出三个具体目标。在目标1中，我们将使用X射线晶体学和单粒子电子显微镜（EM）表征Dsl 1复合物和其他运输因子之间的功能相互作用。此外，我们将利用 我们的完整结构的Dsl 1复杂的突变体设计用于蛋白质组学和合成遗传筛选额外的Dsl 1相互作用的合作伙伴。在目标2中，我们将使用单粒子EM来完成我们将八个不同亚基映射到COG复合物的整体结构中，并辅之以COG子组件内相互作用元素的X射线晶体学研究。此外，我们将确定COG-SNARE复杂的结构，以阐明COG如何指导SNARE组装。最后，在目标3中，我们提出了一个全新的项目，HOPS复合体的结构研究及其与SNARE的相互作用。我们将确定关键HOPS亚基和亚组装体的结构，然后可以作为体内和体外功能研究的蓝图。此外，与前两个目标一样，我们将使用X射线晶体学研究与SNARE的复合物。由于HOPS复合物与Dsl 1和COG复合物无关，因此这项工作应揭示MTCs之间的类特异性差异和共同原则，从而加深我们对这些迷人的细胞内贩运机制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.