ROLE OF THE YEAST VPS15 PROTEIN KINASE IN INTRACELLULAR PROTEIN SORTING

蛋白质的选择性识别、分选和转运， 通过囊泡载体将细胞内细胞器连接到另一个细胞器代表了 所有真核细胞的基本特征。 精确的机制和 指导这些过程的机械还不为人所知。 我们有 分离出大量的突变体，这些突变体在 蛋白质递送到酵母溶酶体样液泡。 缺陷基因 在这些突变体中可能编码细胞蛋白质的成分 分类装置 为了理解分子和 这些细胞中液泡蛋白分选缺陷的生化基础 突变体（vps），我们建议的特点的作用，某些VPS 基因及其产物在液泡的分类和传递中起作用， 从高尔基复合体到酵母中的液泡的酶。 根本 酵母和其他真核细胞在其途径上的相似性 用于蛋白质输送，以及强大的遗传和分子 酵母中可用的方法，使酵母成为理想的生物体， 解决这些问题。 我们分离出的酵母突变体 与许多转化的哺乳动物细胞系相似的表型， 已知异常分泌溶酶体蛋白酶。 的错误定位 已经提出这些蛋白酶增强肿瘤的转移 细胞 这项研究的重点是两个VPS基因，VPS 15和VPS 34。 每个基因的突变导致一组共同的表型， 每一种都可以促进液泡蛋白质递送中的相同步骤 通路 VPS 15基因的序列预测了一种蛋白质， 以下特征：N-末端豆蔻酰化的共有位点， 与蛋白磷酸酶2A调节亚基同源的区域 （PP2A）。 VPS 34基因产物具有显著的序列相似性 与哺乳动物磷脂酰肌醇-3-激酶的催化亚基。 VPS 34 P13-激酶活性可由VPS 15基因产物调节。 我们已经发现Vps 34蛋白的过表达可以抑制 VPS 15基因激酶结构域内突变引起的缺陷 产物，但不会抑制VPS 15的无效等位基因。 同时使用 遗传和生物化学方法，我们建议表征：1） Vps 15激酶结构域的功能作用，以及蛋白质 磷酸化，在液泡蛋白分选，2）的意义， Vps 15 p与PP 2A调节亚基的序列同源性，3） 亚细胞的位置和可能的作用位点的Vps 15和 Vps 34蛋白，以及4）Vps 15和Vps 34的潜在相互作用 蛋白质与彼此以及可能的其它细胞组分（例如， PP2A）。

The selective recognition, sorting, and transport of proteins from one intracellular organelle to another via vesicular carriers 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 mutant that exhibit severe defects in protein delivery to the yeast lysosome-like vacuole. The defective genes in these mutant 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 in yeast. 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 yeast mutants we have isolated exhibit a similar phenotype to many transformed mammalian cell lines that are known to aberrantly secret lysosomal proteases. The mislocalization of these proteases has been proposed to potentiate the metastasis of tumor cells. The proposed studies focus on two of the VPS genes, VPS15 and VPS34. Mutations in each gene result in a common set of phenotypes suggesting that each may facilitate the same step in the vacuolar protein delivery pathway. The sequence of the VPS15 gene predicts a protein with the following features: a concensus site for N-terminal myristoylation, a region of homology with the regulatory subunit of protein phosphatase 2A (PP2A). The VPS34 gene product shares significant sequence similarity with the catalytic subunit of mammalian phosphatidylinositol-3-kinase. VPS34 P13-kinase activity may be regulated by the VPS15 gene product. We have found that overexpression of the Vps34 protein can suppress the defects caused by mutation within the kinase domain of the VPS15 gene product, but will not suppress a null allele of VPS15. Using both genetic and biochemical approaches, we propose to characterize: 1) the functional role of the Vps15 kinase domain, as well as protein phosphorylation, in vacuolar protein sorting, 2) the significance of the sequence homology in Vps15p to the PP2A regulatory subunit, 3) the subcellular location and possible site of action for both the Vps15 and Vps34 proteins, and 4) the potential interaction of Vps15 and Vps34 proteins with each other and possibly other cellular components (e.g., PP2A).