The yeast vacuole strongly resembles an animal cell lysosome, for it is an acid compartment, contains a set of hydrolases, is the final destination of ligands taken up by fluid phase endocytosis and receptormediated endocytosis. Since yeast is amenable to gentical, biochemical and molecular analyses, it affords an excellent model system for studies of the function and assembly of this important organelle. The overall goals of this research are to understand the role of the set of vacuolar hydrolases in the metabolism and differentiation of yeast, to understand how these enzyme activities are generated, to understand how the activities of these enzymes are regulated and integrated into cellular function and to understand the regulatory circuits that govern the genes for these enzymes.
Our specific aims for this period are 1) to genetically dissect the role that protease A plays in effecting processing of hydrolase precursors 2) to delineate the processing pathway for the protease B precursor and test whether our proposed pathway is correct and 3) to analyze the regulatory circuit that effects carbon catabolite repression and expression of the set of vacuolar proteases. To achieve these goals we will isolate and characterize mutants obtained by in vitro mutagenesis of the cloned protease. A structural gene and relate the phenotypic changes to the sequenced mutations. We will delineate the protease B processing pathway by kinetic experiments using antibodies and by engineering derivatives of the structural gene that should be exempt from a processing requirement. To analyze regulation we will place PRB1 upstream regulatory sequences upstream of the SUC2 and lacZ structural genes and use these hybrid genes to isolate regulatory mutations and to define cis regulatory sequences of PRB1.
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