The Role of Polyphosphates in the Transport Mechanism of Glucose in Yeast Cells

Several cations inhibit anaerobic fermentation of glucose by intact yeast cells. Some ions (e.g. Hg⁺⁺) penetrate into the cytoplasm and cause an irreversible inhibition of fermentation. Other ions (e.g. UO₂⁺⁺, Ni⁺⁺, and Co⁺⁺) are reversibly bound to a substance at the outside of the yeast cell identified as polyphosphate. Although the cations are bound to exactly the same extent, their influences on fermentation differ greatly. Thorium ions are bound not only to the polyphosphates, but in addition, to phosphatides in the cell membrane. Under circumstances in which glucose is transported into the cell, the amount of polyphosphate in the outer face of the membrane decreases considerably. If yeast is poisoned with monoiodoacetate, the number of glucose molecules that can still be taken up equals the original number of cation-binding sites at the outer surface of the membrane. These data suggest that one molecule of glucose is taken up in connection with the disappearance of one polyphosphate monomer. The hypothesis is framed that the uptake of glucose into the yeast cell is associated with an enzymic phosphorylation (possibly of the carrier), with polyphosphate as phosphate donor. The inhibition of glucose uptake caused by certain metal ions may be the consequence of induced changes in the spatial arrangement of polyphosphate chains; the greater the change in configuration, the larger is the inhibition.     

Determination of the role of polyphosphate in transport-coupled phosphorylation in the yeast Saccharomyces cerevisiae

The role of polyphosphate in 2-deoxy-D-glucose transport was studied in yeast cells, pulse-labeled with [³²P]orthophosphate, by comparing the concentrations and specific activities of polyphosphate, orthophosphate and 2-dGlc-phosphate. When 2-dGlc transport was measured under aerobic conditions, it appeared that polyphosphate replenished the orthophosphate pool, indicating that polyphosphate has, at least mainly, an indirect role in sugar phosphorylation. Also in cells with a reduced respiratory capacity, due to a treatment with antimycin A, no direct role for polyphosphate in 2-dGlc transport could be detected. Under these conditions, only a very limited breakdown of polyphosphate occurred, probably because of the small decrease in the orthophosphate concentration.Abbreviations 2-dGlc 2-deoxy-D-glucose - P_i orthophosphate - P_n polyphosphate - SP sugar phosphate     

Synthesis and degradation of polyphosphate in the fission yeast Schizosaccharomyces pombe: mutations in phosphatase genes do not affect polyphosphate metabolism

The fission yeast Schizosaccharomyces pombe was found to accumulate large amounts of polyphosphate, particularly when grown on arginine as the nitrogen source. Upon transfer to a medium without phosphate, polyphosphate was degraded and served as an endogenous phosphate reserve. When phosphate was added again after a prolonged period of phosphate starvation, fission yeast cells synthesized more polyphosphate than they had contained before starvation, a phenomenon known as over-compensation. Strains carrying mutated structural genes for three different phosphatases, pho1, pho2 or pho3, degraded polyphosphate at the same rate as the wild-type strain during phosphate starvation and showed the same type of over-compensation when phosphate was added again.     

A kinetic study of the inhibition of yeast AMP deaminase by polyphosphate

Inorganic pyrophosphate and polyphosphates have acted as potent inhibitors of purified AMP deaminase (EC 3.5.4.6) from yeast: the activity fell to a definite limit with the increase in the concentration of the inhibitor. The effect of polyphosphate was largely on the maximal velocity of the enzyme with some decrease in affinity. The cooperative effect of AMP, analyzed in terms of a Hill coefficient, remained at 2 in the absence and presence of polyphosphate. Binding of polyphosphate to the enzyme showed no cooperativity. The inhibition of AMP deaminase by polyphosphate can be qualitatively and quantitatively accounted for by the partial mixed-type inhibition mechanism. Both the Ki value for the inhibitor and the breakdown rate of the enzyme-substrate-inhibitor complex are dependent on the chain length of polyphosphate, suggesting that the breakdown rate of the enzyme-substrate-inhibitor complex is regulated by binding of polyphosphate to a specific inhibitory site.     

Phosphite disrupts the acclimation of Saccharomyces cerevisiae to phosphate starvation.

The influence of phosphite (H2PO3-) on the response of Saccharomyces cerevisiae to orthophosphate (HPO4(2-); Pi) starvation was assessed. Phosphate-repressible acid phosphatase (rAPase) derepression and cell development were abolished when phosphate-sufficient (+Pi) yeast were subcultured into phosphate-deficient (-Pi) media containing 0.1 mM phosphite. By contrast, treatment with 0.1 mM phosphite exerted no influence on rAPase activity or growth of +Pi cells. 31P NMR spectroscopy revealed that phosphite is assimilated and concentrated by yeast cultured with 0.1 mM phosphite, and that the levels of sugar phosphates, pyrophosphate, and particularly polyphosphate were significantly reduced in the phosphite-treated -Pi cells. Examination of phosphite's effects on two PHO regulon mutants that constitutively express rAPase indicated that (i) a potential target for phosphite's action in -Pi yeast is Pho84 (plasmalemma high-affinity Pi transporter and component of a putative phosphate sensor-complex), and that (ii) an additional mechanism exists to control rAPase expression that is independent of Pho85 (cyclin-dependent protein kinase). Marked accumulation of polyphosphate in the delta pho85 mutant suggested that Pho85 contributes to the control of polyphosphate metabolism. Results are consistent with the hypothesis that phosphite obstructs the signaling pathway by which S. cerevisiae perceives and responds to phosphate deprivation at the molecular level.     

Cytochemical staining of a yeast polyphosphate fraction,localized outside the plasma membrane

Summary Primary aldehyde fixation in the presence of Ca²⁺ and Mg²⁺ followed by alkaline Pb²⁺ staining leads to electron microscopical visualization of lead precipitates in the yeastKluyveromyces marxianus. These lead precipitates are found in vacuoles, cytoplasm, and on the outside of the plasma membrane in the periplasmic and inner cell wall regions.X-ray microanalysis shows that the precipitates contain high amounts of Pb and P. The amount of precipitated material appeared to correlate with the cellular polyphosphate content. When Ca²⁺ and Mg²⁺ are omitted from the primary fixative no peripheral Pb/P deposits are observed. In a subsequent washing step a small amount of long chain polyphosphate is liberated. It is concluded that this method leads to visualization of cellular polyphosphate, including a fraction localized outside the plasma membrane ofKluyveromyces marxianus.     

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.     

Transmembrane gradient of K+ ions as an energy source in the yeast Saccharomyces carlsbergensis

In the presence of 100 mM glucose antimycin A inhibits the respiration of the yeast S. carlsbergensis by 94%, but does not affect the K+ efflux, Mn2+ influx or the synthesis of high molecular weight polyphosphate (HPP). Therefore phosphorylation at the respiratory chain level is not involved in HPP synthesis or Mn2+ accumulation. Zn2+ similar to Mn2+ induces K+ efflux and HPP synthesis, while Co2+ and Ni2+ fail to produce these effects. The extracellular K+ (1-5 mM KCl) completely inhibits the HPP synthesis and reduces Mn2+ uptake by 40%. NaCl (60 mM) inhibits the HPP synthesis by 28%. Nigericin, candicidin and FCCP plus valinomycin completely prevent the HPP synthesis. The prolonged accumulation of Zn2+ and Mn2+ is accompanied by HPP conversion into low molecular weight polyphosphate (LPP). The HPP synthesis in response to the K+ efflux may be regarded as a specific regulatory mechanism, which increases the energy efficiency of yeast metabolism.     

The involvement of high-energy phosphate in 2-deoxy-d-glucose transport in Kluyveromyces marxianus

Under aerobic conditions 2-deoxy-d-glucose was accumulated in Kluyveromyces marxianus mainly in a phosphorylated form. During sugar uptake both ATP, polyphosphate and orthophosphate levels decreased. Under anaerobic conditions considerably less sugar was taken up. The intracellular free sugar concentration did not exceed the medium concentration, whereas sugar phosphorylation leveled off at about 3 μmol/g yeast. In response to anaerobic 2-deoxy-d-glucose uptake only ATP and polyphosphate appeared to decrease. Within the experimental error sugar phosphorylation was counterbalanced by the polyphosphate decrease. Pulse labeling experiments revealed transport-associated phosphorylation under these anaerobic conditions, Further, kinetic studies on permeabilized cells showed that cytoplasmic ATP could not be the phosphoryl donor in this transport-associated phosphorylation. These results confirm and extend previous observations, indicating that polyphosphate plays a crucial role in 2-deoxy-d-glucose transport in Kluyveromyces marxianus.     

The crystal structure of the cytosolic exopolyphosphatase from Saccharomyces cerevisiae reveals the basis for substrate specificity

Inorganic long-chain polyphosphate is a ubiquitous linear polymer in biology, consisting of many phosphate moieties linked by phosphoanhydride bonds. It is synthesized by polyphosphate kinase, and metabolised by a number of enzymes, including exo- and endopolyphosphatases. The Saccharomyces cerevisiae gene PPX1 encodes for a 45 kDa, metal-dependent, cytosolic exopolyphosphatase that processively cleaves the terminal phosphate group from the polyphosphate chain, until inorganic pyrophosphate is all that remains. PPX1 belongs to the DHH family of phosphoesterases, which includes: family-2 inorganic pyrophosphatases, found in Gram-positive bacteria; prune, a cyclic AMPase; and RecJ, a single-stranded DNA exonuclease. We describe the high-resolution X-ray structures of yeast PPX1, solved using the multiple isomorphous replacement with anomalous scattering (MIRAS) technique, and its complexes with phosphate (1.6 A), sulphate (1.8 A) and ATP (1.9 A). Yeast PPX1 folds into two domains, and the structures reveal a strong similarity to the family-2 inorganic pyrophosphatases, particularly in the active-site region. A large, extended channel formed at the interface of the N and C-terminal domains is lined with positively charged amino acids and represents a conduit for polyphosphate and the site of phosphate hydrolysis. Structural comparisons with the inorganic pyrophosphatases and analysis of the ligand-bound complexes lead us to propose a hydrolysis mechanism. Finally, we discuss a structural basis for substrate selectivity and processivity.     

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.     

Repression of the acid phosphatase of Saccharomyces bisporus in relation to the polyphosphate content of the cells.

The relationship between the level of stored polyphosphate in growing cells of Saccharomyces bisporus and the repressing or derepression of the synthesis of the enzyme acid phosphatase (EC 3.1.3.2) was investigated. Time-course studies showed that there is no correlation between the cellular concentrations of either polyphosphate or orthophosphate and the ability of the cells to form this enzyme. The only compound investigated that was capable of repressing acid phosphatase synthesis was orthophosphate in the growth medium (i.e. orthophosphate outside the cell).     

Different chain length specificity among three polyphosphate quantification methods

Polyphosphate is ubiquitous among living organisms and has a variety of biochemical functions. Arbuscular mycorrhizal fungi have been known to accumulate polyphosphate as a key compound for their function. However, an enzymatic assay using polyphosphate kinase (PPK) reverse reaction, in which polyphosphate is converted to adenosine triphosphate (ATP) and quantified by luciferase assay, failed to detect accumulation of polyphosphate in some mycorrhizal root. When yeast exopolyphosphatase (PPX) was applied to these samples, a much higher polyphosphate level was detected than when the PPK assay was applied. Detailed analysis of substrate chain length specificity of these methods using polyphosphate chain length standards revealed that the PPX method was the most appropriate to detect short-chain polyphosphate. The average chain length of the shortest polyphosphate fraction that could be quantified with more than 50% efficiency was 3 for the PPX method and 38 for the PPK method. It was also suggested that the ratio of the PPK value to the PPX value may be useful as a simple and relative index to compare polyphosphate chain length distribution in different samples.     

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.     

Polyphosphate synthesis in yeast

Polyphosphate synthesis was studied in phosphate-starved cells of Saccharomyces cerevisiae and Kluyveromyces marxianus. Incubation of these yeasts for a short time with phosphate and either glucose or ethanol resulted in the formation of polyphosphate with a short chain length. With increasing incubation times, polyphosphates with longer chain lengths were formed. Polyphosphates were synthesized faster during incubation with glucose than with ethanol. Antimycin did not affect the glucose-induced polyphosphate synthesis in either yeast. Using ethanol as an energy source, antimycin A treatment blocked both polyphosphate synthesis and accumulation of orthophosphate in the yeast S. cerevisiae. However, in K. marxianus, polyphosphate synthesis and orthophosphate accumulation proceeded normally in antimycin-treated cells, suggesting that endogenous reserves were used as energy source. This was confirmed in experiments, conducted in the absence of an exogenous energy source.     

Isolation and characterization of polyphosphates from the yeast cell surface

When cells of Saccharomyces fragilis are subjected to osmotic shock, they release a limited amount of inorganic polyphosphate into the medium, which represents about 10% of the total cellular content. The osmotic shock procedure causes no substantial membrane damage, as judged from the unimpaired cell viability, limited K⁺ leakage and low percentage of stained cells. It is therefore suggested that this polyphosphate fraction is localized outside the plasma membrane. The released polyphosphate fraction differs from the remaining cellular polyphosphates in two respects: the mean chain length of the shock-sensitive fraction is significantly higher than that of the total cellular polyphosphates and its metabolic turnover rate, subsequent to pulsing with [³²P]orthophosphate is much lower compared to the rest of the cellular polyphosphate. Incubation of intact cells with the anion exchange resin Dowex AG 1-X4 results in the release of high molecular weight polyphosphates. These results suggest that the osmotic shock-sensitive polyphosphate fraction has specific characteristics in both its cellular localization and metabolism.     

Polyphosphates and exopolyphosphatase activities in the yeast Saccharomyces cerevisiae under overexpression of homologous and heterologous PPN1 genes

The role of exopolyphosphatase PPN1 in polyphosphate metabolism in fungi has been studied in strains of Saccharomyces cerevisiae transformed by the yeast PPN1 gene and its ortholog of the fungus Acremonium chrysogenum producing cephalosporin C. The PPN1 genes were expressed under a strong constitutive promoter of the gene of glycerol aldehyde-triphosphate dehydrogenase of S. cerevisiae in the vector pMB1. The yeast strain with inactivated PPN1 gene was transformed by the above vectors containing the PPN1 genes of S. cerevisiae and A. chrysogenum. Exopolyphosphatase activity in the transformant with the yeast PPN1 increased 28- and 11-fold compared to the mutant and parent PPN1 strains. The amount of polyphosphate in this transformant decreased threefold. Neither the increase in exopolyphosphatase activity nor the decrease in polyphosphate content was observed in the transformant with the orthologous PPN1 gene of A. chrysogenum, suggesting the absence of the active form of PPN1 in this transformant.     

KCS1 deletion in Saccharomyces cerevisiae leads to a defect in translocation of autophagic proteins and reduces autophagosome formation

Inositol phosphates are implicated in the regulation of autophagy; however, the exact role of each inositol phosphate species is unclear. In this study, we systematically analyzed the highly conserved inositol polyphosphate synthesis pathway in S. cerevisiae for its role in regulating autophagy. Using yeast mutants that harbored a deletion in each of the genes within the inositol polyphosphate synthesis pathway, we found that deletion of KCS1, and to a lesser degree IPK2, led to a defect in autophagy. KCS1 encodes an inositol hexakisphosphate/heptakisposphate kinase that synthesizes 5-IP 7 and IP 8; and IPK2 encodes an inositol polyphosphate multikinase required for synthesis of IP 4 and IP 5. We characterized the kcs1Δ mutant strain in detail. The kcs1Δ yeast exhibited reduced autophagic flux, which might be caused by both the reduction in autophagosome number and autophagosome size as observed under nitrogen starvation. The autophagy defect in kcs1Δ strain was associated with mislocalization of the phagophore assembly site (PAS) and a defect in Atg18 release from the vacuole membrane under nitrogen deprivation conditions. Interestingly, formation of autophagosome-like vesicles was commonly observed to originate from the plasma membrane in the kcs1Δ strain. Our results indicate that lack of KCS1 interferes with proper localization of the PAS, leads to reduction of autophagosome formation, and causes the formation of autophagosome-like structure in abnormal subcellular locations.     

The Acidocalcisome Vacuolar Transporter Chaperone 4 Catalyzes the Synthesis of Polyphosphate in Insect‐stages of Trypanosoma brucei and T. cruzi

Polyphosphate is a polymer of inorganic phosphate found in both prokaryotes and eukaryotes. Polyphosphate typically accumulates in acidic, calcium‐rich organelles known as acidocalcisomes, and recent research demonstrated that vacuolar transporter chaperone 4 catalyzes its synthesis in yeast. The human pathogens Trypanosoma brucei and T. cruzi possess vacuolar transporter chaperone 4 homologs. We demonstrate that T. cruzi vacuolar transporter chaperone 4 localizes to acidocalcisomes of epimastigotes by immunofluorescence and immuno‐electron microscopy and that the recombinant catalytic region of the T. cruzi enzyme is a polyphosphate kinase. RNA interference of the T. brucei enzyme in procyclic form parasites reduced short chain polyphosphate levels and resulted in accumulation of pyrophosphate. These results suggest that this trypanosome enzyme is an important component of a polyphosphate synthase complex that utilizes ATP to synthesize and translocate polyphosphate to acidocalcisomes in insect stages of these parasites.     

Tol1, a fission yeast phosphomonoesterase, is an in vivo target of lithium, and its deletion leads to sulfite auxotrophy

Lithium is the drug of choice for the treatment of bipolar affective disorder. The identification of an in vivo target of lithium in fission yeast as a model organism may help in the understanding of lithium therapy. For this purpose, we have isolated genes whose overexpression improved cell growth under high LiCl concentrations. Overexpression of tol1(+), one of the isolated genes, increased the tolerance of wild-type yeast cells for LiCl but not for NaCl. tol1(+) encodes a member of the lithium-sensitive phosphomonoesterase protein family, and it exerts dual enzymatic activities, 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase. tol1(+) gene-disrupted cells required high concentrations of sulfite in the medium for growth. Consistently, sulfite repressed the sulfate assimilation pathway in fission yeast. However, tol1(+) gene-disrupted cells could not fully recover from their growth defect and abnormal morphology even when the medium was supplemented with sulfite, suggesting the possible implication of inositol polyphosphate 1-phosphatase activity for cell growth and morphology. Given the remarkable functional conservation of the lithium-sensitive dual-specificity phosphomonoesterase between fission yeast and higher-eukaryotic cells during evolution, it may represent a likely in vivo target of lithium action across many species.