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

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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.