The work is aimed at understanding the sorting and transport of membrane proteins to the yeast lysosome-like vacuole. The simple eukaryote yeast will be used as a model eukaryotic sorting system, since the secretory and vacuole assembly pathways are very similar to the pathways in animal cells. Studies in yeast offer a unique opportunity to investigate the complex processes involved in membrane protein sorting and transport by taking advantage of the ability to exploit the powerful genetic approaches available in yeast. It also appears likely that the basic cellular functions that facilitate sorting of vacuolar/lysosomal membrane proteins will be conserved across all eukaryotic cells. Yeast mutants that mislocalize the vacuolar membrane protein dipeptidyl amino-peptidase (DPAP-B) will be obtained by exploiting a newly developed selection procedure. These mutants will be screened biochemically and by immunogold labeling for the secretion of a large number of soluble and membrane-bound vacuolar proteins. In addition, a major effort will be made towards identifying the vacuolar sorting and transport signals present on the membrane protein DPAP-B. Mutations in the DPAP-B structural gene will be generated and those resulting in missorting of enzymatically active DPAP-B will be identified with the DPAP-B mislocalization selection procedure. The structural genes of the two largest subunits of the yeast vacuolar membrane H+-translocating ATPase will be cloned using the Lambdagtll yeast library. Mutations will be constructed in these genes to elucidate the role of this H+-ATPase in acidification of the vacuole. These mutations will also permit an analysis of the role of acidification in the sorting of newly synthesized vacuolar hydrolases, in fluid-phase and receptor-mediated endocytosis, and in the function of the vacuole. The biosynthesis, assembly, targeting and transport of this vacuolar multi-subunit membrane-bound H+-ATPase complex will be investigated in an effort to understand the relationship between the synthesis and assembly of the subunits and their transport to the vacuole. These studies are likely to increase our basic understanding of diseases that result from missorting of lysosomal hydrolases such as Mucolipidosis II and III and other lysosomal storage diseases.
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