SORTING AND TRANSPORT OF MEMBRANE PROTEINS

膜蛋白的分选和运输

• 批准号: 3293961
• 负责人: Tom Hall Stevens
• 金额: $ 13.62万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3293961
• 关键词: I cell disease; adenosinetriphosphatase; aminopeptidase; eukaryote; gene mutation; genetic manipulation; immunofluorescence technique; immunoprecipitation; inborn lysosomal enzyme disorder; lysosomes; molecular cloning; mucopolysaccharidosis type III; nucleic acid sequence; radionuclide double label; receptor mediated endocytosis; structural genes; vesicle /vacuole; yeasts

The work is aimed at understanding the sorting and transport of membrane proteins to the yeast lysosome-like vacuole. The simple eukaryote yeast will be used as a model eukaryotic sorting system, since the secretory and vacuole assembly pathways are very similar to the pathways in animal cells. Studies in yeast offer a unique opportunity to investigate the complex processes involved in membrane protein sorting and transport by taking advantage of the ability to exploit the powerful genetic approaches available in yeast. It also appears likely that the basic cellular functions that facilitate sorting of vacuolar/lysosomal membrane proteins will be conserved across all eukaryotic cells. Yeast mutants that mislocalize the vacuolar membrane protein dipeptidyl amino-peptidase (DPAP-B) will be obtained by exploiting a newly developed selection procedure. These mutants will be screened biochemically and by immunogold labeling for the secretion of a large number of soluble and membrane-bound vacuolar proteins. In addition, a major effort will be made towards identifying the vacuolar sorting and transport signals present on the membrane protein DPAP-B. Mutations in the DPAP-B structural gene will be generated and those resulting in missorting of enzymatically active DPAP-B will be identified with the DPAP-B mislocalization selection procedure. The structural genes of the two largest subunits of the yeast vacuolar membrane H+-translocating ATPase will be cloned using the Lambdagtll yeast library. Mutations will be constructed in these genes to elucidate the role of this H+-ATPase in acidification of the vacuole. These mutations will also permit an analysis of the role of acidification in the sorting of newly synthesized vacuolar hydrolases, in fluid-phase and receptor-mediated endocytosis, and in the function of the vacuole. The biosynthesis, assembly, targeting and transport of this vacuolar multi-subunit membrane-bound H+-ATPase complex will be investigated in an effort to understand the relationship between the synthesis and assembly of the subunits and their transport to the vacuole. These studies are likely to increase our basic understanding of diseases that result from missorting of lysosomal hydrolases such as Mucolipidosis II and III and other lysosomal storage diseases.

该工作旨在了解膜的分类和运输 蛋白质到酵母溶酶体样液泡。 简单的真核生物酵母 自分泌和 液泡组件路径与动物的途径非常相似 细胞。 酵母研究提供了一个独特的机会来调查 涉及膜蛋白分类和运输的复杂过程 利用利用强大遗传方法的能力 可在酵母中购买。 似乎基本的蜂窝可能也可能 促进液泡/溶酶体膜蛋白的功能 将在所有真核细胞中保守。 将液泡膜蛋白二肽基的酵母突变体错误定位 通过利用新开发的氨基肽酶（DPAP-B）将获得 选择程序。 这些突变体将通过生化和通过 分泌大量可溶性的免疫金标签 膜结合的液泡蛋白。 此外，还将做出重大努力 要识别存在的液泡分类和传输信号 膜蛋白DPAP-B。 DPAP-B结构基因中的突变将 产生和导致酶活性的那些 DPAP-B将通过DPAP-B错误定位选择确定 程序。 酵母液泡的两个最大亚基的结构基因 膜H+translosating ATPase将使用lambdagtll酵母克隆 图书馆。 这些基因将构建突变，以阐明 该H+ATPase在液泡的酸化中的作用。 这些突变 还将允许分析酸化在分类中的作用 在流体相和受体介导的新合成的液泡水解酶 内吞作用，以及液泡的功能。 生物合成， 该液泡多物品的组装，靶向和运输 将研究膜结合的H+-ATPase复合物以进行研究 了解合成和组装之间的关系 亚基及其运输到液泡。 这些研究可能是 提高我们对失误导致疾病的基本理解 溶酶体水解酶，例如粘脂脂病II和III以及其他溶酶体 存储疾病。