THE GENE FOR A MAJOR EXOPOLYPHOSPHATASE OF SACCHAROMYCES-CEREVISIAE

The gene encoding a major exopolyphosphatase (scPPX1) in Saccharomyces cerevisiae (H. Wurst and A. Kornberg, J. Biol. Chem, 269:10996-11001, 1994) has been isolated from a genomic library, The gene, located at 57 kbp from the end of the right arm of chromosome VIII, encodes a pro tein of 396 amino acids, Overexpression in Escherichia coli allowed th e ready purification of a recombinant form of the enzyme. Disruption o f the gene did not affect the growth rate of S. cerevisiae, Lysates fr om the mutants displayed considerably lower exopolyphosphatase activit y than the wild type, The enzyme is located in the cytosol, whereas th e vast accumulation of polyphosphate (polyP) of the yeast is in the va cuole, Disruption of PPX1 in strains with and without deficiencies in vacuolar proteases allowed the identification of exopolyphosphatase ac tivity in the vacuole, This residual activity was strongly reduced in the absence of vacuolar proteases, indicating a dependence on proteoly tic activation, A 50-fold-lower protease-independent activity could be distinguished from this protease-dependent activity by different patt erns of expression during growth and activation by arginine, With rega rd to the levels of polyP in various mutants, those deficient in vacuo lar ATPase retain less than 1% of the cellular polyP, a loss that is n ot offset by additional mutations that eliminate the cytosolic exopoly phosphatase and the vacuolar polyphosphatases dependent on vacuolar pr otease processing.