FUNCTION OF CLATHRIN-COATED MEMBRANES

网格蛋白涂层膜的功能

• 批准号: 3291483
• 负责人: RANDY W. SCHEKMAN
• 金额: $ 13.06万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3291483
• 关键词: antibody; antibody specificity; autoradiography; beta fructofuranosidase; carboxypeptidase; cell growth regulation; electron microscopy; gel electrophoresis; gene expression; gene mutation; immunoprecipitation; membrane activity; membrane reconstitution /synthesis; messenger RNA; molecular cloning; nucleic acid sequence; phagocytosis; radiotracer; secretion; vesicle /vacuole; yeasts

Clathrin-coated vesicles and membranes have been implicated in a variety of intracellular transport processes in eukaryotic cells. The precise funciton of clathrin in cell growth and protein transport will be addressed by evaluation of yeast mutants defective in production of clathrin heavy and light subunits. Polyclonal antiserum was used to identify a clone of the yeast heavy chain gene (CHC1) expressed in a Lambdagtll genomic library. The identity of this insert was confirmed by hybrid-selected translation of heavy chain mRNA followed by immune precipitation. A fragment of the insert was then used to generate deletions of the chromosomal CHC1 locus. Surprisingly, viable deletion mutant strains were produced which have no detectable immunoreactive heavy chain, and which produce vesicles that also lack clathrin light chain. Although clathrin deletion mutant cells grow slowly, secretion of the glycoprotein invertase is nearly normal. Deletion mutant cells will now be examined for the rate and fidelity of transport and sorting of proteins to various destinations. Structural analogues of clathrin will be sought using sensitive nucleic acid hybridization procedures and by examining alternative associations formed with clathrin light chain in heavy chain deletion mutant cells. Cloning and deletion analysis of the light chain gene will be used to detect any independent role that light chains may play in protein transport.

网格蛋白涂层的囊泡和膜已与多种 真核细胞中的细胞内转运过程。 确切的 网细胞生长和蛋白质传输中的clathrin funciton将被解决 通过评估酵母突变体的产生，网状蛋白沉重的产生 和轻度亚基。 多克隆抗血清用于识别 酵母重链基因（CHC1）在lambdagtll基因组中表达 图书馆。 该插入物的身份通过杂种选择 重链mRNA的翻译随后是免疫沉淀。 一个 然后，使用插入物的片段来产生删除 染色体CHC1基因座。 令人惊讶的是，可行的缺失突变菌株是 生产的，没有可检测到的免疫反应性重链，并且 产生也缺乏网状蛋白轻链的囊泡。 虽然是氯化链蛋白 缺失突变细胞生长缓慢，分泌糖蛋白转化酶 几乎正常。 现在将检查缺失突变细胞的速率 以及将蛋白质分类到各个目的地的忠诚度。 使用敏感的核心，将寻求网格蛋白的结构类似物 酸杂交程序并检查替代关联 在重链缺失突变细胞中用网状蛋白轻链形成。 轻链基因的克隆和删除分析将用于 检测光链在蛋白质传输中可能起的任何独立作用。