) 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 protein of 396 amino acids. Overexpression in Escherichia coli allowed the ready purification of a recombinant form of the enzyme. Disruption of the gene did not affect the growth rate of S. cerevisiae. Lysates from the mutants displayed considerably lower exopolyphosphatase activity than the wild type. The enzyme is located in the cytosol, whereas the vast accumulation of polyphosphate (polyP) of the yeast is in the vacuole. Disruption of PPX1 in strains with and without deficiencies in vacuolar proteases allowed the identification of exopolyphosphatase activity in the vacuole. This residual activity was strongly reduced in the absence of vacuolar proteases, indicating a dependence on proteolytic activation. A 50-fold-lower protease-independent activity could be distinguished from this protease-dependent activity by different patterns of expression during growth and activation by arginine. With regard to the levels of polyP in various mutants, those deficient in vacuolar ATPase retain less than 1% of the cellular polyP, a loss that is not offset by additional mutations that eliminate the cytosolic exopolyphosphatase and the vacuolar polyphosphatases dependent on vacuolar protease processing.Inorganic polyphosphates (polyP) are widespread in nature and are particularly abundant in Saccharomyces cerevisiae, accounting for nearly 40% of the total phosphate content (22). The polyP chains of S. cerevisiae vary between three and several thousand P i residues (19). Among the various yeast cell compartments, the bulk is in the vacuoles (38), with lesser amounts and smaller chains found in mitochondria (4); in Saccharomyces fragilis, polyP has also been found on the cell surface (37). The distinct locations and sizes of polyP in yeast cells indicate either different pathways of biosynthesis and degradation or a sophisticated sorting system. These different polyP pools might be involved in a variety of functions, such as energy storage, P i reservoir, metal chelator, buffer against alkali, and regulator. None of these functions for polyP have been established in S. cerevisiae, whereas in Escherichia coli, a requirement for polyP for survival in stationary phase has been demonstrated (8).Polyphosphatase (polyPase) activity in S. cerevisiae is demonstrably high: a cell extract releases up to 130 nmol of P i per min per mg of yeast protein (42). In contrast, biosynthetic activity is rather feeble in vitro. Polyphosphate kinase, the enzyme that can make polyP in a reversible reaction by condensing the terminal phosphate of ATP, catalyzes the transfer of only about 4 nmol of phosphate residues from polyP to ADP per min per mg of yeast protein and is even less active in the synthesis of polyP (12,34). In view of the high level of polyPase activity, effective mechanisms must exist to protect polyP from hydrolytic degradation. Also implied are functions for the polyPase in using polyP as a reservoir...

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