ROLE OF THE YEAST VPS15 PROTEIN KINASE IN INTRACELLULAR PROTEIN SORTING

蛋白质的选择性识别、分类和运输 胞内细胞器通过囊泡载体传输到另一个细胞器代表着一种 所有真核细胞的基本特征。精确的机械装置和 指导这些过程的机制尚不清楚。我们有 分离出大量表现出严重缺陷的突变体 蛋白质输送到酵母溶酶体样液泡。有缺陷的基因 在这些突变体中很可能编码细胞蛋白质的组成部分 分拣设备。对分子和分子的理解 空泡蛋白分选缺陷的生化基础 突变体(VP)，我们建议对VPS的某些角色进行表征 基因及其产物在液泡分选和运输中的作用 酵母菌中从高尔基复合体到液泡的酶。最基本的 酵母与其他真核细胞途径的相似性 用于蛋白质的传递，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 解决这些问题。我们分离的酵母突变株展示了 一种与许多转化的哺乳动物细胞系相似的表型 已知异常秘密的溶酶体蛋白水解酶。错误的本地化 这些蛋白水解酶被认为可以促进肿瘤的转移。 细胞。 拟议的研究集中在VPS基因中的两个，VPS15和Vps34。 每个基因的突变都会导致一组共同的表型 每一个都可能促进液泡蛋白递送的相同步骤 路径。VPS15基因的序列预测一种带有 以下特征：N-末端肉豆蔻酰化的汇合部位，a 与蛋白磷酸酶2A调节亚基同源的区域 (PP2A)。Vps34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-激酶的催化亚单位结合。 VPS34 P13-激酶活性可能受VPS15基因产物的调节。 我们发现，Vps34蛋白的过表达可以抑制 VPS15基因激酶域突变引起的缺陷 产物，但不会抑制VPS15的零等位基因。两者都使用 遗传和生化方法，我们建议表征：1) Vps15蛋白激活区及其蛋白的功能 在空泡蛋白分选中的磷酸化，2) Vps15p中与PP2A调节亚单位的序列同源性，3) VPS15和VPS15的亚细胞位置和可能的作用部位 Vps34蛋白；4)Vps15和Vps34的潜在相互作用 彼此的蛋白质以及可能的其他细胞成分(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).