The yeast vacuole resembles an animal cell lysosome, for it is an acidic compartment, contains a complement of hydrolases, and is the final destination of ligands taken up by fluid phase and receptor-mediated endocytosis. Since yeast is amenable to genetical, biochemical and molecular analysis, it affords an excellent model system for studies of the function and assembly of this important organelle. These studies may contribute to our understanding of the mannose-6-phosphate independent pathway of lysosomal/vacuolar enzyme targeting and of translocation and chaperone functions (including intramolecular chaperones) required for lysosomal/vacuolar and secreted proteins that have a high charge density (like prostromelysin and procollagenase). The overall goals of this research are to understand the role of the set of vacuolar proteases in the metabolism and differentiation of yeast, how the activities of these enzymes are generated, regulated and integrated into cellular function, especially in relation to other regulatory circuits that also respond to glucose levels.
Specific aims are: (1) To determine whether the acidic pH of the vacuole triggers the autocatalytic activation of the proteinase A (PrA) precursor in vivo, the step proposed to initiate the cascade that results in activation of all other hydrolase precursors. Synthesis and maturation of preproPrA will be followed kinetically. (2) To dissect genetically structure-function relationships of the proteinase B(PrB) precursor, including identification of regions responsible for its intramolecular chaperone function and for targeting to the vacuole. Mutations that compromise vacuolar targeting of a PRB1-SUC2 fusion will be selected and analyzed. (3) To identify gene products required for translocation of the highly charged PrB precursor into the lumen of the endoplasmic reticulum. Mutants that fails to translocate a PRB1-URA3 fusion protein will be selected and analyzed. (4) To continue studies on regulation of the vacuolar proteases, with an emphasis on PrB, since it is the most highly regulated of the protease complement. Mutants that fail to express or express constitutively PPB1-URA3, PRB1-lacZ and/or PRB1-SUC2 fusions will be selected or screened for an analyzed. Initially, the focus will be on a PrB (or protease) specific circuit. Analysis of integrating circuitry will follow.
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