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(申请人提供)：高尔基体在真核细胞分泌途径中的蛋白质分选和糖解中起关键作用。高尔基体内囊泡运输的缺陷影响其结构和功能。因此，这种缺陷可能会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。目前的建议集中在最近发现的一种蛋白质上，这种蛋白质对正常的高尔基体形态和功能至关重要。这种被称为保守的寡聚体高尔基体(COG)复合体的异八聚体蛋白似乎对高尔基体内的逆行运输很重要，在高尔基体中，它可能具有将来自反高尔基体的囊泡导向顺式高尔基体池的功能。此外，COG是被称为系留因子的细胞内运输蛋白家族的成员。几条证据表明，系留因子作用于圈套的上游，调解运输小泡与其膜目标之间的最早接触。然而，尽管系留因素具有核心重要性，但人们对它们的理解相对较少。具体地说，包括COG在内的六种大型异寡聚体拴系蛋白的结构几乎一无所知，也没有很好地描述它们的作用机制。因此，这项提案旨在通过对COG进行粗粒度和细粒度的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亚基连接和复合体的整体结构。在第二个目标中，将使用一组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和目标4启动了COG的高分辨率结构分析。在第三个目标中，将使用二维和三维核磁共振来阐明酵母Cog2p亚基的结构。在最终目标中，X射线结晶学将被用来确定较大的COG组件和与其他运输因子的络合物的结构。