SORTING AND TRANSPORT OF MEMBRANE PROTEINS

这项工作的目的是了解膜的分选和运输 蛋白质进入酵母溶酶体样空泡。 简单的真核酵母 将被用作真核生物分选系统的模型，因为分泌和 液泡组装途径与动物中的途径非常相似， 细胞 对酵母的研究提供了一个独特的机会， 涉及膜蛋白分选和转运的复杂过程， 利用强大的遗传学方法， 在酵母中可用。 它也似乎是基本的细胞 促进液泡/溶酶体膜蛋白分选的功能 在所有真核细胞中都是保守的 错误定位液泡膜蛋白二肽基的酵母突变体 氨基肽酶（DPAP-B）是利用新开发的 选择程序。 这些突变体将通过生物化学方法进行筛选 免疫金标记的分泌大量的可溶性和 膜结合液泡蛋白。 此外，还将大力 为了识别液泡分类和运输信号存在于 膜蛋白DPAP-B。 DPAP-B结构基因的突变将 和那些导致酶活性物质的错误分选的物质。 DPAP-B将通过DPAP-B错误定位选择进行识别 procedure. 酵母液泡膜两个最大亚基的结构基因 膜H+-转运ATP酶将使用Lambdagtll酵母克隆 图书馆 将在这些基因中构建突变，以阐明 H+-ATPase在液泡酸化中的作用。 这些突变 还将允许分析酸化在分类中的作用， 新合成的液泡水解酶，在液相和受体介导 内吞作用和液泡的功能。 生物合成， 这种液泡多亚基的组装、靶向和转运 膜结合H+-ATP酶复合物将被研究， 理解合成和组装之间的关系 亚基及其向液泡的运输。 这些研究很可能 增加我们对疾病的基本了解， 溶酶体水解酶如粘脂沉积症II和III，以及其它溶酶体水解酶 贮藏病害

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.