T. V. Kulakovskaya

Exopolyphosphatases of the yeast Saccharomyces cerevisiae

Separate compartments of the yeast cell possess their own exopolyphosphatases differing from each other in their properties and dependence on culture conditions. The low-molecular-mass exopolyphosphatases of the cytosol, cell envelope, and mitochondrial matrix are encoded by the PPX1 gene, while the high-molecular-mass exopolyphosphatase of the cytosol and those of the vacuoles, mitochondrial membranes, and nuclei are presumably encoded by their own genes. Based on recent works, a preliminary classification of the yeast exopolyphosphatases is proposed.

Metabolism and Function of Polyphosphates in Bacteria and Yeast

Inorganic polyphosphate (polyPs) found in living organisms more than 100 years ago by Lieberman (1888) are linear polymers containing 2-1000 residues of orthophosphate linked by the energy-rich phosphoanhydride bond. They are widely spread in various microoganisms and are found in small amounts in the cells of animals and plants (Kulaev 1979; Kulaev and Vagabov 1983; Wood and Clark 1988). PolyPs perform varied biological functions. Recent investigations have shown the polyPs belong to biopolymers, the function of which changes when passing from prokaryotic cells to cells of lower and then higher eukaryotes. The comparative characteristics of the functions and metabolic ways of polyPs in different organisms are of interest from the viewpoint of the evolutionary physiology of the cell.

MEMBRANE-BOUND AND SOLUBLE POLYPHOSPHATASES OF MITOCHONDRIA OF SACCHAROMYCES CEREVISIAE : IDENTIFICATION AND COMPARATIVE CHARACTERIZATION

Isolated mitochondria of Saccharomyces cerevisiae possess polyphosphatases insensitive to a number of inhibitors of ATPase and pyrophosphatase of the same organelles and differing from the last two by neutral pH optima and molecular masses. After subfractionation of mitochondria, the polyphosphatase activity is distributed among the membrane and soluble preparations. The membrane-bound and soluble polyphosphatase activities are represented by different enzymes distinguished by molecular masses, substrate specificity, Km values, and relation to mono- and divalent cations. The membrane-bound polyphosphatases have molecular masses of 120 and 76 kDa, and the soluble one of about 36 kDa. All three enzymes appear to have a monomeric structure. The soluble polyphosphatase activity is stimulated by divalent cations in contrast to the membrane-bound one which is inhibited by the same cations, including Mg2+. Monovalent cations do not actually change the activity of the soluble enzyme, but stimulate it in the membrane preparation. Specific activities for the hydrolysis of polyphosphates with average chain lengths of 9-188 phosphate residues increase under increasing degree of substrate polymerization in the membrane preparation and are actually unchanged in the soluble one. The affinity of the soluble enzyme to polyphosphates is 5-10 times higher than that of the membrane-bound polyphosphatases.

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 (Pi) reserve in the yeast Saccharomyces cerevisiae. In this work, the participation of cytosol polyPs and exopolyphosphatases in maintenance of Pi homeostasis under Pi 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 Pi-deficient medium is not disturbed. All the studied strains are able to maintain relatively constant Pi levels in the cytosol. In Pi-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.

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.

The Yeast Pseudozyma fusiformataVKM Y-2821 Producing an Antifungal Glycolipid

The yeast Pseudozyma fusiformata(Ustilaginales) produces an extracellular low-molecular-weight protease-resistant thermostable fungicide, which is active against more than 80% of the 280 yeast and yeast-like species tested under acidic conditions. The fungicide, extracted with methanol and purified by column and thin-layer chromatography, was found to consist of glucose and saturated fatty acids.

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.

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 Pi. 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.

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.

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.