INTRACELLULAR ROLE OF CLATHRIN AND ITS REGULATION

描述(改编自申请人的摘要)：总体目标 提出的工作之一就是了解被包被蛋白的功能 活细胞中的囊泡和包被的囊泡蛋白。奥哈罗兰医生的 重点是研究笼状蛋白的生理和形态-- 枯竭的网柄网柄菌细胞。这些突变体是有生存能力的，但它们缺乏 内小体和一个可收缩的液泡，这是一个重要的细胞器 对渗透胁迫的适应。此外，细胞表现出严重的 缺乏内吞作用和渗透调节。这样做的目的是 这项研究是为了剖析笼状蛋白对这些生理功能的贡献。 她有两个主要目标。 (1)笼蛋白在细胞功能中的作用机制将是 下定决心。笼状蛋白重链蛋白Will的条件突变体 被创造和研究。这两种情况之间的关系 膜蛋白重链蛋白--收缩空泡的重排 标记物和突变细胞渗透调节活性的丧失将是 下定决心。笼状蛋白直接调节液体相的假说 并将测试受体介导的内吞作用。笼状蛋白的作用 在对溶酶体酶的分选、加工和靶向方面 下定决心。 (2)网状蛋白轻链和适配素在细胞内的作用， 可能的网状蛋白功能调节者将被确定。涂覆的 将从网囊藻中分离出小泡，并将其成分 蛋白质。分子筛轻链的cDNA及其适配/组装 蛋白质复合体将从网柄菌文库中克隆出来。突变者 包含这些基因的缺失或过度表达功能 基因产物的结构域将被构建。这些基因的表型 将对突变体进行详细研究，以检验这些基因 产品在调节网状蛋白的交通方面起作用。

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.