Structural Analysis of Golgi Trafficking Proteins

描述（由申请人提供）：高尔基体在真核生物分泌途径中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缺陷影响其结构和功能。因此，这些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。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形态和功能至关重要。这种异八聚体蛋白，被称为保守低聚高尔基（COG）复合物，似乎对高尔基体内的逆行运输很重要，在那里它可能起着引导从反式高尔基体到顺式高尔基池的囊泡的作用。此外，COG是被称为栓系因子的细胞内运输蛋白家族的一员。一些证据表明，系缚因子在SNAREs的上游起作用，介导转运囊泡与其膜靶之间的最早接触。然而，尽管束缚因素至关重要，但人们对它们的理解却相对较少。具体来说，包括COG在内的六种大型异聚寡聚系栓蛋白的结构几乎一无所知，这些蛋白被认为在分泌途径中起作用，它们的作用机制也没有得到很好的表征。因此，本提案旨在通过开展COG的粗粒度和细粒度结构研究来填补这一空白。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通性和复合物的整体结构。在第二个目标中，将使用免疫沉淀和一组多克隆抗体来鉴定与COG在物理和功能上相互作用的蛋白质。目标3和目标4启动了高分辨率的COG结构分析。在第三个目标中，酵母Cog2p亚基的结构将用二维和三维核磁共振来阐明。在最终目标中，x射线晶体学将用于确定更大的COG子组件和具有其他运输因素的复合物的结构。

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.