STRUCTURAL AND FUNCTIONAL ANALYSIS OF P115

P115的结构和功能分析

• 批准号: 2186298
• 负责人: M GERARD WATERS
• 金额: $ 13.67万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186298
• 关键词: Golgi apparatus; Saccharomyces cerevisiae; affinity chromatography; animal tissue; circular dichroism; electron microscopy; epitope mapping; eukaryote; intracellular transport; laboratory rabbit; lipid transport; membrane proteins; molecular cloning; nucleic acid sequence; polymerase chain reaction; protein structure function; proteolysis; radionuclide double label; transport proteins; vesicle /vacuole; Golgi apparatus; Saccharomyces cerevisiae; affinity chromatography; animal tissue; circular dichroism; electron microscopy; epitope mapping; eukaryote; intracellular transport; laboratory rabbit; lipid transport; membrane proteins; molecular cloning; nucleic acid sequence; polymerase chain reaction; protein structure function; proteolysis; radionuclide double label; transport proteins; vesicle /vacuole

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.

本提案中描述的工作的长期目标是进一步 我们对囊泡运输机制的理解 桉树细胞。这是细胞专门移动的过程 蛋白质和脂质从一个细胞室到另一个细胞室。 了解这个复杂的多步过程很重要，因为它是 对于细胞室的生成和维护至关重要，以及 因此，用于细胞活力。这项工作将集中于功能和 新型传输因子的结构分析称为P115，这是 最近孤立。 P115是一种同性寡聚的外围膜蛋白 这可能在通过高尔基体囊泡运输的早期作用。这 该提案的具体目的如下。 P115的点 运输过程中的行为将通过动力学严格确定 分析与高尔基堆积的形态分析之后 带有和没有P115的运输。一个“囊泡萌芽测定”将是 开发并用于直接评估P115在 传输囊泡形成。确定是否需要P115 除顺式外，多个运输步骤 高尔基，将通过亚细胞定位研究和 确定是否需要P115的体外运输测定法 其他几个运输步骤。与P115相互作用的推定蛋白质， 膜相关和可溶性都将通过 生化和分子遗传技术，并在可能的情况下进行纯化。一个 哺乳动物P115 cDNA将被克隆并测序。努力确定 酿酒酵母的P115同源物通过生化， 将进行免疫学和分子遗传技术。在 存在酵母同源物，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，P115 结构将通过电子显微镜和域分析研究 通过有限的蛋白水解。这项新运输的研究结果 因子应有助于阐明桉树的分子机制 细胞用于影响囊泡流量。