FUNCTION OF CLATHRIN-COATED MEMBRANES

网格蛋白涂层膜的功能

• 批准号: 3291486
• 负责人: RANDY W. SCHEKMAN
• 金额: $ 17.03万
• 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/3291486
• 关键词: antibody; antibody specificity; autoradiography; beta fructofuranosidase; carboxypeptidase; cell growth regulation; clathrin; electron microscopy; gel electrophoresis; gene expression; gene mutation; immunoprecipitation; laboratory mouse; laboratory rabbit; membrane activity; membrane proteins; membrane reconstitution /synthesis; messenger RNA; molecular cloning; nucleic acid hybridization; nucleic acid sequence; phagocytosis; protein biosynthesis; protein structure; protein transport; 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.

网格蛋白包被的囊泡和膜已经涉及多种免疫反应。 真核细胞的胞内转运过程。 的精确 网格蛋白在细胞生长和蛋白质转运中的作用将被讨论 通过评价在网格蛋白重链产生中缺陷的酵母突变体， 和轻亚基。 多克隆抗血清用于鉴定一个克隆， 在LambdagtII基因组中表达的酵母重链基因（CHC 1） 图书馆 该插入片段的身份通过杂交选择性PCR确认。 翻译重链mRNA，然后进行免疫沉淀。 一 然后使用插入片段来产生插入片段的缺失。 染色体CHC1基因座。 令人惊讶的是，存活的缺失突变株被 产生的，其不具有可检测的免疫反应性重链，并且其 产生同样缺乏网格蛋白轻链的囊泡。 虽然网格蛋白 缺失突变细胞生长缓慢，分泌糖蛋白转化酶 几乎是正常的。 现在将检查缺失突变细胞的 和蛋白质到不同目的地的运输和分选的保真度。 网格蛋白的结构类似物将使用敏感的核酸来寻找。 酸杂交程序，并通过检查替代协会 在重链缺失突变细胞中与网格蛋白轻链形成。 轻链基因的克隆和缺失分析将用于 检测轻链在蛋白质转运中可能发挥的任何独立作用。