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）：传输囊泡和其他膜载体建立的交通模式对于真核细胞内的蛋白质定位，修饰和功能至关重要。传输囊泡及其膜靶之间的初始接触似乎需要八个或更多大的异源式“绑扎”配合物之一。该赠款提案的重点是从酵母到哺乳动物保守的两个这样的络合物，称为保守的寡聚高尔基（COG）复合物和DSL1P复合物。两者都介导了早期分泌途径中的Copi囊泡的束缚。 COG在高尔基体内的Copi囊泡运输中起作用，因此对于正常高尔基体的复杂结构和功能至关重要。 COG缺陷导致糖基化的先天性疾病。 DSL1P复合物对于从高尔基体到ER的Copi囊泡运输非常重要，这是回收顺行转运机械和ER居民蛋白质的回收必不可少的途径。对多生育束缚络合物的更深入的机械理解取决于确定其三维结构。在该赠款的最初资助期间，我们绘制了COG和DSL1P复合物的整体体系结构，并确定了其四个亚基的结构或部分结构。现在，我们建议解决更大的子组件，在DSL1P的情况下，整个综合体。该提案的第二个主要目标是启动对齿轮和DSL1P复合物与贩运机制其他组件之间相互作用的结构表征。由于这种努力而产生的模型将为基础，从而产生有关这些以及其他多育种束缚络合物的更敏锐的机械假设。为了实现这些目标，我们提出以下特定目标。在第一个目标中，我们将在我们先前对COG亚基连接性的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通过X射线晶体学和EM的组合阐明。我们还将使用高度优化的质谱法对COG相互作用蛋白进行公正的搜索。将使用生化和结构方法研究COG亚基或子组件与功能验证的伙伴之间的相互作用。在第二个特定目标中，我们将注意力转向DSL1P复合体。我们已经确定了代表DSL1P复合物的50％（质量）的晶体结构。我们建议使用X射线晶体学和EM完成此分析。最后，在第三个特定目的中，我们将对与网罗和涂层蛋白进行DSL1P复合物相互作用进行结构/功能研究。 公共卫生相关性：高尔基体在真核分泌途径内蛋白质分类和糖基化中起关键作用。囊泡运输的缺陷到高尔基体中，以及高尔基体内的缺陷都会影响其结构和功能。因此，这种缺陷可以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。