Effect of a carbon source on polyphosphate accumulation in Saccharomyces cerevisiae

The cells of Saccharomyces cerevisiae accumulate inorganic polyphosphate (polyP) when reinoculated on a phosphate-containing medium after phosphorus starvation. Total polyP accumulation was similar at cultivation on both glucose and ethanol. Five separate fractions of polyP: acid-soluble fraction polyP1, salt-soluble fraction polyP2, weakly alkali-soluble fraction polyP3, alkali-soluble fraction polyP4, and polyP5, have been obtained from the cells grown on glucose and ethanol under phosphate overplus. The dynamics of polyP fractions depend on a carbon source. The accumulation rates for fractions polyP2 and polyP4 were independent of the carbon source. The accumulation rates of polyP1 and polyP3 were higher on glucose, while fraction polyP5 accumulated faster on ethanol. As to the maximal polyP levels, they were independent of the carbon source for fractions polyP2, polyP3, and polyP4. The maximal level of fraction polyP1 was higher on glucose than on ethanol, but the level of fraction polyP5 was higher on ethanol. It was assumed that accumulation of separate polyP fractions has a metabolic interrelation with different energy-providing pathways. The polyphosphate nature of fraction polyP5 was demonstrated for the first time by ³¹P nuclear magnetic resonance spectroscopy, enzymatic assay, and electrophoresis.     

Inorganic polyphosphate in mitochondria of Saccharomyces cerevisiae at phosphate limitation and phosphate excess

Isolated mitochondria of Saccharomyces cerevisiae cells grown on glucose possess acid-soluble inorganic polyphosphate (polyP). Its level strongly depends on phosphate (P(i)) concentration in the culture medium. The polyP level in mitochondria showed 11-fold decrease under 0.8 mM P(i) as compared with 19.3 mM P(i). When spheroplasts isolated from P(i)-starved cells were incubated in the P(i)-complete medium, they accumulated polyP and exhibited a phosphate overplus effect. Under phosphate overplus the polyP level in mitochondria was two times higher than in the complete medium without preliminary P(i) starvation. The average chain length of polyP in mitochondria was of <15 phosphate residues at 19.3 mM P(i) in the culture medium and increased at phosphate overplus. Deoxyglucose inhibited polyP accumulation in spheroplasts, but had no effect on polyP accumulation in mitochondria. Uncouplers (FCCP, dinitrophenol) and ionophores (monensin, nigericin) inhibited polyP accumulation in mitochondria more efficiently than in spheroplasts. Fast hydrolysis of polyP was observed after sonication of isolated mitochondria. Probably, the accumulation of polyP in mitochondria depended on the proton-motive force of their membranes.     

Study of the content of inorganic polyphosphates in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> grown on different carbon sources with different O<Subscript>2</Subscript> concentrations in the medium

The content of different fractions of inorganic polyphosphates (polyP) was studied in Saccharomyces cerevisiae VKM Y-1173 growing on a complete medium with glucose under hypoxia and active aeration as well as on ethanol. The highest growth rate was observed for aerobic fermentation, while the yield of biomass was maximal for cultivation on ethanol. In the mid-log growth phase, the amount of poly P was maximal in the cells grown on glucose under hypoxia and minimal on ethanol. In this latter case, the content of different poly P fractions changed unevenly: polyP3, polyP4, and polyP1 decreased by approximately 60%, 45%, and 30%, respectively; the salt-soluble polyP2 remained at almost the same level; while polyP5 abruptly increased 10-to 15-fold. These findings demonstrate that the metabolic pathways for poly P fractions are different. A significant drop in the amount of the main poly P fractions accompanied by a decrease of the poly P average chain length in the presence of carbon and P_i sources in the medium is evidence of active involvement of poly P as additional energy sources in the flows of energy in actively growing yeast cells.     

Inorganic polyphosphates of different fractions in the mutant yeast Saccharomyces cerevisiae with impaired mitochondrial ATP synthesis

Impaired synthetase function of the mitochondrial ATPase induced by mutation in the ATP22 gene results in decreased accumulation of inorganic polyphosphates in the stationary growth phase of the yeast Saccharomyces cerevisiae grown on glucose. The content of polyphosphates in the mutant strain in this phase is 2.5 times lower than in the parent strain. This difference is most pronounced for the acid-soluble polyP1 fraction and the alkali-soluble polyP3 fraction. Polyphosphate chain length in mutant cells is less than in the parent cells in both the acid-soluble polyP1 and in the salt-soluble polyP2 fractions. The mutation had no effect on polyphosphates content in the mitochondria.     

Polyphosphates as an energy source for growth of Saccharomyces cerevisiae

Cells of the yeast Saccharomyces cerevisiae with a low content of polyphosphates (polyP) are characterized by disturbance of growth in medium with 0.5% glucose. The parent strain with polyP level reduced by phosphate starvation had a longer lag phase. The growth rate of strains with genetically determined low content of polyP due to their enhanced hydrolysis (CRN/pMB1_PPN1 Sc is a superproducer of exopolyphosphatase PPN1) or reduced synthesis (the BY4741 vma2Δ mutant with impaired vacuolar membrane energization) was lower in the exponential phase. The growth of cells with high content of polyP was accompanied by polyP consumption. In cells of strains with low content of polyP, CRN/pMB1_PPN1 Sc and BY4741 vma2Δ, their consumption was insignificant. These findings provide more evidence indicating the use of polyP as an extra energy source for maintaining high growth rate.     

Biotechnological synthesis of water-soluble food-grade polyphosphate with Saccharomyces cerevisiae

Inorganic polyphosphate (polyP) is the polymer of phosphate. Water-soluble polyPs with average chain lengths of 2–40 P-subunits are widely used as food additives and are currently synthesized chemically. An environmentally friendly highly scalable process to biosynthesize water-soluble food-grade polyP in powder form (termed bio-polyP) is presented in this study. After incubation in a phosphate-free medium, generally regarded as safe wild-type baker's yeast (Saccharomyces cerevisiae) took up phosphate and intracellularly polymerized it into 26.5% polyP (as KPO₃, in cell dry weight). The cells were lyzed by freeze-thawing and gentle heat treatment (10 min, 70°C). Protein and nucleic acid were removed from the soluble cell components by precipitation with 50 mM HCl. Two chain length fractions (42 and 11P-subunits average polyP chain length, purity on a par with chemically produced polyP) were obtained by fractional polyP precipitation (Fraction 1 was precipitated with 100 mM NaCl and 0.15 vol ethanol, and Fraction 2 with 1 final vol ethanol), drying, and milling. The physicochemical properties of bio-polyP were analyzed with an enzyme assay, ³¹P nuclear magnetic resonance spectroscopy, and polyacrylamide gel electrophoresis, among others. An envisaged application of the process is phosphate recycling from waste streams into high-value bio-polyP.     

Intracellular accumulation of polyphosphate by the yeast Candida humicola G-1 in response to acid pH

Cells of a newly isolated environmental strain of Candida humicola accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by C. humicola G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.     

Intracellular Accumulation of Polyphosphate by the Yeast Candida humicola G-1 in Response to Acid pH

Cells of a newly isolated environmental strain of Candida humicola accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by C. humicola G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.     

Accumulation of Polyphosphates and Expression of High Molecular Weight Exopolyphosphatase in the Yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The effect of cultivation time and concentration of inorganic phosphate (P(i)) in the culture medium on the accumulation of polyphosphates (polyP) and the activity of two cytosolic exopolyphosphatases of the yeast Saccharomyces cerevisiae was studied: an exopolyphosphatase of 40 kD encoded by PPX1 and a high molecular weight exopolyphosphatase encoded by another gene. Depletion of polyP in the cells on P(i) starvation is a signal factor for the accumulation of polyP after the subsequent addition of 5-20 mM P(i) and glucose to the cells or spheroplasts. A high activity of both exopolyphosphatases does not prevent the accumulation of polyP. The expression of the high molecular weight exopolyphosphatase is due to the acceleration of metabolism in cells that have reached the stage of growth deceleration on the addition of P(i) and glucose or complete culture medium. This process may occur independently from the accumulation of polyP. The activity of exopolyphosphatase PPX1 depends less on the mentioned factors, decreasing 10-fold only under conditions of phosphate surplus at the stationary growth stage.     

Effect of inhibitors on polyphosphate metabolism in the yeast Saccharomyces cerevisiae under hypercompensation conditions

After re-inoculation of the yeast Saccharomyces cerevisiae from phosphate-deficient to complete medium, the total content of polyphosphates increased tenfold during 2 h (hypercompensation), but the content of certain fractions increased differently. The content of acid-soluble polyphosphate increased to the maximal extent. The ratio of the activities of two exopolyphosphatases also changed in the cytosol. Activity of a low molecular weight exopolyphosphatase (40 kD) decreased almost twice, whereas activity of a high molecular weight exopolyphosphatase (830 kD) increased tenfold. Cycloheximide blocks the increase in activity of high molecular weight exopolyphosphatase and hence, under these conditions the latter is synthesized de novo. Inhibitors of energy metabolism and cycloheximide, an inhibitor of protein synthesis, differently influence accumulation of certain polyphosphate fractions under hypercompensation conditions. The effect of iodoacetamide, an inhibitor of glycolysis, on any fraction is negligible, while cycloheximide suppresses accumulation of only polyP4 fraction associated with the cell envelope and bafilomycin A1, an inhibitor of vacuolar H⁺-ATPase, suppresses accumulation of polyP3 fraction. The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) to variable extent inhibits accumulation of all the fractions. Analysis of the effect of inhibitors on accumulation of polyphosphates under hypercompensation conditions confirms various localization, heterogeneity, and multiplicity of the routes of biosynthesis of certain fractions of these macroergic phosphorus compounds and also suggests interrelation between their biosynthesis and the gradient of H⁺ electrochemical potential.     

V-ATPase dysfunction suppresses polyphosphate synthesis in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae accumulates the high levels of inorganic polyphosphates (polyPs) performing in the cells numerous functions, including phosphate and energy storage. The effects of vacuolar membrane ATPase (V-ATPase) dysfunction were studied on polyP accumulation under short-term cultivation in the P_i–excess media after P_i starvation. The addition of bafilomycin A1, a specific inhibitor of V-ATPase, to the medium with glucose resulted in strong inhibition of the synthesis of long-chain polyP and in substantial suppression of short-chain polyP. The addition of bafilomycin to the medium with ethanol resulted in decreased accumulation of high-molecular polyP, while the accumulation of low-molecular polyP was not affected. The levels of polyP synthesis in the mutant strain with a deletion in the vma2 gene encoding a V-ATPase subunit were significantly lower than in the parent strain in the media with glucose and with ethanol. The synthesis of the longest chain polyP was not observed in the mutant cells. The synthesis of only the low-polymer acid-soluble polyP fraction occurred in the cells of the mutant strain. However, the level of polyP1 was nearly tenfold lower than compared to the cells of the parent strain. Both bafilomycin A1 and the mutation in vacuolar ATPase subunit vma2 lead to a considerable decrease of cellular polyP accumulation. Thus, the defects in ΔμH⁺ formation on the vacuolar membrane resulted in the decrease of polyP biosynthesis in S. cerevisiae.     

The content and chain length of polyphosphates from vacuoles of Saccharomyces cerevisiae VKM Y-1173

The content of inorganic polyphosphates (polyP) in vacuoles of the yeast Saccharomyces cerevisiae is 15% of the total cellular polyP. Over 80% of the vacuole polyP are in an acid-soluble fraction. It was first established by ³¹P-NMR spectroscopy that a polymeric degree (n) of two subfractions obtained by precipitation with Ba²⁺ in succession at pH 4.5 and 8.2 was approximately 20 ± 5 and 5 ± 2 residues of ortho-phosphoric acid, respectively. Under a deficit of phosphate (P_i) in the cultivation medium, the polyP content in vacuoles decreased 7-fold with the same drastic reduction of their content in the cell. Unlike intact yeast cells, where polyP overcompensation is observed after their transfer from phosphate-free to phosphate-containing medium, the vacuoles do not show this effect. The data indicate the occurrence of special regulatory mechanisms of polyP synthesis in vacuoles differing from those in the whole cell.     

Effect of cycloheximide, iodoacetamide, and antimycin A on inorganic phosphate synthesis in Saccharomyces cerevisiae VKM Y-1173

The effect of inhibitors of protein synthesis (cycloheximide, CHI), glycolysis (iodoacetamide, IAA), and oxidative phosphorylation (antimycin A, ANM) on inorganic phosphate (polyP) synthesis during the first 0.5 h of their hypercompensation in Saccharomyces cerevisiae VKM Y-l173 grown on 2% glucose-containing media at low (hypoxia) or high aeration rates or in the presence of 1 vol % ethanol under high aeration conditions was studied. PolyP accumulation was highest in the medium with glucose under hypoxia; lower, with glucose at high aeration; and lowest, in the medium with ethanol. CHI had a small effect on the total polyP level but significantly stimulated ATP accumulation, irrespective of the culture growth conditions. The low-polymer acid-soluble polyP1 were synthesized most actively by the cells grown on glucose under hypoxia, alkali-soluble polyP3 were synthesized at en hanced aeration, and the most hig-molecular fraction, polyP5, was actively accumulated along with polyP3 at cultivation on ethanol. Regardless of the growth conditions, CHI inhibited accumulation of polyP4, the synthesis of which is associated with the synthesis of mannoproteins. IAA and ANM largely inhibited synthesis of all fractions at yeast growth under hypoxia and on ethanol, respectively. The results as a whole demonstrate the dependence of polyP formation on the main energy-generating cell processes and, at the same time, the absence of direct dependence of their synthesis on ATP concentration in Saccharomyces cerevisiae VKM Y-l 173.     

Purification and Properties of Exopolyphosphatase from the Cytosol of Saccharomyces cerevisiae Not Encoded by the PPX1 Gene

A novel exopolyphosphatase has been isolated from the cytosol of Saccharomyces cerevisiae grown to the stationary phase after its transfer from phosphate-deficient to complete medium. The PPX1 gene responsible for 40-kD exopolyphosphatase of the cytosol does not encode it. Specific activity of the preparation is 150 U/mg, purification degree is 319, and the yield is 16.9%. The minimal molecular mass of the active but unstable enzyme complex is approximately 125 kD. A stable enzyme complex with a molecular mass of approximately 500 kD is composed of two polypeptides of approximately 32 and 35 kD and apparently polyphosphates (polyP). Unlike the enzyme encoded by PPX1, the high-molecular-mass exopolyphosphatase is slightly active with polyP3, not inhibited by antibodies suppressing the activity of 40-kD exopolyphosphatase, inhibited by EDTA, and stimulated by divalent cations to a lesser extent. The high-molecular-mass exopolyphosphatase hydrolyzes polyP with an average chain length of 208 to 15 phosphate residues to the same extent, but is inactive with ATP, PPi, and p-nitrophenyl phosphate. The activity with polyP3 is 13% of that with polyP208. The Km values for polyP208, polyP15, and polyP3 hydrolysis are 3.5, 75, and 1100 microM, respectively. The enzyme is most active at pH approximately 7. Co2+ at the optimal concentration of 0.1 mM stimulates the activity 6-fold, while Mg2+ at the optimal concentration of 1 mM enhances it 2-fold. The enzyme under study is similar in some properties to an exopolyphosphatase purified earlier from yeast vacuoles.     

Polyphosphates and exopolyphosphatases in cytosol and mitochondria of Saccharomyces cerevisiae during growth on glucose or ethanol under phosphate surplus

Content and chain lengths of inorganic polyphosphates (polyP) as well as exopolyphosphatase activities were compared in cytosol and mitochondria of the yeast Saccharomyces cerevisiae during growth on glucose or ethanol under phosphate surplus. PolyP metabolism in cytosol and mitochondria was substantially dependent upon the carbon source. Acid-soluble polyP accumulated mainly in cytosol using either glucose or ethanol. The level of the accumulation was lower during growth on ethanol compared to that on glucose. Increase in polyP content in mitochondria was observed during growth on glucose, but not on ethanol. In cytosol the activity of exopolyphosphatase PPN1 was increased and the activity of exopolyphosphatase PPX1 was decreased independently of the carbon source under phosphate surplus conditions. Growth on ethanol caused exopolyphosphatase PPN1 to appear in the soluble mitochondrial fraction, while during growth on glucose only exopolyphosphatase PPX1 was present in this fraction.     

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 (∼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.     

Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase.

Recent results revealed that inorganic polyphosphates (polyP), being energy-rich linear polymers of orthophosphate residues known from bacteria and yeast, also exist in higher eukaryotes. However, the enzymatic basis of their metabolism especially in mammalian cells is still uncertain. Here we demonstrate for the first time that alkaline phosphatase from calf intestine (CIAP) is able to cleave polyP molecules up to a chain length of about 800. The enzyme acts as an exopolyphosphatase degrading polyP in a processive manner. The pH optimum is in the alkaline range. Divalent cations are not required for catalytic activity but inhibit the degradation of polyP. The rate of hydrolysis of short-chain polyP by CIAP is comparable to that of the standard alkaline phosphatase (AP) substrate p-nitrophenyl phosphate. The specific activity of the enzyme decreases with increasing chain length of the polymer both in the alkaline and in the neutral pH range. The K(m) of the enzyme also decreases with increasing chain length. The mammalian tissue non-specific isoform of AP was not able to hydrolyze polyP under the conditions applied while the placental-type AP and the bacterial (Escherichia coli) AP displayed polyP-degrading activity.     

Inorganic Polyphosphate in Escherichia coli: the Phosphate Regulon and the Stringent Response

Escherichia coli transiently accumulates large amounts of inorganic polyphosphate (polyP), up to 20 mM in phosphate residues (P_i), in media deficient in both P_i and amino acids. This transient accumulation is preceded by the appearance of nucleotides ppGpp and pppGpp, generated in response to nutritional stresses. Mutants which lack PhoB, the response regulator of the phosphate regulon, do not accumulate polyP even though they develop wild-type levels of (p)ppGpp when subjected to amino acid starvation. When complemented with a phoB-containing plasmid, phoB mutants regain the ability to accumulate polyP. PolyP accumulation requires high levels of (p)ppGpp independent of whether they are generated by RelA (active during the stringent response) or SpoT (expressed during P_i starvation). Hence, accumulation of polyP requires a functional phoB gene and elevated levels of (p)ppGpp. A rapid assay of polyP depends on its adsorption to an anion-exchange disk on which it is hydrolyzed by a yeast exopolyphosphatase.     

Direct labeling of polyphosphate at the ultrastructural level in Saccharomyces cerevisiae by using the affinity of the polyphosphate binding domain of Escherichia coli exopolyphosphatase

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate and has many biological functions in prokaryotic and eukaryotic organisms. To investigate polyP localization, we developed a novel technique using the affinity of the recombinant polyphosphate binding domain (PPBD) of Escherichia coli exopolyphosphatase to polyP. An epitope-tagged PPBD was expressed and purified from E. coli. Equilibrium binding assay of PPBD revealed its high affinity for long-chain polyP and its weak affinity for short-chain polyP and nucleic acids. To directly demonstrate polyP localization in Saccharomyces cerevisiae on resin sections prepared by rapid freezing and freeze-substitution, specimens were labeled with PPBD containing an epitope tag and then the epitope tag was detected by an indirect immunocytochemical method. A goat anti-mouse immunoglobulin G antibody conjugated with Alexa 488 for laser confocal microscopy or with colloidal gold for transmission electron microscopy was used. When the S. cerevisiae was cultured in yeast extract-peptone-dextrose medium (10 mM phosphate) for 10 h, polyP was distributed in a dispersed fashion in vacuoles in successfully cryofixed cells. A few polyP signals of the labeling were sometimes observed in cytosol around vacuoles with electron microscopy. Under our experimental conditions, polyP granules were not observed. Therefore, it remains unclear whether the method can detect the granule form. The method directly demonstrated the localization of polyP at the electron microscopic level for the first time and enabled the visualization of polyP localization with much higher specificity and resolution than with other conventional methods.     

Inorganic polyphosphate in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with a mutation disturbing the function of vacuolar ATPase

A mutation in the vma2 gene disturbing V-ATPase function in the yeast Saccharomyces cerevisiae results in a five- and threefold decrease in inorganic polyphosphate content in the stationary and active phases of growth on glucose, respectively. The average polyphosphate chain length in the mutant cells is decreased. The mutation does not prevent polyphosphate utilization during cultivation in a phosphate-deficient medium and recovery of its level on reinoculation in complete medium after phosphate deficiency. The content of short chain acid-soluble polyphosphates is recovered first. It is supposed that these polyphosphates are less dependent on the electrochemical gradient on the vacuolar membrane.