The selective recognition, sorting and vesicle-mediated transport of proteins through the secretory pathway 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 yeast mutants that exhibit severe and specific defects in protein delivery to the lysosome-like vacuole in yeast. The defective genes in these mutants 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. 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 medical importance of this sorting pathway is exemplified by the serious lysosomal storage diseases (e.g. I-cell disease, pseudo-Hurler polydystrophy as well as other diseases (e.g. osteoporosis and the progression of certain types of cancer) that result from, or are correlated with, mislocalization of lysosomal hydrolases. Eight VPS genes have been cloned and sequenced in the lab. To determine where and how the products of these genes act to direct the sorting and delivery of vacuolar hydrolases, we propose to: 1) use indirect immunofluorescence and cell fractionation techniques to determine the subcellular location of the VPS gene products, 2) isolate temperature- conditional vps alleles to analyze the onset kinetics of the vacuolar protein sorting defects and to identify potential intermediates in the sorting process, 3) use genetic suppression and chemical cross-linking agents to identify and characterize interactions among VPS gene products, 4) employ an in vitro assay that reconstitutes vacuolar protein delivery to characterize the stage specific requirements and functions of Vps proteins, and 5) clone and sequence functional homologs for certain VPS genes from cDNA libraries of Schizosaccharomyces pombe as well as mammalian (human) cell types to begin an analysis of the functional role VPS gene products play in other eukaryotes. It is anticipated that these studies will result in a detailed view of the organization and control of protein sorting and vesicular traffic between the Golgi complex and the vacuole/lysosome in yeast and other eukaryotes.
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