STRUCTURAL AND FUNCTIONAL ANALYSIS OF P115

本提案所述工作的长期目标是进一步 我们对微泡运输机制的理解 真核细胞这是细胞特定移动的过程 蛋白质和脂质从一个细胞区室转移到另一个细胞区室。 理解这个复杂的多步骤过程很重要，因为它是 对细胞区室的产生和维持至关重要， 从而影响细胞活力。工作将侧重于功能和 一种新的转运因子p115的结构分析， 最近被孤立。p115是一种同源寡聚外周膜蛋白 这可能在通过高尔基体的囊泡运输的早期起作用。的 这项建议的具体目标如下。在P115 运输过程中的行为将通过动力学严格确定， 结合高尔基体堆积的形态学分析， 带和不带P115的运输。将进行“囊泡出芽测定”， 开发并用于直接评估p115在 运输囊泡形成。确定是否需要P115 多个转运步骤，除了顺位至内侧转运外， 高尔基体，将通过亚细胞定位研究进行， 确定P115是否需要在体外转运试验中用于 其他运输步骤。与P115相互作用的推定蛋白质， 膜相关的和可溶的，将通过 生物化学和分子遗传技术，如果可能的话，进行纯化。一 哺乳动物P115 cDNA将被克隆和测序。努力查明 P115同源物在酿酒酵母中通过生物化学， 免疫学和分子遗传学技术。中 酵母同源物存在，该基因将被克隆和测序，并用于 启动酵母中P115功能遗传分析。最后，P115 结构将通过电子显微镜和域分析进行研究 通过有限的蛋白质水解。对这种新运输方式的研究结果 因子应该有助于阐明真核生物的分子机制， 细胞用于影响囊泡运输。

The long-term goal of the work described in this proposal is to further our understanding of the mechanism of vesicular transport in the eucaryotic cell. This is the process by which the cell specifically moves proteins and lipids from one cellular compartment to another. Understanding this complex, multistep process is important because it is crucial for the generation and maintenance of cellular compartments, and hence for cellular viability. The work will focus on functional and structural analyses of a novel transport factor, termed p115, which was recently isolated. p115 is a homo-oligomeric peripheral membrane protein that may act early in vesicular transport through the Golgi apparatus. The specific aims of this proposal are as follows. The point at which P115 acts during transport will be rigorously determined through kinetic analyses in conjunction with morphological analysis of Golgi stacks after transport with and without P115. A "vesicle budding assay" will be developed and used to directly assess p115's possible function in transport vesicle formation. Determination of whether P115 is required at multiple transport steps, besides cis to medial transport through the Golgi, will be undertaken via subcellular localization studies and by determining whether P115 is required in in vitro transport assays for several other transport steps. Putative proteins that interact with P115, both membrane associated and soluble, will be identified through biochemical and molecular genetic techniques, and purified if possible. A mammalian P115 cDNA will be cloned and sequenced. Efforts to identify a P115 homolog in Saccharomyces cerevisiae through biochemical, immunological, and molecular genetic techniques will be undertaken. In a yeast homolog exists, the gene will be cloned and sequenced, and used to initiate a genetic analysis of P115 function in yeast. Finally, P115 structure will be studied via electron microscopy and domain analysis through limited proteolysis. The results of studies of this new transport factor should help illuminate the molecular mechanisms that eucaryotic cells use to effect vesicular traffic.