The endopolyphosphatase gene: Essential in Saccharomyces cerevisiae

Endopolyphosphatases (Ppn1) from yeast and animal cells hydrolyze inorganic polyphosphate (poly P) chains of many hundreds of phosphate residues into shorter lengths. The limit digest consists predominantly of chains of 60 (P -60) and 3 (P-3) P-i residues. Ppn1 of Saccharomyces cerevisiae, a homodime r of 35-kDa subunits (about 352-aa) is of vacuolar origin and requires the protease activation of a 75-kDa (674-aa) precursor polypeptide. The Ppn1 ge ne (PPN1) now has been cloned, sequenced, overexpressed, and deleted. That PPN1 encodes Ppn1 was verified by a 25-fold increase in Ppn1 when overexpre ssed under a GAL promoter and also by several peptide sequences that match exactly with sequences in a yeast genome ORF, the mutation of which abolish es Ppn1 activity. Null mutants in Ppn1 accumulate long-chain poly P and are defective in growth in minimal media. A double mutant of PPN1 and PPX1 (th e gene encoding a potent exopolyphosphatase) loses viability rapidly in sta tionary phase. Whether this loss is a result of the excess of long-chain po ly P or to the lack of shorter chains (i.e., poly P-60 and P-3) is unknown. Overexpression of the processed form of Ppn1 should provide a unique and p owerful reagent to analyze poly P when the chain termini are unavailable to the actions of polyPase and poly P kinase.