Structural Analysis of Golgi Trafficking Proteins

高尔基体运输蛋白的结构分析

• 批准号: 7373599
• 负责人: FREDERICK M HUGHSON
• 金额: $ 27.14万
• 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 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7373599
• 关键词: Affect; Applications Grants; Architecture; Baculoviruses; Biochemical; Capillary Electrophoresis; Cell Surface Proteins; Cells; Cereals; Coat Protein Complex I; Complex; Data; Defect; Depth; Eukaryotic Cell; Family; Foundations; Goals; Golgi Apparatus; Immunoprecipitation; In Vitro; Insecta; Lead; Mammals; Maps; Mediating; Membrane; Membrane Protein Traffic; Methods; Modeling; Modification; Morphology; NOESY; Pathway interactions; Pattern; Personal Satisfaction; Play; Protein Binding; Protein Sortings; Proteins; Protons; Range; Recombinants; Research Personnel; Research Proposals; Resolution; Roentgen Rays; Role; Side; Structural Models; Structure; Techniques; Testing; Thinking; Translations; Transport Vesicles; Vesicle; Work; X-Ray Crystallography; Yeasts; analytical ultracentrifugation; glycosylation; human disease; intracellular protein transport; light scattering; member; polyclonal antibody; polypeptide; protein function; protein localization location; protein transport; research study; retrograde transport; size; stoichiometry; three dimensional structure; trafficking; yeast two hybrid system

DESCRIPTION (provided by applicant): The Golgi apparatus plays a key role in protein sorting and glycoslyation within the eukaryotic secretory pathway. Defects in vesicular trafficking 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. The current proposal focuses on a recently discovered protein that is essential for normal Golgi morphology and function. This hetero-octameric protein, known as the Conserved Oligomeric Golgi (or COG) complex, appears to be important for retrograde trafficking within the Golgi apparatus, where it likely functions to direct vesicles originating from the trans-Golgi to cis Golgi cisternae. Furthermore, COG is a member of a family of intracellular trafficking proteins known as tethering factors. Several lines of evidence indicate that tethering factors act upstream of SNAREs, mediating the earliest contact between transport vesicles and their membrane targets. Nonetheless tethering factors, despite their central importance, are relatively poorly understood. Specifically, almost nothing is known about the structure of any of the six large hetero-oligomeric tethering proteins, including COG, thought to function in the secretory pathway, nor has their mechanism of action been well characterized. This proposal, therefore, aims to begin filling in this gap by undertaking coarse- and fine-grained structural studies of COG. In the first aim, a complementary set of biochemical methods will be used to elucidate the subunit connectivity and overall architecture of the complex. In the second aim, proteins that interact physically and functionally with COG will be identified using immunoprecipitation with a battery of polyclonal antibodies. Aims 3 and 4 initiate high-resolution structural analysis of COG. In the third aim, the structure of the yeast Cog2p subunit will be elucidated using two- and three-dimensional NMR. In the final aim, X-ray crystallography will be used to determine the structures of larger COG subassemblies and complexes with other trafficking factors.

描述（由申请人提供）：高尔基体在真核分泌途径内的蛋白质分选和糖基化中起关键作用。高尔基体内囊泡运输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。目前的提案重点关注最近发现的一种对正常高尔基体形态和功能至关重要的蛋白质。这种异源八聚体蛋白，被称为保守寡聚体高尔基体（或COG）复合物，似乎是重要的逆行运输内的高尔基体，在那里它可能的功能，指导囊泡起源于反式高尔基体顺高尔基池。此外，COG是称为系链因子的细胞内运输蛋白家族的成员。一些证据表明，系留因子的SNARE的上游，介导运输囊泡和它们的膜靶之间的最早接触。尽管束缚因素至关重要，但人们对它们的了解相对较少。具体来说，几乎没有什么是已知的结构的任何六个大的异源寡聚拴系蛋白，包括COG，认为在分泌途径中发挥作用，也没有很好地表征其作用机制。因此，本提案旨在通过对COG进行粗粒度和细粒度结构研究来开始填补这一空白。在第一个目标中，将使用一套互补的生化方法来阐明复合物的亚基连接性和整体结构。在第二个目标中，将使用免疫沉淀与一组多克隆抗体鉴定与COG物理和功能相互作用的蛋白质。目的3和4启动COG的高分辨率结构分析。在第三个目标中，酵母Cog2p亚基的结构将使用二维和三维NMR来阐明。在最后的目标，X射线晶体学将用于确定结构的较大的COG双和复合物与其他交通因素。