High molecular mass exopolyphosphatase from the cytosol of the yeast Saccharomyces cerevisiae is encoded by the PPN1 gene

It has been shown that the high molecular mass exopolyphosphatase localized in cytosol of the yeast Saccharomyces cerevisiae is encoded by the PPN1 gene. This enzyme is expressed under special culture conditions when stationary phase cells are passing on to new budding on glucose addition and phosphate excess. The enzyme under study releases orthophosphate from the very beginning of polyphosphate hydrolysis.     

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.     

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.     

Nuclear exopolyphosphatase of Saccharomyces cerevisiae is not encoded by the PPX1 gene encoding the major yeast exopolyphosphatase

Intact nuclei from a parental strain CRY and a PPX1-mutant CRX of Saccharomyces cerevisiae were isolated and found to be essentially free of cytoplasmic, mitochondrial and vacuolar marker enzymes. The protein-to-DNA ratios of the nuclei were 22 and 30 for CRY and CRX nuclei, respectively. An exopolyphosphatase (exopolyPase) with molecular mass of approximately 57 kDa and a pyrophosphatase (PPase) of approximately 41 kDa were detected in the parental strain CRY. Inactivation of PPX1 encoding a major exopolyPase (PPX1) in S. cerevisiae did not result in considerable changes in the content and properties of nuclear exopolyPase as compared to the parental strain of S. cerevisiae. Consequently, the nuclear exopolyPase was not encoded by PPX1. In the CRX strain, the exopolyPase was stimulated by bivalent metal cations. Co2+, the best activator, stimulated it by approximately 2.5-fold. The exopolyPase activity was nearly the same with polyphosphate (polyP) chain lengths ranging from 3 to 208 orthophosphate when measured with Mg2+. With Co 2+, the exopolyPase activity increased along with the increase in polymerization degree of the substrate.     

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.     

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.     

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.     

The patterns of utilization and accumulation of polyphosphates in the cytosol of the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under inactivation of exopolyphosphatase genes <Emphasis Type="Italic">PPX1</Emphasis> and <Emphasis Type="Italic">PPN1</Emphasis>

The effect of inactivation of the PPX1 and PPN1 genes encoding the yeast exopolyphosphatases on the activities of these enzymes and polyphosphate content in the cytosol of Saccharomyces cerevisiae was studied under P_i deficit and P_i excess in the cultivation medium. Under P_i deficit, exopolyphosphatase activity in strain CRN (with inactivated PPN1 gene) and in the parent strain CRY increased 3- and 1.5-fold, respectively. In the strain CRX (with inactivated PPX1 gene), exopolyphosphatase activity did not change under P_i deficit. Transfer from P_i-deficient to P_i-rich medium was accompanied by an ～1.7-fold increase of exopolyphosphatase activities in the cytosol preparations of strains CRY, CRX, and CRN. In the cytosol of the double mutant, exopolyphosphatase activity was practically absent under all of the above cultivation conditions. The content of polyphosphates in the cytosol preparations of all strains under study substantially decreased under P_i deficit. Transfer from P_i-deficient to P_i-rich medium was accompanied by polyphosphate over-accumulation only in the cytosol preparations of stains CRX and CNX, where their levels increased ～1.3 and 3.5-fold, respectively. No over-accumulation was observed in the parent strain CRY and in the PPN1-deficient strain CRN. These data suggest that the exopolyphosphatases encoded by the PPX1 and PPN1 genes are not involved in polyphosphate synthesis.     

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 (∼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 ∼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. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 11, pp. 1445–1450.     

Two Exopolyphosphatases of the Cytosol of the Yeast S. cerevisiae: Comparative Characteristics

In cell-free extracts of the yeast Saccharomyces cerevisiae that had been transferred from phosphate-deficient (–P) medium to complete (+P) medium (hypercompensation conditions), the specific and the total polyphosphatase activities increased (by 50 and 60%, respectively) compared to the control that was transferred from (+P) medium to (+P) medium. Specific and total polyphosphatase activities under hypercompensation conditions increased by 25 and 43% in cytosol, by 33 and 100% in vacuoles, and by 50 and 50% in the total membrane fraction, respectively. In contrast, the polyphosphatase activity in the cell envelope somewhat decreased compared to the control. Under the growth conditions indicated above, a novel high molecular weight polyphosphatase was revealed in the cytosol fraction along with the previously studied 40-kD polyphosphatase. Unlike the 40-kD polyphosphatase, which is most active with tripolyphosphate, this novel enzyme has a molecular mass of more than 440 kD and is most active with high molecular weight polyphosphates. This polyphosphatase is insensitive to antibodies that suppress the activity of the 40-kD polyphosphatase of the cytosol. In a number of properties, the high molecular weight polyphosphatase of the cytosol resembles the polyphosphatase of vacuoles, but it differs from the polyphosphatases of nuclei and mitochondria of S. cerevisiae. The ratio of the low and high molecular weight polyphosphatases depends on the culture growth conditions. Under hypercompensation conditions, the total activity of the high molecular weight polyphosphatase in the cytosol is five times higher than that of the 40-kD polyphosphatase. During growth without re-inoculation, the 40-kD polyphosphatase is predominant in the cytosol; its total activity in dependence on the growth stage is 3.5-12.5 times higher than the activity of the high molecular weight form.     

The effects of vanadate on the yeast Saccharomyces cerevisiae

Growth of Saccharomyces cerevisiae on non-fermentable medium was more sensitive to inhibition by vanadate than growth of fermentable medium. The frequency of petite mutants increased in cultures grown for 18 hours in fermentable medium containing vanadate. However, oxygen uptake markedly increased in yeast cultures grown in the presence of vanadate, a similar effect being produced by phosphate. It was also found that oligomycin toxicity was relieved by vanadate. These results suggest that vanadate may interact with the mitochondria of S. cerevisiae.     

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 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.     

Inorganic Polyphosphates and Exopolyphosphatases in Cell Compartments of the Yeast Saccharomyces cerevisiae Under Inactivation of PPX1 and PPN1 Genes

Purified fractions of cytosol, vacuoles, nuclei, and mitochondria of Saccharomyces cerevisiae possessed inorganic polyphosphates with chain lengths characteristic of each individual compartment. The most part (80–90%) of the total polyphosphate level was found in the cytosol fractions. Inactivation of a PPX1 gene encoding ~40-kDa exopolyphosphatase substantially decreased exopolyphosphatase activities only in the cytosol and soluble mitochondrial fraction, the compartments where PPX1 activity was localized. This inactivation slightly increased the levels of polyphosphates in the cytosol and vacuoles and had no effect on polyphosphate chain lengths in all compartments. Exopolyphosphatase activities in all yeast compartments under study critically depended on the PPN1 gene encoding an endopolyphosphatase. In the single PPN1 mutant, a considerable decrease of exopolyphosphatase activity was observed in all the compartments under study. Inactivation of PPN1 decreased the polyphosphate level in the cytosol 1.4-fold and increased it 2- and 2.5-fold in mitochondria and vacuoles, respectively. This inactivation was accompanied by polyphosphate chain elongation. In nuclei, this mutation had no effect on polyphosphate level and chain length as compared with the parent strain CRY. In the double mutant of PPX1 and PPN1, no exopolyphosphatase activity was detected in the cytosol, nuclei, and mitochondria and further elongation of polyphosphates was observed in all compartments.     

Effect of some heavy metal ions on copper-induced metallothionein synthesis in the yeast Saccharomyces cerevisiae

Copper-induced metallothionein (MT) synthesis in Saccharomyces cerevisiae was investigated in order to associate this exclusively with Cu²⁺ in vivo, when cultured in nutrient medium containing other heavy metal ions. Expression of the CUP1 promoter/lacZ fusion gene was inhibited by all heavy metal ions tested, especially Cd²⁺ and Mn²⁺. By adding Cd²⁺ and Mn²⁺ at 10 M concentration, the -galactosidase activity decreased by about 80% and 50% of the maximum induction observed with 1 mM CuSO₄, respectively. Furthermore, cell growth was markedly inhibited by combinations of 1 mM-Cu²⁺ and 1 M-Cd²⁺. Therefore, the yeast S. cerevisiae could not rely on MT synthesis as one of the copper-resistance mechanisms, when grown in a Cd²⁺ environment. In contrast, the presence of Mn²⁺ in the nutrient medium showed alleviation rather than growth inhibition by high concentrations of Cu²⁺. The recovery from growth inhibition by Mn²⁺ was due to decreased Cu²⁺ accumulation. Inhibitory concentrations of Co²⁺, Ni²⁺ and Zn²⁺ on expression of the CUP1p/lacZ fusion gene were at least one order of magnitude higher than that of Cd²⁺ and Mn²⁺. These results are discussed in relation to Cu²⁺ transport and Cu-induced MT synthesis in the copper-resistance mechanism of the yeast S. cerevisiae.     

Localization of trehalase in vacuoles and of trehalose in the cytosol of yeast (Saccharomyces cerevisiae)

Protoplasts of Saccharomyces cerevisiae synthesized and degraded trehalose when they were incubated in a medium containing traces of glucose and acetate. Such protoplasts were gently lyzed by the polybase method and a particulate and soluble fraction was prepared. Trehalose was found in the soluble fraction and the trehalase activity mostly in the particulate fraction which also contained the vacuoles besides other cell organelles. Upon purification of the vacuoles, by density gradient centrifugation, the specific activity of trehalase increased parallel to the specific content of vacuolar markers. This indicates that trehalose is located in the cytosol and trehalase in the vacuole. It is suggested that trehalose, in addition to its role as a reserve may also function as a protective agent to maintain the cytosolic structure under conditions of stress.Non Standard Abbreviations AMPD 2-amino-2-methyl-1,3-propanediol - DTT dithiothreitol - MES 2-N-morpholinoethanesulfonic acid - PIPES piperazine-N,N-bis-2-ethanesulfonic acid - PMSF phenylmethylsulfonylfluoride     

Genetic analysis of inducible sexual agglutination ability in the yeast Saccharomyces cerevisiae

Genetic regulation of the inducibility of sexual agglutination ability in the yeast Saccharomyces cerevisiae was studied. Detailed analysis of the degree of sexual agglutination was carried out; it showed that a greater number of genes are involved in the regulation of inducible sexual agglutination in strain H1-0 than previously assumed. Although dominancy of inducible phenotype over constitutive was confirmed, the effectiveness of one gene changing the constitutive phenotype to the inducible seemed to be somewhat low. Quantity per cell of agglutination substances responsible for sexual agglutination increased as the agglutination ability became greater.     

Metabolic flux analysis of the sterol pathway in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model system for examining the biosynthesis of sterols in eukaryotic cells. To investigate underlying regulation mechanisms, a flux analysis of the ergosterol pathway was performed. A stoichiometric model was derived based on well known biochemistry of the pathway. The model was integrated in the Software COMPFlux which uses a global optimization algorithm for the estimation of intracellular fluxes. Sterol concentration patterns were determined by gas chromatography in aerobic and anaerobic batch cultivations, when the sterol metabolism was suppressed due to the absence of oxygen. In addition, the sterol concentrations were observed in a cultivation which was shifted from anaerobic to aerobic growth conditions causing the sterol pools in the cell to be filled. From time-dependent flux patterns, possible limitations in the pathway could be localized and the esterification of sterols was identified as an integral part of regulation in ergosterol biosynthesis.     

Immunologically related proteins in cytoplasmic and mitochondrial ribosomes of yeast Saccharomyces cerevisiae

Summary Ribosomal proteins from the cytoplasm and mitochondria of the yeast Saccharomyces cerevisiae were compared by immunoblotting techniques. Antibodies raised against cytoplasmic ribosomal proteins cross-react with five mitochondrial ribosomal proteins, four of which are located in the large and one in the small mitochondrial subunits. The possible existence of common ribosomal proteins for cytoplasmic and mitochondrial ribosomes is discussed.Abbreviations cyto cytoplasmic - mito mitochondrial     

Effects of beta-amyloid on proliferation and morphology of yeast Saccharomyces cerevisiae

Summary Deposition of beta-amyloid peptide (1–42) (βAP) in the brain is an early event linked with pathogenesis of cell injury and death in Alzheimer disease. Previous studies have demonstrated that βAP induces cytotoxicity in several types of human cells. Surprisingly, the peptide was found not only to be non toxic for yeast cells, but to stimulate growth of yeast culture. The results are consistent with βAP binding to yeast cell as illustrated by binding isotherms with the apparent dissociation constant of 8×10⁻⁷ M and B_max of 4.7×10⁴ molecules/cell.