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。运输囊泡和它们的膜靶之间的初始接触似乎需要一组八个或更多个大的异源寡聚体“拴系”复合物中的一个。这项资助计划的重点是两个这样的复合物，从酵母到哺乳动物，称为保守的寡聚高尔基体（COG）复合物和Dsl 1 p复合物。两者都介导COPI囊泡在早期分泌途径中的束缚。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缺陷引起先天性糖基化障碍。Dsl 1 p复合物对于COPI囊泡从高尔基体运输到ER是重要的，这是一种对于顺行运输机械的再循环和ER驻留蛋白的检索至关重要的途径。多亚基拴系复合物的更深层次的机械理解关键取决于确定其三维结构。在最初的资助期间，我们绘制了COG和Dsl 1 p复合物的整体架构，并确定了它们四个亚单位的结构或部分结构。我们现在建议解决更大的问题，在Dsl 1 p的情况下，解决整个复杂的问题。该提案的第二个主要目标是开始对COG和Dsl 1 p复合物与贩运机制的其他组成部分之间的相互作用进行结构表征。从这些努力产生的模型将提供一个基础，产生更深刻的机制假说，这些，也许其他，多亚基拴系复合物。为实现这些目标，我们提出以下具体目标。在第一个目标中，我们将进行主要COG的结构分析，我们以前的COG亚基连接分析的指导下。将使用X射线晶体学和EM的组合来阐明这些晶体的结构。我们还将使用高度优化的质谱方法对COG相互作用蛋白进行公正的搜索。将使用生物化学和结构方法研究COG亚基或coc与功能验证的合作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Dsl 1 p复合物。我们已经确定了晶体结构，代表约50%（按质量计）的Dsl 1 p复合物。我们建议使用X射线晶体学和EM完成此分析。最后，在第三个具体目标中，我们将进行Dsl 1 p复合物与SNARE和外壳蛋白相互作用的结构/功能研究。 公共卫生相关性：高尔基体在真核分泌途径中的蛋白质分选和糖基化中起关键作用。囊泡运输到高尔基体，从高尔基体，和在高尔基体内的缺陷影响其结构和功能。因此，这种缺陷可能对细胞表面蛋白的糖基化和稳定性具有多效性效应，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.