Polyphosphate synthesis in yeast

Schuddemat, J.; Boo, R. de; Leeuwen, C. C. M. Van; Broek, Peter J.A. Van den; Steveninck, J. Van

doi:10.1016/0167-4889(89)90160-2

Cited by 49 publications

(24 citation statements)

References 16 publications

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“…In contrast, cells harvested at a culture OD 650 of 0.8 contained high-molecular-weight polyP containing up to 150 residues in addition to the shorter-chain polyP. This is in accord with the results reported by Schuddemat et al (51) for polyP synthesis in the yeasts S. cerevisiae and Kluyceromyces marxianus, during which polyP was formed progressively as chains of increasing length.…”

Section: Accumulation Of Polypsupporting

confidence: 80%

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

McGrath

Quinn

2000

Appl Environ Microbiol

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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.Inorganic polyphosphate (polyP) is a linear polymer of phosphate residues linked together by high-energy phosphoanhydride bonds (34). PolyP, ranging in length from 3 to greater than 1,000 orthophosphate residues, has been detected in almost all organisms studied, including bacteria, yeasts, fungi, plants, and animals (12,35,36). Under optimal conditions polyP may amount to 10 to 20% of the cellular dry weight and thus greatly exceed the phosphate requirement of the cell, suggesting metabolic roles other than simply as a phosphate reserve (42). Microbial polyP synthesis is primarily carried out by the enzyme polyphosphate kinase (PPK) (32), which catalyzes the reversible transfer of the gamma phosphate from ATP to polyP (1,21,29,53). PolyP hydrolysis is mediated by exopolyphosphatases (PPX) (2, 60), endopolyphosphatases (38), and specific kinases (59).The exact physiological function of polyP remains uncertain, although various biological functions have been demonstrated, including as a reservoir of energy and phosphate (32), a chelator of divalent cations (30), a capsule material (54), and a "channelling" agent in the phenomenon of bacterial transformation (32, 48). Moreover, extensive accumulation of polyP has been detected in Escherichia coli in response to osmotic stress or nutritional stress imposed by either nitrogen, amino acid, or phosphate limitation (3,46). Similarly, Pseudomonas aeruginosa mucoid strain 8830 accumulates intracellular polyP particularly during stationary phase and in response to phosphate and amino acid limitations (3, 31), while both the unicellular alga Dunaliella salina and Saccharomyces cerevisiae utilize their intracellular polyP reserves to provide a pH-stat mechanism to counterbalance alkaline stress (4,11,42,43). PolyP accumulation has also been observed upon exposure of the freshwater sponge Ephydatia muelleri to various organic pollutants (27). Of greatest economical significance is the accumulation of polyP by some Acinetobacter spp. and other undefined organisms when they are exposed to alternating anaerobic/aerobic cycles; this phenomenon is the basis of the biotechnologically important wastewater treatment process designated Enhanced Biological Phosphate Removal (reviewed by VanLoosdrecht et al. [56] and Mino et a...

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Section: Accumulation Of Polypsupporting

confidence: 80%

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

McGrath

Quinn

2000

Appl Environ Microbiol

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“…In contrast, ⌬pho84 cells, which initially had a slightly lower polyphosphate content, had, at an OD 600 of 1.5, accumulated a high level of polyphosphates which, at an OD 600 of 3, had been reduced to a level comparable to that of wildtype and ⌬pho89 cells. The P i acquisition by ⌬pho84 cells following the initial rapid efflux was during prolonged growth (OD 600 of 1.5) paralleled by a pronounced synthesis of intracellular polyphosphate known to occur under conditions where phosphate and metabolic energy are available, especially when P i is added to cells previously starved for P i , resulting in intracellular P i levels of up to 20 mol/g (wet weight) of cells (2,13). It has been suggested that when, with continued growth, the metabolic requirements of the cells exceed the extracellular supply of P i which can be taken up via the P i transporters, vacuolar polyphosphate is mobilized to replenish the cytosolic phosphate pool (1,6).…”

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confidence: 99%

Regulation of Cation-Coupled High-Affinity Phosphate Uptake in the Yeast Saccharomyces cerevisiae

Pattison-Granberg

Persson

2000

J Bacteriol

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Studies of the high-affinity phosphate transporters in the yeast Saccharomyces cerevisiae using mutant strains lacking either the Pho84 or the Pho89 permease revealed that the transporters are differentially regulated. Although both genes are induced by phosphate starvation, activation of the Pho89 transporter precedes that of the Pho84 transporter early in the growth phase in a way which may possibly reflect a fine tuning of the phosphate uptake process relative to the availability of external phosphate.Saccharomyces cerevisiae has over the years provided a model for studies of how a cell makes a coordinated response in adapting to environmental changes in phosphate levels (7,11). This and other microorganisms have evolved complex mechanisms to efficiently take up this essential nutrient, which is often present in low amounts in the environment. When the cells meet a limitation in external phosphate, a high-affinity transport system with a K m for external phosphate of 0.5 to 10 M is mobilized (3,8). Of the proteins responsible for the high-affinity uptake, one is an H ϩ -coupled phosphate cotransporter encoded by the PHO84 gene (3). The activity of the Pho84p transporter has been shown to be regulated by the external phosphate level through expression of the gene, sorting of the synthesized protein to the plasma membrane, and degradation by rerouting of the protein to the vacuole (9, 12). The other high-affinity phosphate transporter is encoded by the PHO89 gene (8). The Pho89p transporter is largely inactive at the pH optimum for Pho84p-mediated transport, suggesting that this transporter has a complementary role in cellular phosphate acquisition. In this study, we have characterized the regulation and activity of the Pho89p transporter by use of mutants lacking either the Pho84p or the Pho89p transporter.The S. cerevisiae strains used were MB191 (MATa pho3-1 ade2 leu2-3,112 his3-532 trp1-289 ura3-1,2 can1) (3), MB192 (MATa pho3-1 ⌬pho84::HIS3 ade2 leu2-3,112 his3-532 trp1-289 ura3-1,2 can1) (3), PAM1 (MATa pho3-1 ⌬pho89::TRP1 ade2 leu2-3,112 his3-532 trp1-289 ura3-1,2 can1) (8), and PAM2 (MATa pho3-1 ⌬pho84::HIS3 ⌬pho89::TRP1 ade2 leu2-3,112 his3-532 trp1-289 ura3-1,2 can1) (8). Cells were routinely grown in shaking Erlenmayer flasks at 30°C in low-phosphate (LP i ) medium (5), pH 4.5, to an optical density at 600 nm (OD 600 ) ranging from 0.1 to 4.5. Cells were harvested by centrifugation at 2,300 ϫ g for 10 min and washed either once with 25 mM Tris-succinate (for P i uptake assays), at a different pH for each experiment, or twice with ice-cold bidistilled water (for 31 P nuclear magnetic resonance [NMR] measurements). The supernatants were subjected to phosphate concentration measurements spectrophotometrically essentially as described previously (10).Phosphate uptake in ⌬pho84 cells was assayed by the addition of 1-l volumes of [ 32 P]orthophosphate (0.18 Ci/mol; 1 mCi ϭ 37 MBq; Amersham-Pharmacia Biotech) to 30-l aliquots containing (each) 3 mg (wet weight) of cells suspended in 25 mM Tris-succinate buf...

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“…Poly(P) has been implicated as a reservoir of energy (1,3) and phosphate (3)(4)(5), as a kinase donor for sugars (1,6,7), in the chelation ofcations (4,8,9), in the entry of DNA into bacterial cells (10), and in the regulation of gene expression and enzyme activity (1,3,11). Although some of these functions remain largely unproven, the ubiquity and dynamic features of poly(P) suggest a variety of important roles in cellular metabolism and organismal development.…”

mentioning

confidence: 99%

Guanosine pentaphosphate phosphohydrolase of Escherichia coli is a long-chain exopolyphosphatase.

Keasling

Bertsch²,

Kornberg³

1993

Proc. Natl. Acad. Sci. U.S.A.

134

112

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An exopolyphosphatase [exopoly(P)ase; EC 3.6.1.11] activity has recently been purified to homogeneity from a mutant strain of Escherichia coi which lacks the principal exopoly(P)ase. The second exopoly(P)ase has now been identified as guanosine pentaphosphate phosphohydrolase (GPP; EC 3.6.1.40) by three lines of evidence: (i) the sequences of five btptic digestion fragments of the purified protein are found in the translated gppA gene, (u) the size of the protein (100 kDa) agrees with published values for GPP, and (iu) the ratio of exopoly(P)ase activity to GPP activity remains constant throughout a 300-fold purification in the last steps of the procedure. Inorganic polyphosphate [poly(P)] is a linear chain of nearly 1000 phosphoanhydride-bonded residues of inorganic phosphate (Pi) found in abundance in bacteria, fungi, plants, and animals (1, 2). Poly(P) has been implicated as a reservoir of energy (1, 3) and phosphate (3-5), as a kinase donor for sugars (1, 6, 7), in the chelation ofcations (4, 8, 9), in the entry of DNA into bacterial cells (10), and in the regulation of gene expression and enzyme activity (1,3,11). Although some of these functions remain largely unproven, the ubiquity and dynamic features of poly(P) suggest a variety of important roles in cellular metabolism and organismal development. Guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) have long been implicated in regulation of bacterial adjustments to stress. In response to amino acid starvation in Escherichia coli, the cellular content of ppGpp increases, with a concomitant shift in transcription from ribosomal genes to biosynthetic genes, a phenomenon known as the stringent response (12, 13). RNA polymerase has been suggested as a target for regulation by ppGpp (12,14).Recently, poly(P) kinase (PPK), which reversibly transfers the terminal phosphate of ATP to form poly(P), was purified from E. coli (15). In the course of cloning and overexpressing theppk gene, exopolyphosphatase (PPX; EC 3.6.1.11), which hydrolyzes the terminal residues of poly(P), was discovered in the same operon (16). From a ppx mutant a second poly(P)ase was purified and characterized. We report here its identification as guanosine pentaphosphate phosphohydrolase (GPP; EC 3.6.1.40), the protein which hydrolyzes the 5'-yphosphate of pppGpp to form ppGpp. Abbreviations: DTT, dithiothreitol; GPP, guanosine 5'-triphosphate 3'-diphosphate phosphohydrolase; PEI, polyethyleneimine; poly(P), polyphosphate; poly(P)ase, polyphosphatase; PPK, poly(P) kinase; ppGpp, guanosine 5'-diphosphate 3'-diphosphate (guanosine tetraphosphate); pppGpp, guanosine 5'-triphosphate 3'-diphosphate (guanosine pentaphosphate); PPX, exopolyphosphatase [exopoly(P)ase].ITo whom reprint requests should be addressed. 7029

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Polyphosphate synthesis in yeast

Cited by 49 publications

References 16 publications

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

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

Regulation of Cation-Coupled High-Affinity Phosphate Uptake in the Yeast Saccharomyces cerevisiae

Guanosine pentaphosphate phosphohydrolase of Escherichia coli is a long-chain exopolyphosphatase.

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