INTRACELLULAR ROLE OF CLATHRIN AND ITS REGULATION

描述（改编自申请人摘要）：总体目标 提出的工作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。 奥哈洛伦医生 重点是研究网格蛋白的生理学和形态学- 耗尽的网骨藻细胞。 这些突变体是可行的，但他们缺乏 内体和收缩泡，一个重要的细胞器， 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功能的贡献， 她有两个主要目标。 (1)网格蛋白在细胞功能中的作用机制将是 测定 网格蛋白重链蛋白的条件突变体将 创造和研究。 缺乏的关系 网格蛋白重链蛋白，收缩泡重排 标志物和突变细胞中β-内酰胺酶调节活性的丧失将被 测定 网格蛋白直接介导液相的假说 并测试受体介导的内吞作用。 网格蛋白的作用 在溶酶体酶的分选、加工和靶向方面， 测定 (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。 涂覆 将从网骨藻中分离出囊泡， proteins. 网格蛋白轻链和衔接蛋白/组装的cDNA 蛋白复合物将从网柄藻文库中克隆。 突变体 其在这些基因中含有缺失或过表达功能性 将构建基因产物的结构域。 这些表型 将详细研究突变体，以验证这些基因 产物在网格蛋白运输的调节中起作用。

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.