STRUCTURAL AND FUNCTIONAL ANALYSIS OF P115

本提案所述工作的长期目标是进一步 我们对囊泡转运机制的认识 真核细胞。这是细胞具体移动的过程 蛋白质和类脂从一个细胞室到另一个细胞室。 理解这个复杂的、多步骤的过程很重要，因为它 对细胞隔室的产生和维护至关重要，以及 因此，对于细胞的生存能力。这项工作将集中在功能和 一种新的运输因子p115的结构分析 最近被隔离了。P115是一种同源低聚外周膜蛋白 这可能在囊泡通过高尔基体运输的早期起作用。这个 这项建议的具体目标如下。P115的时间点 运输过程中的行为将通过动力学严格确定 高尔基体堆积后的形态分析 带P115和不带P115的运输。一项“囊泡发芽试验”将是 开发并用于直接评估p115‘S可能的功能 运输囊泡的形成。确定是否需要P115 多个运输步骤，除了通过 高尔基人，将通过亚细胞定位研究和通过 确定在体外转运试验中是否需要P115 其他几个运输步骤。可能与P115相互作用的蛋白质， 膜的缔合和可溶的，将通过 生物化学和分子遗传学技术，如果可能的话，进行纯化。一个 哺乳动物的P115基因将被克隆和测序。努力确定一个 P115在酿酒酵母中的同源物 将采用免疫学和分子遗传学技术。在一个 酵母同源基因的存在，该基因将被克隆和测序，并用于 启动酵母中P115功能的遗传分析。最后，第115页 将通过电子显微镜和结构域分析来研究结构 通过有限的蛋白质分解。对这一新交通工具的研究结果 因子有助于阐明真核生物的分子机制 细胞用来实现囊泡的运输。

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.