Role for Protein Phosphorylation in Yeast Vacuolar Protein Sorting

拟议的研究集中在酵母空泡蛋白分类(VPS)基因之一VPS15。预测的VPS15基因产物与丝氨酸/苏氨酸蛋白激酶家族的催化结构域具有显著的序列相似性。使用遗传学和生物化学方法，将分析以下内容：(1)肉豆蔻酸化对Vps15p功能和定位的功能作用；(2)Vps15蛋白的亚细胞位置和可能的作用部位；(3)VPS15基因产物的可能的激酶活性。预计这些研究将对真核细胞高尔基体和液泡之间的囊泡运输的组织和控制，以及可能发生的其他细胞器间运输事件有一个详细的认识。蛋白质的选择性识别、分选和通过囊泡载体将蛋白质从一个细胞内细胞器转移到另一个细胞器的过程是所有真核细胞的一个基本特征。指导这些过程的确切机制和机制尚不清楚。该实验室已经分离出大量酵母突变株，这些突变株在向溶酶体样液泡运送蛋白质方面存在严重缺陷。这些突变体中的缺陷基因很可能编码细胞蛋白质分选装置的组件。为了了解这些突变体中液泡蛋白分离缺陷的分子和生化基础，本研究将表征某些VPS基因及其产物在酵母中液泡酶从高尔基复合体到液泡的分选和运输中所起的作用。酵母与其他真核细胞在蛋白质传递途径上的基本相似之处，再加上酵母强大的遗传学和分子方法，使酵母成为解决这些问题的理想生物。

The proposed studies focus on one of the yeast vacuolar protein sorting (VPS) genes, VPS15. The predicted product of the VPS15 gene shares significant sequence similarity with the catalytic domains of the family of serine/threonine protein kinases. Using both genetic and biochemical approaches, the following will be analyzed: (1) the functional role of myristoylation on Vps15p function and localization, (2) the subcellular location and possible site of action of the Vps15 protein, and (3) the presumed kinase activity of the VPS15 gene product. It is anticipated that these studies will result in a detailed view of the organization and control of vesicular traffic between the Golgi and the vacuole, and possibly other inter-organelle transport events in eukaryotic cells. The process of 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. This laboratory has isolated a large collection of yeast mutants that exhibit severe defects in protein delivery to the lysosome-like vacuole. The defective genes in these mutants are likely to encode components of the cell's protein sorting apparatus. Toward an understanding of the molecular and biochemical basis of the vacuolar protein sorting ("vps") defects in these mutants, this research will 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 genetics and molecular approaches available in yeast, make yeast an ideal organism for addressing these problems.