Structural Analysis of Golgi Trafficking Proteins

描述（由申请人提供）：高尔基体在真核分泌途径中的蛋白质分选和糖基化中起着关键作用。高尔基体内囊泡运输的缺陷会影响其结构和功能。因此，此类缺陷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，被称为保守寡聚高尔基体（或COG）复合物，似乎对于高尔基体内的逆行运输很重要，它可能具有将源自反式高尔基体的囊泡引导至顺式高尔基体池的功能。此外，COG 是称为束缚因子的细胞内运输蛋白家族的成员。多项证据表明，束缚因子在 SNARE 上游发挥作用，介导转运囊泡与其膜靶标之间的最早接触。尽管如此，尽管束缚因素至关重要，但人们对它们的了解却相对较少。具体来说，人们对六种大型异源寡聚束缚蛋白（包括 COG）的结构几乎一无所知，这些蛋白被认为在分泌途径中发挥作用，它们的作用机制也没有得到很好的表征。因此，该提案旨在通过对 COG 进行粗粒度和细粒度的结构研究来填补这一空白。第一个目标是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，将使用一组多克隆抗体进行免疫沉淀来鉴定与 COG 发生物理和功能相互作用的蛋白质。目标 3 和 4 启动 COG 的高分辨率结构分析。第三个目标是利用二维和三维 NMR 阐明酵母 Cog2p 亚基的结构。最终目标是，X 射线晶体学将用于确定较大的 COG 子组件以及与其他贩运因子的复合物的结构。

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.