INTRACELLULAR ROLE OF CLATHRIN AND ITS REGULATION

网格蛋白的细胞内作用及其调控

• 批准号: 2022667
• 负责人: THERESA Joyce O'HALLORAN
• 金额: $ 18.93万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022667
• 关键词: Dictyostelium; clathrin; endocytosis; intracellular transport; molecular cloning; osmosis; phenotype; protein biosynthesis; protein purification; protein structure function; protein transport; receptor mediated endocytosis; vesicle /vacuole

DESCRIPTION (adapted from applicant's abstract): The overall objective of the work proposed is to understand the function of clathrin coated vesicles and coated vesicle proteins in living cells. Dr. O'Halloran's focus has been to study the physiology and morphology of clathrin- depleted Dictyostelium cells. These mutants are viable, but they lack endosomes and a contractile vacuole, an organelle important for adaptation to osmotic stress. In addition, the cells exhibit severe deficiencies in endocytosis and osmoregulation. The purpose of this study is to dissect the contribution of clathrin to these physiological processes.She has two major goals. (1) The mechanism for clathrin's role in cellular function will be determined. Conditional mutants for clathrin heavy chain protein will be created and studied. The relationship between the absence of clathrin heavy chain protein, the rearrangements of contractile vacuole markers and the loss of osmoregulatory activity in mutant cells will be determined. The hypothesis that clathrin directly mediates fluid-phase and receptor-mediated endocytosis will be tested. The role of clathrin in the sorting, processing and targeting of lysosomal enzymes will be determined. (2) The intracellular role of clathrin light chains and adaptins, possible regulators of clathrin function will be determined. Coated vesicles from Dictyostelium will be isolate and the constituent proteins. cDNAs for clathrin light chains and the adaptin/assembly protein complex will be cloned from a Dictyostelium library. Mutants that contain deletions in these genes or which overexpress functional domains of the gene products will be constructed. The phenotype of these mutants will be studied in detail to test the hypothesis that these gene products function in the regulation of clathrin traffic.

描述（改编自申请人的摘要）：总体目标 提议的工作之一是了解网格蛋白涂层的功能 活细胞中的囊泡和包被囊泡蛋白。 奥哈洛伦博士的 重点是研究网格蛋白的生理学和形态学 耗尽的盘基网柄菌细胞。 这些突变体是可行的，但它们缺乏 内体和收缩液泡，一种重要的细胞器 对渗透压的适应。此外，细胞表现出严重的 内吞作用和渗透调节的缺陷。 这样做的目的 研究的目的是剖析网格蛋白对这些生理学的贡献 她有两个主要目标。 (1)网格蛋白在细胞功能中的作用机制是 决定。 网格蛋白重链蛋白的条件突变体将 被创造和研究。 不存在之间的关系 网格蛋白重链蛋白，收缩液泡的重排 突变细胞中的标记物和渗透压调节活性的丧失将 决定。 网格蛋白直接介导液相的假设 并将测试受体介导的内吞作用。 网格蛋白的作用 溶酶体酶的分选、加工和靶向将 决定。 (2)网格蛋白轻链和适应素的细胞内作用， 将确定网格蛋白功能的可能调节剂。 涂层 来自盘基网柄菌的囊泡将被分离，其成分 蛋白质。 网格蛋白轻链和适应素/组装体的 cDNA 蛋白质复合物将从盘基网柄菌文库中克隆。 突变体 这些基因中含有缺失或过度表达功能 将构建基因产物的结构域。 这些的表型 将详细研究突变体，以检验这些基因的假设 产品具有调节网格蛋白运输的功能。