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.     

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.     

Effects of excess thymidylate on thymidylate low-requiring strains of Saccharomyces cerevisiae: high mutagenicity and absence of DNA strand breaks

dTMP exposure concentrations of 0.1 mM or higher are genotoxic in exponentially growing cells of thymidylate low-requiring mutants of Saccharomyces cerevisiae. Mutagenicity of excess dTMP is highest in an exposure concentration 10-fold of that needed for external supplementation of endogenously blocked thymidylate synthesis. Still higher dTMP concentrations are primarily cytotoxic. The canavanine forward-mutation system shows excess dTMP to be as potent a mutagen as irradiation by ultraviolet light. Mutagenicity of excess dTMP, however, differs from that of direct DNA-attacking mutagens in that it is highest in the absence of significant toxicity. Alkaline sucrose gradient centrifugation shows that excess dTMP does not induce significant numbers of DNA single- or double-strand breaks, while conditions of thymidylate deprivation lead to DNA-strand breaks and thymineless death.     

Substrate preference is altered by mutations in the fifth transmembrane domain of Ptr2p,the di/tri-peptide transporter of Saccharomyces cerevisiae

The integral membrane protein Ptr2p transports di/tri-peptides into the yeast Saccharomyces cerevisiae. The sequence FYXXINXG (FYING motif) in the 5th transmembrane domain (TM5) is invariably conserved among the members of the PTR (Peptide TRansport) family ranging from yeast to human. To test the role of TM5 in Ptr2p function, Ala-scanning mutagenesis of the 22 residues comprising TM5 was completed. All mutated transporters, with the exception of the Y248A mutant, were expressed as determined by immunoblots. In peptide-dependent growth assays, ten mutants of the non-FYING residues grew as well as wild-type Ptr2p on all twelve different peptides tested. All of the FYING motif mutants, except the non-expressed Y248A, plus seven other mutants in TM5 exhibited differential growth on peptides including Leu-Leu and Met-Met-Met indicating that these mutations conferred substrate preference. In assays measuring direct uptake of the radioactive peptides ³H-Leu-Leu or ¹⁴C-Met-Met-Met, the F, I and G mutants of the FYING motif did not demonstrate accumulation of these peptides over a ten minute interval. The mutation N252A of the FYING motif, along with L240A, M250A, and L258A, exhibited differential substrate preference for Met-Met-Met over Leu-Leu. Other mutations (T239A, Q241A, N242A, M245A, and A260) resulted in preference for Leu-Leu over Met-Met-Met. These data demonstrate that TM5, in particular its conserved FYING motif, is involved in substrate preference of Ptr2p.     

Oscillatory metabolism of Saccharomyces cerevisiae in continuous culture

Short-period (40-50 min) synchronized metabolic oscillation was found in a continuous culture of yeast Saccharomyces cerevisiae under aerobic conditions at low-dilution rates. During oscillation, many parameters changed cyclically, such as dissolved oxygen concentration, respiration rate, ethanol and acetate concentrations in the culture, glycogen, ATP, NADH, pyruvate and acetate concentrations in the cells. These changes were considered to be associated with glycogen metabolism. When glycogen was degraded, the respiro-fermentative phase was observed, in which ethanol was produced and the respiration rate decreased. In this phase, the levels of intracellular pyruvate and acetate became minimum, ATP became high and intracellular pH at its lowest level. When glycogen metabolism changed from degradation to accumulation, the respiratory phase started, during which ethanol was re-assimilated from the culture and the respiration rate increased. Intracellular pyruvate and acetate became maximum, ATP decreased and the intracellular pH appeared high. These findings may indicate new aspects of the control mechanism of glycogen metabolism and how respiration and ethanol fermentation are regulated together under aerobic conditions.     

Identification, cloning and characterization of a derepressible Na+-coupled phosphate transporter in Saccharomyces cerevisiae

Based on the high sequence homology between the yeast ORF YBR296c (accession number P38361 in the SWISS-PROT database) and the PHO4 gene of Neurospora crassa, which codes for a Na⁺/P_i cotransporter with twelve putative transmembrane domains, the YBR296c ORF was considered to be a promising candidate gene for a plasma membrane-bound phosphate transporter in Saccharomyces cerevisiae. Therefore, this gene, here designated PHO89, was cloned and a set of deletion mutants was constructed. We then studied their P_i uptake activity under different conditions. We show here that a transport activity displayed by PHO89 strains under alkaline conditions and in the presence of Na⁺ is absent in pho89 null mutants. Moreover, when the pH was lowered to pH 4.5 or when Na⁺ was omitted, this activity decreased significantly, reaching values close to those exhibited by the Δpho89 mutant. Studies of the acid phosphatase activity of these strains, as well as promoter sequence analysis, suggest that expression of the PHO89 gene is under the control of the PHO regulatory system. Northern analysis shows that this gene is only transcribed under conditions of P_i limitation. This is, to our knowledge, the first demonstration that the PHO89 gene codes for the Na⁺/P_i cotransporter previously characterized by kinetic studies, and represents the only Na+-coupled secondary anion transport system so far identified in S. cerevisiae. Pho89p has been shown to have an apparent K_m of 0.5 μM and a pH optimum of 9.5, and is highly specific for Na⁺; activation of transport is maximal at a Na⁺ concentration of 25 mM. Received: 2 November 1997 / Accepted: 20 February 1998     

Investigations on phosphate uptake and polyphosphate metabolism by mycorrhized and nonmycorrhized roots of beech and pine as investigated by in vivo 31P-NMR

Comparative in vivo ³¹P-NMR studies of mycorrhized and nonmycorrhized roots of Fagus sylvatica and Pinus sylvestris and of the fungus Suillus bovinus in pure culture have produced interesting new data. With respect to intracellular compartments and pH, ³¹P-NMR spectroscopy showed that the spectrum of the mycorrhiza results from simple superimposition of the spectra of its symbionts. A special method of cyclic phosphate supply followed by block averaging of the NMR spectra was used to determine the kinetic behaviour of phosphate uptake and storage and its incorporation into polyphosphate at a constant external pH of 5.5. Mycorrhized roots and pure fungus showed transformation of accumulated inorganic phosphate into mobile polyphosphate with a medium chain length. Transformation of mobile into immobile polyphosphate either with a long chain length or in a granular state was also observed. Thus, two different types of fungal polyphosphate could be verified. Deficiency of external phosphate initiated the mobilization of internal phosphate, transforming stored polyphosphate into phosphate. It could be shown that a high fungal mass renders mycorrhizal phosphate metabolism less sensitive to external variation in nutrient concentration. The central role of the fungus in regulating mycorrhizal phosphate metabolism is discussed.     

cAMP-dependent phosphoprotein changes in the yeast Saccharomyces cerevisiae

Abstract cAMP-dependent phosphoprotein changes were determined using 1-dimensional SDS-gel electrophoresis in a cAMP-requiring yeast mutant ( Saccharomyces cerevisiae AM18). During cAMP starvation, the yeast cells accumulated 3 ³²P-labeled bands with M _r/ 72000, 54000, and 37000. The M _r/ 72000 protein was the most prominent phosphorylated protein. After the readdition of cAMP, these phosphoproteins lost their ³²P-label while phosphoproteins with M _r/ 76000, 65000, 56000 and 30000 were accumulated. Similar phosphoprotein changes were also detected in cdc35 at the nonpermissive temperature, but not in wildtype (A363A) or cdc7 strains of S. cerevisiae .     

Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

New aspects on phosphate sensing and signalling in Saccharomyces cerevisiae

The mechanism involved in the cellular phosphate response of Saccharomyces cerevisiae forms part of the PHO pathway, which upon expression allows a co-ordinated cellular response and adaptation to changes in availability of external phosphate. Although genetic studies and analyses of the S. cerevisiae genome have produced much information on the components of the PHO pathway, little is known about how cells sense the environmental phosphate level and the mechanistic regulation of phosphate acquisition. Recent studies emphasize different levels in phosphate sensing and signalling in response to external phosphate fluctuations. This review integrates all these findings into a model involving rapid and long-term effects of phosphate sensing and signalling in S. cerevisiae. The model describes in particular how yeast cells are able to adjust phosphate acquisition by integrating the status of the intracellular phosphate pools together with the extracellular phosphate concentration.     

Sequential inactivation of ammonium and glucose transport in Saccharomyces cerevisiae during fermentation

Abstract Ethanol at concentrations above 12% (v/v) in mineral medium with glucose and with ammonium as the only nitrogen source induced rapid inactivation of the ammonium transport system in the strain IGC 3507 of Saccharomyces cerevisiae terminating protein synthesis. Subsequently, when glucose was present, the glucose transport system was irreversibly inactivated. This two-step mechanism may play a decisive role when ethanol stops fermentation by S. cerevisiae , before all the fermentable sugar has been consumed.     

Transcriptional changes associated with ethanol tolerance in Saccharomyces cerevisiae

Saccharomyces spp. are widely used for ethanol production; however, fermentation productivity is negatively affected by the impact of ethanol accumulation on yeast metabolic rate and viability. This study used microarray and statistical two-way ANOVA analysis to compare and evaluate gene expression profiles of two previously generated ethanol-tolerant mutants, CM1 and SM1, with their parent, Saccharomyces cerevisiae W303-1A, in the presence and absence of ethanol stress. Although sharing the same parentage, the mutants were created differently: SM1 by adaptive evolution involving long-term exposure to ethanol stress and CM1 using chemical mutagenesis followed by adaptive evolution-based screening. Compared to the parent, differences in the expression levels of genes associated with a number of gene ontology categories in the mutants suggest that their improved ethanol stress response is a consequence of increased mitochondrial and NADH oxidation activities, stimulating glycolysis and other energy-yielding pathways. This leads to increased activity of energy-demanding processes associated with the production of proteins and plasma membrane components, which are necessary for acclimation to ethanol stress. It is suggested that a key function of the ethanol stress response is restoration of the NAD⁺/NADH redox balance, which increases glyceraldehyde-3-phosphate dehydrogenase activity, and higher glycolytic flux in the ethanol-stressed cell. Both mutants achieved this by a constitutive increase in carbon flux in the glycerol pathway as a means of increasing NADH oxidation.     

Systems of l-leucine transport into Saccharomyces cerevisiae protoplasts

l-[¹⁴C]Leucine transport into Saccharomyces cerevisiae protoplasts involves two systems (1 and 2) with different kinetic parameters. The K_T values for these systems are of the same order as those for intact yeast cells. These results suggest that the proteins related to the affinity constants are located in the cytoplasmic membrane.     

Localization of polyphosphate in vacuoles of Saccharomyces cerevisiae

Virtually all of the polyphosphate (PP) present in yeast protoplasts can be recovered in a crude particulate fraction if polybase-induced lysis is used for disrupting the protoplasts. This fraction contains most of the vacuoles, mitochondria and nuclei. Upon the purification of vacuoles the PP is enriched to the same extent as are the vacuolar markers. The amount of PP per vacuole is comparable to the amount of PP per protoplast.The possibility that PP is located in the cell wall is also considered. In the course of the incubation necessary for preparing protoplasts, 20% of the cellular PP is broken down. As this loss of PP occurs to the same extent in the absence of cell wall degrading enzymes, it is inferred that internal PP is metabolically degraded, no PP being located in the cell walls.It is concluded that in Saccharomyces cerevisiae most if not all of the PP is located in the vacuoles, at least under the growth conditions used.Non-Standard Abbreviations PIPES piperazine-N,N-bis-2-ethanolsulfonic acid - DEAE-dextran diethylaminoethyl-dextran     

Enhanced arsenate uptake in Saccharomyces cerevisiae overexpressing the Pho84 phosphate transporter

Arsenate is a major toxic constituent in arsenic-contaminated water supplies. Saccharomyces cerevisiae was engineered as a potential biosorbent for enhanced arsenate accumulation. The phosphate transporter, Pho84p, known to import arsenate, was overexpressed using a 2μ-based vector carrying PHO84 under the control of the late-phase ADH2 promoter. Arsenate uptake was then evaluated using a resting cell system. In buffer solutions containing high arsenate concentrations (12,000 and 30,000 ppb), the engineered strains internalized up to 750 μg of arsenate per gram of cells, a 50% improvement over control strains. Increasing the cell mass 2.5-fold yielded a proportional increase in the volumetric arsenate uptake, while maintaining the same level of specific uptake. At high levels of arsenate, loss from the intact cells to the medium was observed with time; knockouts of two known arsenic extrusion genes, ACR3 and FPS1, did not prevent this loss. At trace level concentrations (120 ppb), rapid and total arsenate removal was observed. The presence of 50 μM phosphate reduced uptake by approximately 15% in buffer containing 80 μM (6,000 ppb) arsenate. At trace levels of arsenate (70 ppb), the phosphate reduced the initial rate of uptake, but not the total amount removed. PHO84 mRNA levels were nearly 30 times higher in the engineered strains relative to the control strains. Uptake may no longer be a limiting factor in the engineered system and further increases should be possible by upregulating the downstream reduction and sequestration pathways.     

31P-NMR saturation transfer measurements of phosphate consumption in Saccharomyces cerevisiae

³¹P-NMR measurements of saturation transfer have been used to measure phosphate consumption in respiratory competent cells of the yeast Saccharomyces cerevisiae. Measurements of oxygen consumption and maintenance of the cells in a metabolic steady state during the NMR experiments were facilitated by immobilisation of the cells in an agarose gel matrix which could be perfused in the NMR spectrometer. The contribution of glycolysis to the observed rate of phosphate consumption was estimated by simultaneously measuring glucose consumption and ethanol production in the perfusion buffer. The remaining phosphate consumption, which was attributed to flux through the reaction catalysed by the mitochondrial ATP synthase, combined with measurements of oxygen consumption allowed estimation of a P:O ratio (mol ATP synthesised:atoms oxygen consumed) which was close to 3.     

Glucose metabolism and cell size in continuous cultures of Saccharomyces cerevisiae

A detailed analysis of the cell size, monitored as protein content, has been performed in glucose-limited continuous cultures, so as to obtain the values of the average protein content for various subpopulations at different cell cycle stages, as a function of the growth rate. Glucose metabolism appears to affect cell size, since there is an increase of the average protein content of the population when cells produce ethanol above the critical dilution rate. If the production of ethanol is forced at low growth rates by the addition of formate, the average protein content increases. These results indicate a link between glucose metabolism and cell size in budding yeast, as observed for mammalian cells.