GENETIC ANALYSIS OF YPTL A SMALL GTP-BINDING PROTEIN

小 GTP 结合蛋白 YPTL 的遗传分析

• 批准号: 3304928
• 负责人: Nava Segev
• 金额: $ 20.24万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304928
• 关键词: Golgi apparatus; alleles; endoplasmic reticulum; gene expression; gene interaction; gene mutation; genetic mapping; genetic regulation; guanine nucleotide binding protein; immunoelectron microscopy; intracellular transport; laboratory rabbit; nucleic acid sequence; phenotype; polymerase chain reaction; protein structure function; protein transport; site directed mutagenesis; yeasts

A central question in understanding cell growth concerns the mechanism by which proteins are sorted into the correct compartments. It is believed that protein transport between intracellular compartments is mediated by membranous vesicles and that GTP-binding proteins are involved in the regulation of the directionality and/or accuracy of this vesicular traffic. Our long-term goals are to understand the mechanism of action of the GTP- binding Ypt1 protein in secretion and to identify and study other compartments of the vesicular transport machinery. We plan first to take advantage of the sophisticated classical and molecular genetics of the yeast system to isolate mutations in the Ypt1 gene and identify other genes and gene products with which it interacts. The mutations in the Ypt1 gene and selected genes with which it interacts will be then characterized by cellular and biochemical approaches. The role of the mammalian Ypt1 protein, which shares 70% identify with the yeast protein, will also be investigated by similar means. This proposal is focussed on two specific aims: 1) definition of the different domains of the Ypt1 protein and their function in protein transport, and 2) identification and characterization of other components of the vesicular transport machinery that interact with the Ypt1 protein. The experimental approach will involve generation of mutations in the Ypt1 gene by random and site specific mutagenesis. Genetic, biochemical and physiological analyses of mutations throughout the Ypt1 gene will used to examine the structure-function relationship of the Ypt1 protein and its various activities and interactions. For example, the role of the cycling of Ypt1 protein between GTP and GDP-bound forms in secretion will be examined. Specific functional regions will be determined by fine mapping of ypt1 mutations and identification of sites that are involved in interactions with other proteins. Genetic approaches will be used to detect other genes whose products interact with, or participate in the same pathway as the Ypt1 protein. Approaches similar to those used in the analysis of Ypt1 will be applied to study these other components. Finally, dominant mutations in the Ypt1 gene identified in yeast will be introduced into mammalian cells to investigate their effect on protein transport and growth of these cells. In summary, these studies will help to elucidate the molecular basis of the vesicular transport machinery and the role that GTP-binding proteins play in its regulation. The ability to study both the yeast and the mammalian systems provides a unique opportunity to approach these issues genetically in both systems and to extend the principles learned with yeast to higher cells.

理解细胞生长的一个核心问题涉及该机制 哪些蛋白质被分类为正确的隔室。 相信 细胞内室之间的蛋白质转运是由 膜囊泡和GTP结合蛋白参与 调节该囊泡流量的方向性和/或准确性。 我们的长期目标是了解GTP的作用机理 在分泌中结合YPT1蛋白，并识别和研究其他 囊泡运输机械的隔室。 我们计划首先服用 复杂的经典和分子遗传学的优势 酵母系统分离YPT1基因中的突变并鉴定其他基因 以及与之相互作用的基因产品。 YPT1基因中的突变 与之相互作用的选定基因将以以下特征 细胞和生化方法。 哺乳动物YPT1的作用 蛋白质共享70％与酵母蛋白相同的蛋白质也将是 通过类似方式调查。 该建议的重点是两个具体目的：1） YPT1蛋白的不同结构域及其在蛋白质中的功能 运输和2）其他组件的识别和表征 与YPT1蛋白相互作用的囊泡转运机制的。 实验方法将涉及YPT1中的突变产生 通过随机和位点特异性诱变的基因。 遗传，生化和 整个YPT1基因突变的生理分析将用于 检查YPT1蛋白及其的结构功能关系 各种活动和互动。 例如，骑自行车的作用 GTP和GDP结合形式之间的YPT1蛋白的分泌物将是 检查。 特定的功能区域将通过精细映射确定 YPT1突变和涉及的地点的识别 与其他蛋白质的相互作用。 遗传方法将用于 检测其产品与之相互作用或参与相同的其他基因 途径为YPT1蛋白。 与在 YPT1的分析将用于研究这些其他组件。 最后， 将引入在酵母中鉴定的YPT1基因中的主要突变 进入哺乳动物细胞，研究其对蛋白质转运的影响， 这些细胞的生长。 总而言之，这些研究将有助于阐明 囊泡运输机械和GTP结合蛋白起的作用 在其监管中。 研究酵母和哺乳动物的能力 系统提供了一个独特的机会来遗传处理这些问题 在这两个系统中，并将酵母学学到的原则扩展到更高 细胞。