Structural Analysis of Golgi Trafficking Proteins

高尔基体运输蛋白的结构分析

• 批准号: 6919577
• 负责人: FREDERICK M HUGHSON
• 金额: $ 27.12万
• 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/6919577
• 关键词: Golgi apparatus; X ray crystallography; affinity chromatography; analytical ultracentrifugation; antibody; capillary electrophoresis; eukaryote; fungal proteins; glycosylation; immunoprecipitation; light scattering; membrane proteins; membrane transport proteins; nuclear magnetic resonance spectroscopy; physical model; protein structure function; protein transport; scanning transmission electron microscopy; secretory protein; 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）：高尔基体在真核生物分泌途径内的蛋白质分类和糖脂中起关键作用。高尔基体内囊泡运输的缺陷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。当前的建议着重于最近发现的蛋白质，这对于正常的高尔基形态和功能至关重要。这种杂氨酸蛋白质，称为保守的寡聚性高尔基体（或COG）配合物，对于高尔基体内部的逆行运输似乎很重要，在那里可能起作用，可以将源自从反式高尔基体到Cis Golgi Cisternae的囊泡引导。此外，COG是一个被称为绑定因子的细胞内运输蛋白家族的成员。几条证据表明，束缚因子在网res上游起作用，从而介导了囊泡及其膜靶的最早接触。尽管如此，束缚因素尽管具有重要意义，但知识却相对较少。具体而言，几乎一无所知。因此，该提案旨在通过进行COG的粗粒结构研究来开始填补这一空白。在第一个目标中，将使用一组互补的生化方法来阐明该复合物的亚基连接性和整体体系结构。在第二个目标中，将使用带有多克隆抗体的免疫沉淀来鉴定与COG在物理和功能上相互作用的蛋白质。目标3和4启动COG的高分辨率结构分析。在第三个目标中，将使用二维和三维NMR阐明酵母COG2P亚基的结构。在最终目的中，X射线晶体学将用于确定与其他运输因子的较大齿轮亚组件和复合物的结构。