RUI: Characterization of a Yeast Gene (ENV7) Involved in Vacuole Structure and Function

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。溶酶体是真核细胞中的主要降解区室，负责清除不需要的细胞物质以及下调细胞表面的细胞通讯蛋白。因此，正确分选和递送分子到溶酶体是所有真核细胞的基本特征。晚期内体是两种不同的运输途径的交汇点到溶酶体：内吞途径和生物合成途径。因此，不同的运输事件发生在晚期内体，因为它与三个隔室：跨高尔基体网络，内吞内体和溶酶体。长期目标是确定晚期内体与溶酶体界面的调节过程。酵母酿酒酵母的晚期内体和液泡在功能上等同于哺乳动物晚期内体和溶酶体;其液泡递送的羧肽酶Y（CPY）途径与哺乳动物细胞中的甘露糖-6-磷酸途径平行。酵母提供了传统和分子遗传工具的优势;在溶酶体运输中鉴定的绝大多数酵母基因在人类中具有直向同源物。主要研究者将一种新型免疫检测筛选应用于基因组方法，以分离在递送和加工的内体至液泡阶段有缺陷的突变体（env突变体）。到目前为止，已经发现了几个新的ENV基因，包括三个以前没有被鉴定的孤儿基因。该项目涉及孤儿基因YPL 236 C（ENV 7）及其产物的分子表征，以及通过生物化学，显微镜和分子方法表征缺乏ENV 7基因的突变体。在缺乏Env 7蛋白的情况下，观察到高度碎片化的空泡，并且空泡酶的前体形式在细胞内积累。这些结果表明液泡功能和生物发生可能存在缺陷。基于已发表的基因组和生物信息学方法，Env 7蛋白似乎是锚定在液泡膜上的棕榈酰化蛋白;它含有丝氨酸/苏氨酸激酶结构域，并且在酵母中是独特的，可能是STK 16的同源物，STK 16是肉豆蔻酰化和棕榈酰化的哺乳动物丝氨酸/苏氨酸激酶。激酶是细胞中重要的调节酶，棕榈酰化是唯一已知的将蛋白质锚定到脂质膜的可逆机制。总之，这些可能性使得Env 7蛋白能够具有重要的调节功能。这个项目的智力价值是它的潜力，以确定一个独特的棕榈酰化激酶在液泡结构，运输和/或功能的作用。该项目的更广泛的影响是继续本科生和硕士生的生产性研究培训，使高中和社区学院的学生继续夏季研究培训，并保持一个全面的西班牙裔服务机构内的主要研究者的研究活力。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).The lysosome is the major degradative compartment in eukaryotic cells and is responsible for clearing of unwanted cellular material as well as down regulation of cellular communication proteins on the cell surface. Thus, correct sorting and delivery of molecules to the lysosome is an essential feature of all eukaryotic cells. The late endosome is the convergence point of two different trafficking pathways to the lysosome: the endocytic pathway and the biosynthetic pathway. As such, distinct trafficking events occur at the late endosome as it interfaces with three compartments: the trans golgi network, endocytic endosomes, and lysosomes. The long term goal is to define the regulatory processes at the late endosome to lysosome interface. The late endosome and vacuole of the yeast Saccharomyces cerevisiae are functionally equivalent to the mammalian late endosome and lysosome; its carboxy peptidase Y (CPY)-pathway of vacuolar delivery parallels the mannose-6-phosphate pathway in mammalian cells. Yeast offers the advantage of both conventional and molecular genetic tools; the large majority of yeast genes identified in lysosomal trafficking have orthologues in humans. The principal investigator has applied a novel immunodetection screen to a genomic approach for isolation of mutants defective at the endosome to vacuole stage of delivery and processing (env mutants). To date, several new ENV genes have been uncovered, including three orphan genes that have not been previously characterized. This project involves the molecular characterization of the orphan gene YPL236C (ENV7) and its product, and characterization of a mutant lacking the ENV7 gene by biochemical, microscopic, and molecular approaches. In the absence of Env7 protein, highly fragmented vacuoles are observed and a precursor form of a vacuole enzyme accumulates inside cells. These results indicate possible defects in vacuolar function and biogenesis. Based on published genomic and bioinformatic approaches, the Env7 protein appears to be a palmitoylated protein anchored to the vacuolar membrane; it contains serine/threonine kinase domains and is unique in yeast as a possible homologue of STK16, a myristoylated and palmitoylated mammalian serine/threonine kinase. Kinases are important regulatory enzymes in cells and palmitoylation is the only known reversible mechanism for anchoring proteins to lipid membranes. Together, these possibilities render Env7 protein capable of important regulatory functions. The intellectual merit of this project is its potential to identify the role of a unique palmitoylated kinase in vacuole structure, trafficking, and/or function. The broader impact of this project is to continue productive research training of undergraduate and masters students, enable continued summer research training of high school and community college students, and maintain the research vibrancy of the principal investigator within a comprehensive Hispanic Serving Institution.