Identify Gene Functions at late endosome & lysosome interface in yeast

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DESCRIPTION (provided by applicant): Correct sorting and vesicular trafficking of molecules to the degradative lysosome is an essential feature of all eukaryotic cells. The late endosome is the convergence point of the endocytic pathway and the biosynthetic pathway of trafficking to the lysosome. As such, distinct trafficking events occur at the late endosome as it interfaces with three compartments: TGN, endocytic endosomes, and Iysosomes. Our 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; large majority of yeast genes identified in lysosomal trafficking have orthologues in humans. Using a novel immunodetection screen, we have isolated four mutants that are defective at endosome to vacuole stage of CPY delivery and processing (env mutants). Characterizations of the mutants have established defects in both late endocytic steps and vacuolar structure/function. The first gene of the collection has been cloned; env1 is a unique allele of VPS35 that defines a second role for its conserved gene product. In this proposal, our specific aims are to complete a genomic approach to directly identify additional ENV genes from a yeast deletion library and to clone and molecularly characterize ENV3. Inherent in this AREA proposal, is the aim to continue productive research training of undergraduate and masters students within a comprehensive, Hispanic Serving Institute. Mislocalization of lysosomal proteases and cargo is associated with Lysosomal Storage Diseases and Alzheimer's Disease (AD). Defects specifically at the late endosome to lysosome stage of trafficking have emerged as the possible underlying mechanism in both juvenile and aging neurodegeneration diseases. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health issues associated with neurodegeneration of aging as manifested in Alzheimer's Disease. It is also relevant to public health issues associated with the progressive neurodegeneration of juvenile patients with lysosomal storage diseases.