PPX1 gene overexpression has no influence on polyphosphates in Saccharomyces cerevisiae.

The role of exopolyphosphatase PPX1 in polyphosphate metabolism in yeasts has been studied in strains of Saccharomyces cerevisiae with inactivated PPX1 and PPN1 genes transformed by the expression vector carrying the yeast PPX1 gene. Exopolyphosphatase activity in transformant strains increased 90- and 40-fold compared to the ΔPPX1 and ΔPPN1 strains, respectively. The purified recombinant exopolyphosphatase PPX1 was similar to the PPX1 of wild strains in its substrate specificity and requirement for divalent metal cations. It was more active with tripolyphosphate and low molecular mass polyphosphates than with high molecular mass polyphosphates and required Mg2+ for its activity. The high level of recombinant PPX1 expression caused no decrease in polyphosphate content in the cells of the transformant. This fact suggests the restricted role of PPX1 in polyphosphate metabolism in yeasts.

Diversity of phosphorus reserves in microorganisms.

Phosphorus compounds are indispensable components of the Earth's biomass metabolized by all living organisms. Under excess of phosphorus compounds in the environment, microorganisms accumulate reserve phosphorus compounds that are used under phosphorus limitation. These compounds vary in their structure and also perform structural and regulatory functions in microbial cells. The most common phosphorus reserve in microorganism is inorganic polyphosphates, but in some archae and bacteria insoluble magnesium phosphate plays this role. Some yeasts produce phosphomannan as a phosphorus reserve. This review covers also other topics, i.e. accumulation of phosphorus reserves under nutrient limitation, phosphorus reserves in activated sludge, mycorrhiza, and the role of mineral phosphorus compounds in mammals.

Extracellular phosphomannan as a phosphate reserve in the yeast Kuraishia capsulata.

We have found that extracellular phosphomannan is the main phosphate reserve in the yeast Kuraishia capsulata, in contrast to other yeast species effectively absorbing Pi. Under nitrogen starvation, K. capsulata absorbed essentially all Pi from the medium containing 240 mM glucose, 2.5 mM MgSO4, and 11 mM KH2PO4. Inorganic polyphosphate level in the cells was about 14% of the Pi absorbed. Most of the Pi (~60%) was found in the fraction of extracellular phosphomannan that can be used as a carbon and phosphorus source by this yeast in deficient media.

Inorganic polyphosphates in mitochondria.

Current data concerning the crucial role of inorganic polyphosphates (polyP) in mitochondrial functions and dysfunctions in yeast and animal cells are reviewed. Biopolymers with short chain length (approximately 15 phosphate residues) were found in the mitochondria of Saccharomyces cerevisiae. They comprised 7-10% of the total polyP content of the cell. The polyP are located in the membranes and intermembrane space of mitochondria. The mitochondrial membranes possess polyP/Ca2+/polyhydroxybutyrate complexes. PolyP accumulation is typical of promitochondria but not of functionally active mitochondria. Yeast mitochondria possess two exopolyphosphatases splitting P(i) from the end of the polyP chain. One of them, encoded by the PPX1 gene, is located in the matrix; the other one, encoded by the PPN1 gene, is membrane-bound. Formation of well-developed mitochondria in the cells of S. cerevisiae after glucose depletion is accompanied by decrease in the polyP level and the chain length. In PPN1 mutants, the polyP chain length increased under glucose consumption, and the formation of well-developed mitochondria was blocked. These mutants were defective in respiration functions and consumption of oxidizable carbon sources such as lactate and ethanol. Since polyP is a compound with high-energy bonds, its metabolism vitally depends on the cell bioenergetics. The maximal level of short-chain acid-soluble polyP was observed in S. cerevisiae under consumption of glucose, while the long-chain polyP prevailed under ethanol consumption. In insects, polyP in the mitochondria change drastically during ontogenetic development, indicating involvement of the polymers in the regulation of mitochondrial metabolism during ontogenesis. In human cell lines, specific reduction of mitochondrial polyP under expression of yeast exopolyphosphatase PPX1 significantly modulates mitochondrial bioenergetics and transport.

Properties of partially purified endopolyphosphatase of the yeast Saccharomyces cerevisiae.

Partially purified endopolyphosphatase from cytosol of the yeast Saccharomyces cerevisiae with inactivated genes PPX1 and PPN1 encoding exopolyphosphatases was obtained with ion-exchange and affinity chromatography. The enzyme activity was estimated by decrease of polyphosphate chain length determined by PAGE. The enzyme cleaved inorganic polyphosphate without the release of orthophosphate (P(i)) and was inhibited by heparin and insensitive to fluoride. Mg2+, Mn2+, and Co2+ (1.5 mM) stimulated the activity, and Ca2+ was ineffective. The molecular mass of the endopolyphosphatase determined by gel filtration was of ~20 kDa.

Finding of endopolyphosphatase activity in the yeast Saccharomyces cerevisiae.

Endopolyphosphatase activity has been revealed in cytosol preparations of the yeast Saccharomyces cerevisiae with inactivated PPX1 and PPN1 genes encoding exopolyphosphatases. The enzyme cleaves inorganic polyphosphates with chain length of 15 to 208 phosphate residues to shorter chains without the release of orthophosphate (P(i)). The long chain polyphosphates are cleaved with preference over the short ones. Heparin, a known inhibitor of exopolyphosphatases, represses this activity. The endopolyphosphatase activity is not stimulated by Mg(2+) or Co(2+), in contrast to exopolyphosphatases. This activity along with a pyrophosphatase is supposed to be responsible for polyphosphate utilization as a phosphate reserve in a mutant devoid of exopolyphosphatases.

Inactivation of PPX1 and PPN1 genes encoding exopolyphosphatases of Saccharomyces cerevisiae does not prevent utilization of polyphosphates as phosphate reserve.

Cytosol polyphosphates (polyPs) are the main phosphate (P(i)) reserve in the yeast Saccharomyces cerevisiae. In this work, the participation of cytosol polyPs and exopolyphosphatases in maintenance of P(i) homeostasis under P(i) deficit in the cultivation medium has been studied in different strains of S. cerevisiae. The growth of yeast strains with inactivated genes PPX1 and PPN1 encoding the yeast exopolyphosphatases and a strain with double mutations in these genes in a P(i)-deficient medium is not disturbed. All the studied strains are able to maintain relatively constant P(i) levels in the cytosol. In P(i)-deficient medium, polyP hydrolysis in the cytosol of the parent and PPN1-deficient strains seems to be performed by exopolyphosphatase Ppx1 and proceeds without any change of the spectrum of polyP chain lengths. In the PPX1-deficient strain, long-chain polyPs are depleted first, and only then short-chain polyPs are hydrolyzed. In the double PPX1 and PPN1 mutant having low exopolyphosphatase activity, polyP hydrolysis in the cytosol starts with a notable delay, and about 20% of short-chain polyPs still remain after the polyP hydrolysis in other strains has almost been completed. This fact suggests that S. cerevisiae possesses a system, which makes it possible to compensate for inactivation of the PPX1 and PPN1 genes encoding exopolyphosphatases of the yeast cells.

Inorganic polyphosphates and exopolyphosphatases in different cell compartments of Saccharomyces cerevisiae.

The cytosol, nuclei, vacuoles, and mitochondria of the yeast Saccharomyces cerevisiae possess inorganic polyphosphates (polyPs). PolyP levels, spectra of polyP chain lengths, and their dependence on the growth phase are distinguished in the mentioned compartments. Inactivation of the PPX1 gene has no effect on the polyP metabolism under cultivation of the yeast in medium with glucose and 5-7 mM P(i). Inactivation of the PPN1 gene results in elimination of the high-molecular-mass exopolyphosphatases (approximately 120 to 830 kD) of the cytosol, nuclei, vacuoles, and mitochondria of S. cerevisiae suggesting that it is just PPN1 that encodes these enzymes. Expression of the low-molecular-mass exopolyphosphatase of approximately 45 kD encoded by the PPX1 gene decreases under PPN1 inactivation as well. While PPN1 inactivation has negligible effect on polyP levels, it results in increase in the long-chain polyPs in all the compartments under study.

[Inactivation of the ppn1 gene exerts different effects on the metabolism of inorganic polyphosphates in the cytosol and the vacuoles of the yeast Saccharomyces cerevisiae].

Inactivation of the PPN1 gene, encoding one of the enzymes involved in polyphosphate metabolism in the yeast Saccharomyces cerevisiae, was found to decrease exopolyphosphatase activity in the cytosol and vacuoles. This effect was more pronounced in the stationary growth phase than in the phase of active growth. The gene inactivation resulted in elimination of a approximately 440-kDa exopolyphosphatase in the vacuoles but did not influence a previously unknown vacuolar exopolyphosphatase with a molecular mass of >1000 kDa, which differed from the former enzyme in the requirement for bivalent cations and sensitivity to heparin. Inactivation of the PPN1 gene did not influence the level of polyphosphates in the cytosol but increased it more than twofold in the vacuoles. In this case, the polyphosphate chain length in the cytosol increased from 10-15 to 130 phosphate residues both in the stationary and active growth phases. In the vacuoles, the polyphosphate length increased only in the stationary growth phase. A conclusion can be made that the PPN1 gene product has different effects on polyphosphate metabolism in the cytosol and the vacuoles.

[The effect of inactivation of the exo- and endopolyphosphatase genes PPX1 and PPN1 on the level of different polyphosphates in the yeast Saccharomyces cerevisiae].

The inactivation of the PPX1 and PPN1 genes, which encode the major enzymes of polyphosphate degradation (exopolyphosphatase and endopolyphosphatase, respectively), was found to exert different effects on the content of different polyphosphates in the yeast Saccharomyces cerevisiae. The content of relatively low-molecular-weight acid-soluble polyphosphates in mutant yeast strains is inversely proportional to the exopolyphosphatase activity of the cytosol. At the same time, the mutation of these genes exerts no effect on salt-soluble polyphosphates. The content of high-molecular-weight alkali-soluble polyphosphates increases twofold in a mutant with inactivated genes of both exopolyphosphatase and endopolyphosphatase. The data obtained confirm the earlier suggestion that the metabolic pathways of particular polyphosphates in yeasts are different.

[Peculiarities of metabolism and functions of high-molecular inorganic polyphosphates in yeasts as representatives of lower eukaryotes].

The review presents the recent data demonstrating the important role high-molecular inorganic polyphosphates in regulatory processes in a yeast cell. It has been shown that polyphosphates are localized in different cell compartments, where they are metabolized by a special set of enzymes. The review presents the evidence in favor of the concept of multiple functions of these biopolymers in a cell, as well as the data on the pleiotropic effects of mutations in the genes encoding the enzymes of polyphosphate metabolism.

Partial purification and characterization of nuclear exopolyphosphatase from Saccharomyces cerevisiae strain with inactivated PPX1 gene encoding a major yeast exopolyphosphatase.

Inactivation of PPX1 encoding the major cytosolic exopolyphosphatase PPX1 in Saccharomyces cerevisiae did not alter exopolyphosphatase activity of the isolated nuclei compared with that in the parent strain. The nuclear exopolyphosphatase of the S. cerevisiae strain deficient in the PPX1 gene was purified 10-fold. According to gel filtration on Superose 6, this enzyme has a molecular mass of approximately 200 kD, and it hydrolyzes polyphosphates with an average chain length of 15 and 208 phosphate residues to the same extent. Its activity is much lower with tripolyphosphate. In the presence of 2.5 mM Mg2+, Km values are 133 and 25 microM in the hydrolysis of polyphosphates with chain lengths of 15 and 208 phosphate residues, respectively. The enzyme activity is stimulated by 2.5 mM Mg2+ and 0.1 mM Co2+ 15- and 31-fold, respectively. RNA does not alter the nuclear exopolyphosphatase activity, while polylysine increases it 2-fold.

Effect of PPX1 inactivation on the exopolyphosphatase spectra in cytosol and mitochondria of the yeast Saccharomyces cerevisiae.

Inactivation of PPX1 encoding exopolyphosphatase PPX1 in Saccharomyces cerevisiae results in a change in the exopolyphosphatase spectrum in the yeast cells. In the PPX1-deficient strain, elimination of an approximately 45 kD exopolyphosphatase is observed in the cytosol, and activity of an exopolyphosphatase with molecular mass of approximately 830 kD increases fivefold. The latter activity differs greatly in properties from the low-molecular-mass enzyme of the parent strain. In the soluble fraction of the mutant mitochondria, exopolyphosphatase of approximately 45 kD characteristic of the soluble mitochondrial fraction in the parent strain is eliminated, and exopolyphosphatase with a molecular mass of approximately 440 to approximately 830 kD is found. On PPX1 inactivation, a membrane-bound form of mitochondrial exopolyphosphatase is unaffected in its activity level and properties. Therefore, the membrane-bound exopolyphosphatase of mitochondria and the high-molecular-mass enzyme of the cytosol of S. cerevisiae are not encoded by the PPX1 gene, unlike the soluble low-molecular-mass exopolyphosphatase of mitochondria, which is probably a product of this gene with a posttranslational modification. In the PPX1 mutant, exopolyphosphatase properties in the cell as a whole undergo modifications including the ability to hydrolyze polyphosphates (polyP) with different polymer degree.

The development of A. N. Belozersky's ideas in polyphosphate biochemistry.

This review covers some trends and approaches to the study of inorganic polyphosphates that originated from the fruitful ideas and pioneering works of A. N. Belozersky. This is, first of all, the elucidation of a close relationship between these biopolymers and nucleic acids in organisms at different evolutionary stages; second, the study of "fossil" reactions in polyphosphate metabolism that permit an understanding of their role in the evolution of phosphorus turnover and cell bioenergetics; third, the possible use of the conservative enzymes of polyphosphate metabolism, e.g., exopolyphosphatases, as molecular chronometers for obtaining additional data concerning the theory of the endosymbiotic origin of eukaryotic cells from prokaryotes.

Purification and characterization of a soluble polyphosphatase from mitochondria of Saccharomyces cerevisiae.

A polyphosphatase with the specific activity 2.2 U/mg was purified to apparent homogeneity from a soluble preparation of mitochondria of Saccharomyces cerevisiae. The polyphosphatase is a monomeric protein of approximately 41 kD. The purified enzyme hydrolyzes polyphosphates with an average chain length of 9 to 208 phosphate residues to the same extent, but its activity is approximately 2-fold higher with tripolyphosphate. ATP, PPi, and p-nitrophenyl phosphate are not substrates of this enzyme. The apparent Km values are 300, 18, and 0.25 microM obtained at hydrolysis of polyphosphates with a chain length of 3, 15, and 188 phosphate residues, respectively. Several divalent cations stimulated the enzyme activity 1.2-27-fold (Mg2+ = Co2+ = Mn2+ > Zn2+). Determination of the protein N-terminal sequence and its comparison with the EMBL data library indicates that the soluble polyphosphatase of mitochondria of S. cerevisiae is not encoded by the gene of the major yeast polyphosphatase PPX1.

Comparison of exopolyphosphatases of different yeast cell compartments.

Purified cell-envelope polyphosphatase as well as polyphoshatase activities of cytosol and isolated vacuoles, of nuclei and mitochondria of the yeast Saccharomyces cerevisiae were compared. The polyphosphatases of cell envelope and cytosol are similar, the polyphosphatases of nuclei, vacuoles and mitochondria differ in their kinetic properties, substrate specificity, requirements in divalent cations and in some effector actions both from these and from each other.

Detection and some properties of membrane-bound and soluble polyphosphatases in mitochondria of the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae mitochondria possess polyphosphatases that are tightly bound to the membranes and differ from soluble polyphosphatase of these organelles in a number of properties. Molecular weights of the membrane-bound polyphosphatases are 120 and 76 kD, and the molecular weight of the soluble polyphosphatase is about 36 kD. All three enzymes are evidently monomers, since antibodies against purified cell-envelope polyphosphatase of S. cerevisiae reacted with 115, 78, and 37 kD polypeptides in immunoblotting. The activities of membrane-bound and soluble polyphosphatase are maximal at neutral pH. The soluble polyphosphatase activity is stimulated by divalent cations, unlike the membrane-bound enzymes which are inhibited by the same cations including Mg2+. Monovalent cations do not affect the activity of the soluble enzyme but stimulate polyphosphatases in the membrane preparation. The specific activities for hydrolysis of polyphosphates with average chain lengths of 9 to 188 phosphate residues are enhanced by increasing the degree of substrate polymerization in the case of the membrane preparation and are unchanged in case of the soluble enzyme. Affinity of the soluble enzyme to polyphosphates is 5-10 times higher than that of the membrane-bound polyphosphatases. In the soluble fraction of mitochondria, high tripolyphosphatase activity is detected which is approximately 80% of that in isolated mitochondria.