CHARACTERIZATION OF NEW VACUOLAR SEGREGATION MUTANTS, ISOLATED BY SCREENING FOR LOSS OF PROTEINASE-B SELF-ACTIVATION

Part of the vacuole in the mother cell of Saccharomyces cerevisiae is segregated early in the cell cycle to establish a new vacuole in the b ud, Investigation of the molecular mechanism of vacuolar segregation h as previously been limited by the lack of an efficient screen for muta nts defective in this process. We developed a new screening procedure based on a cascade for activation of vacuolar proteases, Carboxypeptid ase Y (CPY) is activated by proteinase A (PrA), However, upon PrA depl etion, CPY continues to be activated, supposedly by self-sustaining pr oteinase B (PrB) activity that is transferred from one generation to t he next generation through vacuolar segregation. In this study fourtee n mutants were isolated that failed to sustain CPY activation upon PrA depletion. While these mutants had altered vacuolar protease-activity levels, two mutants showed specific vacuolar segregation defects, The y formed large-budded cells that contained no vacuole or extremely sma ll vacuoles in the bud. These mutants represented two complementation groups, named VAC6 and VAC7. The data indicate that constitutive vacuo lar segregation mutants are viable, but that they are unable to transf er proteolytic activities from mother vacuole to the bud, Surprisingly , despite the apparent lack of quantitative vacuolar inheritance, all daughter cells of vac6 and vac7 had obtained a vacuole before cell div ision.