PROTEIN SORTING TO THE LYSOSOME-LIKE VACUOLE IN YEAST

细胞膜的选择性识别、分选和囊泡介导的转运 蛋白质通过分泌途径代表了一个基本特征， 所有的真核细胞。 精确的机制和机械， 这些方法尚不为人所知。 我们分离出了大量的 在蛋白质递送方面表现出严重和特定缺陷的酵母突变体 到酵母中的溶酶体样空泡。 这些基因的缺陷 突变体可能编码细胞蛋白质分选的成分 设备. 为了理解分子和生物化学基础 对于这些突变体（vps）中的液泡蛋白分选缺陷，我们提出， 描述某些VPS基因及其产物的作用， 在从高尔基复合体到 液泡 酵母与其他酵母之间的基本相似之处 真核细胞在其蛋白质递送途径中的作用，以及 强大的遗传和分子方法，使酵母， 解决这些问题的理想生物。 的医学重要性 这种分选途径的例子是严重的溶酶体储存 疾病（例如，I-细胞疾病、假性Hurler多营养不良以及其他 疾病（例如骨质疏松症和某些类型癌症的进展） 这是由溶酶体的错误定位引起的，或与之相关， 水解酶 实验室已克隆并测序了八个VPS基因。 以确定 这些基因的产物在哪里以及如何起作用来指导分选， 递送液泡水解酶，我们建议：1）使用间接 免疫荧光和细胞分级分离技术，以确定 VPS基因产物的亚细胞定位，2）分离温度- 条件VPS等位基因分析空泡的发病动力学 蛋白质分选缺陷，并确定潜在的中间体， 分选过程，3）使用遗传抑制和化学交联 鉴定和表征VPS基因产物之间相互作用的试剂， 4)采用体外测定，其重建液泡蛋白递送， 表征Vps蛋白的阶段特异性需求和功能， 和5）克隆和测序某些VPS基因的功能同源物， 粟酒裂殖酵母以及哺乳动物（人）的cDNA文库 开始分析VPS基因产物的功能作用 在其他真核生物中发挥作用。 预计这些研究将导致 在组织和蛋白质分选控制的详细视图中， 高尔基复合体和液泡/溶酶体之间的囊泡运输 酵母和其他真核生物。

The selective recognition, sorting and vesicle-mediated transport of proteins through the secretory pathway represents an essential feature of all eukaryotic cells. The precise mechanisms and machinery that direct these processes are not yet known. We have isolated a large collection of yeast mutants that exhibit severe and specific defects in protein delivery to the lysosome-like vacuole in yeast. The defective genes in these mutants are likely to encode components of the cells' protein sorting apparatus. Toward an understanding of the molecular and biochemical basis of the vacuolar protein sorting defects in these mutants (vps), we propose to characterize the roles certain of the VPS genes and their products play in the sorting and delivery of vacuolar enzymes from the Golgi complex to the vacuole. The fundamental similarities between yeast and other eukaryotic cells in their pathways for protein delivery, together with the powerful genetic and molecular approaches available in yeast, make yeast an ideal organism for addressing these problems. The medical importance of this sorting pathway is exemplified by the serious lysosomal storage diseases (e.g. I-cell disease, pseudo-Hurler polydystrophy as well as other diseases (e.g. osteoporosis and the progression of certain types of cancer) that result from, or are correlated with, mislocalization of lysosomal hydrolases. Eight VPS genes have been cloned and sequenced in the lab. To determine where and how the products of these genes act to direct the sorting and delivery of vacuolar hydrolases, we propose to: 1) use indirect immunofluorescence and cell fractionation techniques to determine the subcellular location of the VPS gene products, 2) isolate temperature- conditional vps alleles to analyze the onset kinetics of the vacuolar protein sorting defects and to identify potential intermediates in the sorting process, 3) use genetic suppression and chemical cross-linking agents to identify and characterize interactions among VPS gene products, 4) employ an in vitro assay that reconstitutes vacuolar protein delivery to characterize the stage specific requirements and functions of Vps proteins, and 5) clone and sequence functional homologs for certain VPS genes from cDNA libraries of Schizosaccharomyces pombe as well as mammalian (human) cell types to begin an analysis of the functional role VPS gene products play in other eukaryotes. It is anticipated that these studies will result in a detailed view of the organization and control of protein sorting and vesicular traffic between the Golgi complex and the vacuole/lysosome in yeast and other eukaryotes.