Structural Analysis of Golgi Trafficking Proteins

高尔基体运输蛋白的结构分析

• 批准号: 8059674
• 负责人: FREDERICK M HUGHSON
• 金额: $ 32.71万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8059674
• 关键词: Affect; Applications Grants; Architecture; Attention; Binding; Biochemical; Biological Assay; Capsid Proteins; Cell Surface Proteins; Cells; Coat Protein Complex I; Complex; Congenital Disorders; Crystallography; Defect; Eukaryotic Cell; Family; Foundations; Funding; Goals; Golgi Apparatus; Grant; Human; Mammals; Maps; Mass Spectrum Analysis; Mediating; Membrane; Methods; Modeling; Modification; Molecular Weight; Pathway interactions; Pattern; Play; Proteins; Recycling; Reporting; Retrieval; SNAP receptor; Sorting - Cell Movement; Structure; Testing; Transport Vesicles; Vesicle; Work; Yeasts; anterograde transport; base; glycosylation; human disease; in vivo; protein transport; public health relevance; three dimensional structure; trafficking

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.

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。运输囊泡和它们的膜目标之间的初始接触似乎需要一组8个或更多的大的异聚寡聚“系带”复合物中的一个。这项拨款提案的重点是两个这样的复合体，从酵母到哺乳动物，称为保守的低聚高尔基（COG）复合体和Dsl1p复合体。两者都在早期分泌途径中介导COPI囊泡的栓系。COG在高尔基体内的COPI囊泡运输中起作用，因此对正常的高尔基复合体结构和功能至关重要。先天性糖基化缺陷引起先天性糖基化障碍。Dsl1p复合物对于从高尔基体到内质网的COPI囊泡运输是重要的，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。对多亚基系链复合物的更深入的机理理解关键取决于确定它们的三维结构。在这项拨款的初始资助期间，我们绘制了COG和Dsl1p复合物的整体结构，并确定了其中四个亚基的结构或部分结构。我们现在建议处理更大的子组件，在Dsl1p的情况下，处理整个复合体。本建议的第二个主要目标是开始对COG和Dsl1p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。由这些努力产生的模型将为产生关于这些，也许还有其他的，多亚基拴系复合体的更深刻的机制假设提供基础。为实现这些目标，我们提出以下具体目标。在第一个目标中，我们将在之前对COG亚单元连通性分析的指导下，对主要COG子组件进行结构分析。这些子组件的结构将使用x射线晶体学和EM的组合来阐明。我们还将使用高度优化的质谱方法对cog相互作用蛋白进行无偏搜索。COG亚基或亚组件与功能验证的伙伴之间的相互作用将使用生化和结构方法进行研究。在第二个具体目标中，我们将注意力转向Dsl1p复合体。我们已经确定了约50%（按质量计）的Dsl1p配合物的晶体结构。我们建议使用x射线晶体学和EM来完成这一分析。最后，在第三个具体目标中，我们将进行Dsl1p复合物与SNAREs和外壳蛋白相互作用的结构/功能研究。