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。转运囊泡与其膜靶标之间的初始接触似乎需要八个或更多个大型异寡聚“系留”复合体中的一个。这项拨款建议集中在两个这样的复合体上，从酵母到哺乳动物都是保守的，称为保守的寡聚体高尔基(COG)复合体和Dsl1p复合体。两者都介导了早期分泌途径中COPI囊泡的拴系。COG在高尔基体内COPI囊泡的运输中起作用，因此对于正常的高尔基体复合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。Dsl1p复合体对于COPI囊泡从高尔基体到内质网的运输是重要的，这是顺行运输机制和内质网驻留蛋白回收所必需的途径。对多亚基连接复合体的更深层次的机械理解关键取决于确定它们的三维结构。在这笔赠款的初始资助期内，我们绘制了COG和DSL1P复合体的总体结构图，并确定了它们四个亚单位的结构或部分结构。我们现在提议处理更大的部件，在DSL1P的情况下，处理整个建筑群。这项提议的第二个主要目标是开始确定COG和DSL1P复合体与贩运机制其他组成部分之间的相互作用的结构特征。这些努力产生的模型将为产生关于这些或其他多亚单位系留复合体的更深刻的机制假说提供基础。为实现这些目标，我们提出了以下具体目标。在第一个目标中，我们将在之前对COG亚单位连接性的分析的指导下，对主要的COG亚基进行结构分析。将结合X射线结晶学和EM来阐明这些亚组分的结构。我们还将使用高度优化的质谱学方法，对COG相互作用蛋白进行无偏见的搜索。将使用生化和结构方法研究COG亚基或亚组分与功能验证的伙伴之间的相互作用。在第二个具体目标中，我们将注意力转向Dsl1p情结。我们已经确定了约50%(质量)的Dsl1p络合物的晶体结构。我们建议使用X射线结晶学和EM来完成这一分析。最后，在第三个具体目标中，我们将开展Dsl1p与SNARS和外壳蛋白相互作用的结构/功能研究。 公共卫生相关性：高尔基体在真核细胞分泌途径中的蛋白质分选和糖基化中起着关键作用。囊泡运输到高尔基体、来自高尔基体和高尔基体内部的缺陷会影响高尔基体的结构和功能。因此，这种缺陷可能会对细胞表面蛋白的糖基化和稳定性产生多效性影响，从而导致人类疾病。

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. The initial contact between transport vesicles and their membrane targets appears to require one of a set of eight or more large hetero-oligomeric `tethering' complexes. This grant proposal focuses on two such complexes, conserved from yeast to mammals, called the conserved oligomeric Golgi (COG) complex and the Dsl1p complex. Both mediate the tethering of COPI vesicles in the early secretory pathway. COG functions in the transport of COPI vesicles within the Golgi apparatus and is therefore essential for normal Golgi complex structure and function. COG defects give rise to congenital disorders of glycosylation. The Dsl1p complex is important for COPI vesicle transport from the Golgi to the ER, a pathway essential for the recycling of the anterograde transport machinery and the retrieval of ER resident proteins. A deeper mechanistic understanding of multisubunit tethering complexes depends critically on determining their three-dimensional structures. During the initial funding period for this grant, we mapped the overall architecture of the COG and Dsl1p complexes and determined the structures or partial structures of four of their subunits. We now propose to tackle larger subassemblies and, in the case of Dsl1p, the entire complex. A second major goal of this proposal is to initiate structural characterization of the interactions between the COG and Dsl1p complexes and other components of the trafficking machinery. The models resulting from such efforts will provide a foundation for generating more incisive mechanistic hypotheses regarding these, and perhaps other, multisubunit tethering complexes. To accomplish these goals, we propose the following specific aims. In the first aim, we will undertake structural analysis of major COG subassemblies, guided by our previous analysis of COG subunit connectivity. Structures of these subassemblies will be elucidated using a combination of x-ray crystallography and EM. We will also conduct an unbiased search for COG-interacting proteins using a highly optimized mass spectrometry approach. Interactions between COG subunits or subassemblies and functionally validated partners will be investigated using biochemical and structural methods. In the second specific aim, we turn our attention to the Dsl1p complex. We have determined crystal structures representing about 50% (by mass) of the Dsl1p complex. We propose to complete this analysis using x-ray crystallography and EM. Finally, in the third specific aim, we will carry out structure/function studies of Dsl1p complex interactions with SNAREs and coat proteins. PUBLIC HEALTH RELEVANCE: The Golgi apparatus plays a key role in protein sorting and glycosylation within the eukaryotic secretory pathway. Defects in vesicular trafficking to, from, and within the Golgi affect both its structure and function. As a consequence, such defects can have pleiotropic effects on the glycosylation and stability of cell surface proteins, leading to human disease.