Plc1p, Arg82p, and Kcs1p, Enzymes Involved in Inositol Pyrophosphate Synthesis, Are Essential for Phosphate Regulation and Polyphosphate Accumulation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the phosphate signal transduction PHO pathway is involved in regulating several phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase. In this pathway, a cyclin-dependent kinase inhibitor (Pho81p) regulates the kinase activity of the cyclin-cyclin-dependent kinase complex Pho80p-Pho85p, which phosphorylates the transcription factor Pho4p in response to intracellular phosphate levels. However, how cells sense phosphate availability and transduce the phosphate signal to Pho81p remains unknown. To identify additional components of the PHO pathway, we have screened a collection of yeast deletion strains. We found that disruptants of PLC1, ARG82, and KCS1, which are involved in the synthesis of inositol polyphosphate, and ADK1, which encodes adenylate kinase, constitutively express PHO5. Each of these factors functions upstream of Pho81p and negatively regulates the PHO pathway independently of intracellular orthophosphate levels. Overexpression of KCS1, but not of the other genes, suppressed PHO5 expression in the wild-type strain under low phosphate conditions. These results raise the possibility that diphosphoinositol tetrakisphosphate and/or bisdiphosphoinositol triphosphate may be essential for regulation of the PHO pathway. Furthermore, the Δplc1, Δarg82, and Δkcs1 deletion strains, but not the Δipk1 deletion strain, had significantly reduced intracellular polyphosphate levels, suggesting that enzymes involved in inositol pyrophosphate synthesis are also required for polyphosphate accumulation. In Saccharomyces cerevisiae, the phosphate signal transduction PHO pathway is involved in regulating several phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase. In this pathway, a cyclin-dependent kinase inhibitor (Pho81p) regulates the kinase activity of the cyclin-cyclin-dependent kinase complex Pho80p-Pho85p, which phosphorylates the transcription factor Pho4p in response to intracellular phosphate levels. However, how cells sense phosphate availability and transduce the phosphate signal to Pho81p remains unknown. To identify additional components of the PHO pathway, we have screened a collection of yeast deletion strains. We found that disruptants of PLC1, ARG82, and KCS1, which are involved in the synthesis of inositol polyphosphate, and ADK1, which encodes adenylate kinase, constitutively express PHO5. Each of these factors functions upstream of Pho81p and negatively regulates the PHO pathway independently of intracellular orthophosphate levels. Overexpression of KCS1, but not of the other genes, suppressed PHO5 expression in the wild-type strain under low phosphate conditions. These results raise the possibility that diphosphoinositol tetrakisphosphate and/or bisdiphosphoinositol triphosphate may be essential for regulation of the PHO pathway. Furthermore, the Δplc1, Δarg82, and Δkcs1 deletion strains, but not the Δipk1 deletion strain, had significantly reduced intracellular polyphosphate levels, suggesting that enzymes involved in inositol pyrophosphate synthesis are also required for polyphosphate accumulation. Inorganic phosphate is an essential nutrient for all organisms, being required for many metabolic processes such as the biosynthesis of nucleic acids and phospholipids, energy metabolism, and signal transduction. Organisms therefore need efficient regulatory mechanisms for the acquisition, storage, and utilization of phosphate (1Torriani-Gorini A. Silver S. Yagil E. Phosphate in Microorganisms: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1994Google Scholar). In Saccharomyces cerevisiae, the phosphate signal transduction PHO pathway regulates the expression of a set of phosphate-responsive genes, including PHO5, which encodes repressible acid phosphatase (rAPase), 1The abbreviations used are: rAPase, repressible acid phosphatase; IP4, inositol tetrakisphosphate; IP5, inositol pentakisphosphate; PP-IP4, diphosphoinositol tetrakisphosphate; (PP)2-IP3, bisdiphosphoinositol triphosphate; IP6, inositol hexakisphosphate; IP3, inositol trisphosphate; PP-IP5, diphosphoinositol pentakisphosphate; (PP)2-IP4 bisdiphosphoinositol tetrakisphosphate. in response to changes in intracellular phosphate levels (2Lenburg M.E. O'Shea E.K. Trends Biochem. Sci. 1996; 21: 383-387Abstract Full Text PDF PubMed Scopus (221) Google Scholar, 3Oshima Y. Genes Genet. Syst. 1997; 72: 323-334Crossref PubMed Scopus (210) Google Scholar, 4Persson B.L. Lagerstedt J.O. Pratt J.R. Pattison-Granberg J. Lundh K. Shokrollahzadeh S. Lundh F. Curr. Genet. 2003; 43: 225-244Crossref PubMed Scopus (128) Google Scholar, 5Auesukaree C. Homma T. Tochio H. Shirakawa M. Kaneko Y. Harashima S. J. Biol. Chem. 2004; 279: 17289-17294Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The PHO pathway mediates this response by controlling the activity and localization of the transcription factor Pho4p through phosphorylation by the cyclin-cyclin-dependent kinase complex Pho80p-Pho85p. Under conditions of high phosphate, the Pho80p-Pho85p complex phosphorylates and inactivates the transcription factor Pho4p by triggering the association of phosphorylated Pho4p with the nuclear export receptor Msn5p (6Kaffman A. Herskowitz I. Tjian R. O'Shea E.K. Science. 1994; 263: 1153-1156Crossref PubMed Scopus (318) Google Scholar, 7Kaffman A. Rank N.M. O'Neill E.M. Huang L.S. O'Shea E.K. Nature. 1998; 396: 482-486Crossref PubMed Scopus (288) Google Scholar). This association leads to the rapid export of Pho4p from the nucleus to the cytoplasm and thus to repression of PHO5 expression. By contrast, when yeast cells are starved of phosphate, the cyclin-dependent kinase inhibitor Pho81p inactivates Pho80p-Pho85p (8Schneider K.R. Smith R.L. O'Shea E.K. Science. 1994; 266: 122-126Crossref PubMed Scopus (209) Google Scholar, 9Ogawa N. Noguchi K. Sawai H. Yamashita Y. Yompakdee C. Oshima Y. Mol. Cell. Biol. 1995; 15: 997-1004Crossref PubMed Scopus (77) Google Scholar), thereby allowing unphosphorylated Pho4p to associate with the nuclear import receptor Pse1p and to re-enter the nucleus to induce expression of PHO5 (10Kaffman A. Rank N.M. O'Shea E.K. Genes Dev. 1998; 12: 2673-2683Crossref PubMed Scopus (206) Google Scholar). Pho81p forms a stable complex with Pho80p-Pho85p under both high and low phosphate conditions, but inhibits the kinase activity of Pho85p only under low phosphate conditions, suggesting that the inhibitory activity of Pho81p is regulated post-translationally (8Schneider K.R. Smith R.L. O'Shea E.K. Science. 1994; 266: 122-126Crossref PubMed Scopus (209) Google Scholar). In addition, Pho81p has been demonstrated to be phosphorylated by Pho80p-Pho85p, and phosphorylation of Pho81p itself seems to be required for its stable interaction with the Pho80p-Pho85p complex (11Waters N.C. Knight J.P. Creasy C.L. Bergman L.W. Curr. Genet. 2004; 46: 1-9Crossref PubMed Scopus (18) Google Scholar, 12Knight J.P. Daly T.M. Bergman L.W. Curr. Genet. 2004; 46: 10-19Crossref PubMed Scopus (10) Google Scholar). A recent study has shown that PHO5 expression is regulated by intracellular phosphate levels, especially intracellular orthophosphate levels, but not by extracellular phosphate levels (5Auesukaree C. Homma T. Tochio H. Shirakawa M. Kaneko Y. Harashima S. J. Biol. Chem. 2004; 279: 17289-17294Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Moreover, both inositol polyphosphates and inositol pyrophosphates have been reported to play a role in controlling PHO5 expression, but in different ways. Inositol tetrakisphosphate (IP4) and inositol pentakisphosphate (IP5) are required for modulating the ability of the SWI/SNF and INO80 chromatin-remodeling complexes to induce PHO5 expression under low phosphate conditions (13Steger D.J. Haswell E.S. Miller A.L. Wente S.R. O'Shea E.K. Science. 2003; 299: 114-116Crossref PubMed Scopus (315) Google Scholar), whereas inositol pyrophosphates are necessary to maintain the repression of PHO5 expression under high phosphate conditions (14El Alami M. Messenguy F. Scherens B. Dubois E. Mol. Microbiol. 2003; 49: 457-468Crossref PubMed Scopus (60) Google Scholar). In an effort to understand the mechanism underlying phosphate sensing and signal transduction upstream of the Pho81p-Pho80p-Pho85p complex, we analyzed the Research Genetics collection of yeast deletion mutants. Here, we report that the additional factors Plc1p, Arg82p, Kcs1p, and Adk1p are involved in regulating the PHO pathway upstream of Pho81p. We provide evidence that Plc1p, Arg82p, Kcs1p, and Adk1p negatively regulate the PHO pathway independently of the intracellular orthophosphate levels, raising the possibility that diphosphoinositol tetrakisphosphate (PP-IP4) and/or bisdiphosphoinositol triphosphate ((PP)2-IP3) is important for phosphate regulation. In addition, we show that Plc1p, Arg82p, and Kcs1p are also required for the accumulation of polyphosphate. Strains and Media—The 4828 nonessential haploid S. cerevisiae deletion strains generated by the Saccharomyces Genome Deletion Project (15Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3212) Google Scholar) were obtained from Research Genetics. These strains are on a BY4742 (MATα) background. The isogenic strain BY4741 (MATa) was used for genetic analysis. The double and triple disruption strains used in this study as well as complete genotype descriptions are listed in Table I. Disruptions of PHO3 in strains BY4741 and BY4742 were generated by a previously described PCR-mediated gene disruption method (16Sakumoto N. Mukai Y. Uchida K. Kouchi T. Kuwajima J. Nakagawa Y. Sugioka S. Yamamoto E. Furuyama T. Mizubuchi H. Ohsugi N. Sakuno T. Kikuchi K. Matsuoka I. Ogawa N. Kaneko Y. Harashima S. Yeast. 1999; 15: 1669-1679Crossref PubMed Scopus (95) Google Scholar) using Candida glabrata HIS3 or LEU2, respectively, as a template, resulting in the deletion of the entire open reading frame. Gene disruptions were verified by colony PCR. The other double and triple disruptants were generated by standard genetic crossing, sporulation, and tetrad dissection (17Burke D. Dawson D. Stearns T. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000Google Scholar). Nutrient (yeast extract-peptone-dextrose-adenine (YPDA)), yeast nitrogen base without amino acids and glucose added, with appropriate nutrients (17Burke D. Dawson D. Stearns T. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000Google Scholar), and synthetic high and low phosphate (containing 11 mm and 0.22 mm Pi, respectively) media were prepared as described previously (18Yoshida K. Kuromitsu Z. Ogawa N. Oshima Y. Mol. Gen. Genet. 1989; 217: 31-39Crossref PubMed Scopus (65) Google Scholar).Table IS. cerevisiae strains used in this studyStrainRelevant genotypeSourceBY4741MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0Ref. 20Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2646) Google ScholarBY4742MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0Ref. 20Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2646) Google ScholarHaploidAs BY4742, orf::kanMX4aorf, open reading frame; CgLEU2, C. glabrata LEU2; CgHIS3, C. glabrata HIS3.Ref. 15Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3212) Google ScholarPHY77MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pho3::CgLEU2This studyPHY253MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 pho3::CgHIS3This studyPHY86MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pho3::CgLEU2 pho81::kanMX4This studyPHY94MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pho3::CgLEU2 pho4::kanMX4This studyPHY104MATα his3Δ1 leu2Δ0 ura3Δ0 pho3::CgLEU2 pho80::kanMX4This studyPHY116MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pho3::CgLEU2 plc1::kanMX4This studyPHY121MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pho3::CgLEU2 arg82::kanMX4This studyPHY129MATα his3Δ1 leu2Δ0 ura3Δ0 pho3::CgLEU2 kcs1::kanMX4This studyPHY174MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pho3::CgLEU2 ipk1::kanMX4This studyPHY179MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pho3::CgLEU2 adk1::kanMX4This studyPHY204MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pho3::CgLEU2 pho81::kanMX4 plc1::kanMX4This studyPHY240MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pho3::CgLEU2 pho81::kanMX4 arg82::kanMX4This studyPHY209MATa his3Δ1 leu2Δ0 ura3Δ0 pho3::CgLEU2 pho81::kanMX4 kcs1::kanMX4This studyPHY211MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 pho3::CgLEU2 pho81::kanMX4 adk1::kanMX4This studyPHY275MATa his3Δ1 leu2Δ0 ura3Δ0 pho3::CgLEU2 phm3::kanMX4This studyPHY274MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pho3::CgLEU2 pho84::kanMX4This studyPHY276MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pho3::CgLEU2 pho84::kanMX4 phm3::kanMX4This studyPHY246MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pho3::CgLEU2 adk1::kanMX4 phm3::kanMX4This studya orf, open reading frame; CgLEU2, C. glabrata LEU2; CgHIS3, C. glabrata HIS3. Open table in a new tab Plasmids—Plasmids pHB92 (expressing PLC1), pHB93 (ARG82), pHB94 (KCS1), and pHB95 (IPK1) were constructed in the same way using a 5′-HindIII PCR primer and a 3′-HindIII PCR primer with gene-specific sequences that included the entire open reading frame. PCR products were digested with HindIII and inserted into the HindIII gap of pGAD424 (Clontech), which contains the ADH1 promoter. To construct pHB96 (ADK1), the ADK1 open reading frame was amplified by PCR using appropriate oligonucleotides as primers with a HindIII restriction site. PCR products and pGAD424 were digested with HindIII, blunt-ended with the Klenow fragment, and ligated together. Screening for Phosphate Signaling-defective Deletion Mutants—Cells were transferred from thawed 96-well microtiter plate stocks to YPD medium plates supplemented with 200 mg/liter Geneticin in 96 place grids using a TK-CP96 96-pin replicator (Tokken Inc.). After incubation at 30 °C for 2 days, strains were stamped onto high and low phosphate medium plates and grown at 30 °C for an additional 2 days. The APase activity of the yeast strains was determined by staining colonies using α-naphthyl phosphate as a phosphatase substrate as described previously (19Toh-e A. Ueda Y. Kakimoto S.I. Oshima Y. J. Bacteriol. 1973; 113: 727-738Crossref PubMed Google Scholar). Acid Phosphatase Assay in Cell Suspension—Cells were precultured to log phase in synthetic high phosphate medium at 30 °C. Log-phase cultures were inoculated into a specified medium to give an A600 of 0.1 and cultivated with shaking at 30 °C until an A600 of 1.0 was reached. The APase activity was measured by determining the amount of p-nitrophenyl phosphate cleaved during a 10-min incubation at 35 °C. Cleavage was determined by monitoring the absorbance at 420 nm, and the APase activity was calculated as described previously (19Toh-e A. Ueda Y. Kakimoto S.I. Oshima Y. J. Bacteriol. 1973; 113: 727-738Crossref PubMed Google Scholar). RNA Purification and Northern Blot Analysis—Extraction of total RNA and Northern blot analysis were performed as described previously (17Burke D. Dawson D. Stearns T. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000Google Scholar). Cells were precultivated in synthetic high phosphate medium to log phase at 30 °C. Log-phase cultures were inoculated into a specified medium to give an A600 of 0.1 and shaken at 30 °C until an A600 of 1.0 was reached. Total RNA was extracted, and 15 μg of RNA was loaded per lane. DNA fragments containing the PHO5 open reading frame (+1 to +1404) synthesized by PCR and the 1.0-kb HindIII-XhoI fragment carrying ACT1 prepared from pYA301 (22Gallwitz D. Sures I. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2546-2550Crossref PubMed Scopus (267) Google Scholar) were labeled using a random primer DNA labeling kit (Version 2, Takara) with [α-32P]dCTP. Prehybridization, hybridization, and detection were carried out by standard methods. 31P NMR Spectroscopy—Yeast strains grown to log phase in synthetic high phosphate medium were inoculated into 100 ml of a specified medium to give an A600 of 0.1 and then cultivated at 30 °C until an A600 of 1.0 was reached. The cells were harvested by centrifugation, and excess medium was removed to obtain a cell suspension volume of 0.5 ml. The intracellular phosphate concentration in yeast cells was measured by 31P NMR spectroscopy as described previously (5Auesukaree C. Homma T. Tochio H. Shirakawa M. Kaneko Y. Harashima S. J. Biol. Chem. 2004; 279: 17289-17294Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). 31P NMR spectra were obtained at 202.496 MHz using a Bruker DRX-500 NMR spectrometer at 25 °C. The spectral width was 10 kHz. All spectra were generated from a collection of 512 scans, each with a 0.8-s acquisition time and 1-s delay. Methylene diphosphonate was used as an internal reference with no apparent distortion of yeast metabolism. The intracellular phosphate concentration was estimated on the basis of the integrated area of the resonance relative to methylene diphosphate. A haploid cell volume of 70 μm3 (23Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2545) Google Scholar) and a cell density of 1.85 × 107 cells/ml at an A600 of 1 were assumed. Polyphosphate concentration is given in terms of phosphate residues. Genome-wide Screening for Deletion Mutants Defective in Phosphate Signaling—To identify additional factors involved in the PHO pathway, we systematically screened a collection of 4828 haploid yeast deletion mutants of nonessential genes for mutants that showed defects in phosphate signal transduction. The yeast deletion strains were plated onto high and low phosphate media and scored by the APase assay. We expected to observe two different mutant phenotypes for this signaling pathway. For mutants of positive regulators of the PHO pathway, the expression of phosphate-responsive genes, including PHO5, would be repressed, irrespective of the phosphate concentration, resulting in a negative APase phenotype. By contrast, the expression of PHO5 in mutants of negative regulators would be induced constitutively; thus, these mutants should show a constitutive APase phenotype. From the three screens, we identified 12 mutants exhibiting the constitutive APase phenotype and 10 mutants exhibiting the negative APase phenotype (Fig. 1B and Table II). Of the mutants identified, seven were disruptants of genes encoding known components of the PHO pathway, viz. PHO2, PHO4, PHO5, PHO80, PHO81, PHO84, and PHO85, and 11 were disruptants of genes involved in general transcription or translation. Of the remaining four mutants, three were disruptants of genes involved in the synthesis of soluble inositol polyphosphate, viz. PLC1 encoding phospholipase C, ARG82 encoding inositol-polyphosphate kinase, and KCS1 encoding inositol hexakisphosphate (IP6) kinase. The fourth mutant was a disruptant of ADK1, which encodes adenylate kinase.Table IIGenes whose deletion affects APase activityGeneORFaORF, open reading frame; pol II, polymerase II.FunctionConstitutive APase mutantsPHO80YOL001wCyclinPHO85YPL031cCyclin-dependent kinasePHO84YML123cPhosphate transporterPLC1YPL268wPhospholipase CARG82YDR173cInositol-polyphosphate kinaseKCS1YDR017cInositol-polyphosphate kinaseADK1YDR226wAdenylate kinaseSPT5YML010wpol II transcription elongation factorSPT10YJL127cpol II transcription regulatorSPT21YMR179wpol II transcription regulatorRPD3YNL330cHistone deacetylaseRPO41YFL036wMitochondrial RNA polymeraseNegative APase mutantsPHO2YDL106cDNA-binding transcriptional activatorPHO4YFR034cDNA-binding transcriptional activatorPHO5YBR093cAcid phosphatasePHO81YGR233cCyclin-dependent kinase inhibitorGCN5YGR252wPart of histone acetyltransferase complexesSPT7YBR081cPart of histone acetyltransferase complexesSNF5YBR289wPart of SWI/SNF global transcription activator complexSNF6YHL025wPart of SWI/SNF global transcription activator complexRPL21AYBR191wRibosomal proteinRPL34BYIL052cRibosomal proteina ORF, open reading frame; pol II, polymerase II. Open table in a new tab In the synthetic pathway for soluble inositol polyphosphate, Plc1p hydrolyzes membrane-bound phosphatidylinositol bisphosphate to inositol trisphosphate (IP3) (24Yoko-o T. Matsui Y. Yagisawa H. Nojima H. Uno I. Toh-e A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1804-1808Crossref PubMed Scopus (139) Google Scholar, 25Flick J.S. Thorner J. Mol. Cell. Biol. 1993; 13: 5861-5876Crossref PubMed Scopus (171) Google Scholar), and Arg82p phosphorylates IP3 to IP4 and also converts IP4 to IP5 (26Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar), whereas Kcs1p is required to convert IP5 to PP-IP4, PP-IP4 to (PP)2-IP3, IP6 to diphosphoinositol pentakisphosphate (PP-IP5), and PP-IP5 to bisdiphosphoinositol tetrakisphosphate ((PP)2-IP4) (Fig. 1A) (27Saiardi A. Erdjument-Bromage H. Snowman A.M. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 28Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). By contrast, Adk1p catalyzes the phosphorylation of AMP by ATP to form two ADP molecules (29Abele U. Schulz G.E. Protein Sci. 1995; 4: 1262-1271Crossref PubMed Scopus (129) Google Scholar). The Δplc1, Δarg82, Δkcs1, and Δadk1 strains all exhibited the constitutive APase phenotype. Moreover, it should be noted that the APase activities of the Δplc1, Δarg82, and Δadk1 mutants were higher under the low phosphate conditions than under the high phosphate conditions, whereas the APase activity of the Δkcs1 mutant was almost the same as that in the Δpho80 strain under both high and low phosphate conditions (Fig. 1B). Interestingly, among the genes known to be involved in the synthesis of soluble inositol polyphosphate, deletion of only the IPK1 gene, which encodes IP5 kinase, did not result in the constitutive APase phenotype (Fig. 1, A and B). We therefore focused the following studies on the role of Plc1p, Arg82p, Kcs1p, and Adk1p in regulating the PHO pathway. Plc1p, Arg82p, Kcs1p, and Adk1p Are Required for the Regulation of PHO5 Expression—In S. cerevisiae, there are two types of APase: one is a constitutive APase encoded by PHO3, and the other is an rAPase encoded by PHO5 and its homologs PHO11 and PHO12 (19Toh-e A. Ueda Y. Kakimoto S.I. Oshima Y. J. Bacteriol. 1973; 113: 727-738Crossref PubMed Google Scholar, 30Toh-e A. Kakimoto S. Mol. Gen. Genet. 1975; 143: 65-70Crossref PubMed Scopus (37) Google Scholar). Expression of rAPase is controlled by the PHO pathway in response to intracellular phosphate concentrations (2Lenburg M.E. O'Shea E.K. Trends Biochem. Sci. 1996; 21: 383-387Abstract Full Text PDF PubMed Scopus (221) Google Scholar, 3Oshima Y. Genes Genet. Syst. 1997; 72: 323-334Crossref PubMed Scopus (210) Google Scholar, 4Persson B.L. Lagerstedt J.O. Pratt J.R. Pattison-Granberg J. Lundh K. Shokrollahzadeh S. Lundh F. Curr. Genet. 2003; 43: 225-244Crossref PubMed Scopus (128) Google Scholar, 5Auesukaree C. Homma T. Tochio H. Shirakawa M. Kaneko Y. Harashima S. J. Biol. Chem. 2004; 279: 17289-17294Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). To investigate whether Plc1p, Arg82p, Kcs1p, and Adk1p function as negative regulators in the PHO pathway, we knocked out the PHO3 gene in the Δplc1, Δarg82, Δkcs1, and Δadk1 strains and examined the resulting double disruptants for rAPase activity. We found that deletion of PHO3 in the Δplc1, Δarg82, Δkcs1, and Δadk1 strains did not affect the increased APase activity in these mutants (Fig. 1B). To determine whether the increased rAPase activity in these disruptants is a consequence of an increase in transcription of the PHO5 gene, we examined PHO5 transcript levels in the Δplc1Δpho3, Δarg82Δpho3, Δkcs1Δpho3, and Δadk1Δpho3 strains by Northern blot analysis. We found that the PHO5 transcript levels in these double disruptants were increased even under the high phosphate conditions compared with the wild-type strain (Δpho3) and correlated with the levels of rAPase activity (Fig. 1C), indicating that Plc1p, Arg82p, Kcs1p, and Adk1p are all involved in negative regulation at the level of PHO5 transcription. It should be noted that the PHO5 transcript levels in the Δadk1Δpho3 strain, but not in the other double disruptants, was higher under the low phosphate conditions than under the high phosphate conditions, indicating considerable residual regulation by the phosphate concentration in this strain. These observations suggest that Adk1p is only indirectly involved in regulating the PHO pathway. We also examined PHO5 transcription in the Δipk1Δpho3 strain and found that PHO5 transcription in this strain, which differed from that in the Δplc1Δpho3, Δarg82Δpho3, and Δkcs1Δpho3 strains, was induced only under the low phosphate conditions, as in the wild-type strain (Δpho3) (Fig. 1, B and C). Plc1p, Arg82p, Kcs1p, and Adk1p Function Upstream of the Pho81p-Pho80p-Pho85p Complex—Because the signal transduction mechanisms that function upstream of the Pho81p-Pho80p-Pho85p complex are still unknown, we tested whether Plc1p, Arg82p, Kcs1p, and Adk1p function upstream or downstream of Pho81p by analyzing the epistasis relationship between pho81 and the deletion mutants of these candidate regulators of the PHO pathway. If the deletion mutants are epistatic to a pho81 mutation, then deletion of PHO81 in the disruptants should result in an uninducible PHO5 expression phenotype. However, if the deletion mutants are hypostatic to a pho81 mutation, then deletion of PHO81 should result in the disruptants still displaying a constitutive PHO5 expression phenotype. We therefore generated triple disruptants of Δpho81 and Δplc1Δpho3, Δarg82Δpho3, Δkcs1Δpho3, and Δadk1Δpho3 and examined them for expression of PHO5 by the rAPase assay. The rAPase activity in each of the triple disruptants was diminished, similar to what was observed in the Δpho81Δpho3 strain (Fig. 2), indicating that pho81 is epistatic to each of the mutants. These results suggest that Plc1p, Arg82p, Kcs1p, and Adk1p function as negative regulators of the PHO pathway upstream of Pho81p. Plc1p, Arg82p, Kcs1p, and Adk1p Regulate the PHO Pathway Independently of the Intracellular Orthophosphate Concentration—Previous studies demonstrated that PHO5 expression is closely correlated with intracellular orthophosphate concentrations (5Auesukaree C. Homma T. Tochio H. Shirakawa M. Kaneko Y. Harashima S. J. Biol. Chem. 2004; 279: 17289-17294Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 31Bun-ya M. Nishimura M. Harashima S. Oshima Y. Mol. Cell. Biol. 1991; 11: 3229-3238Crossref PubMed Scopus (340) Google Scholar). To examine whether the induction of PHO5 expression in the Δplc1Δpho3, Δarg82Δpho3, Δkcs1Δpho3, and Δadk1Δpho3 strains under the high phosphate conditions was due to a reduction in intracellular orthophosphate, we measured the intracellular orthophosphate concentration in these double disruptants by in vivo 31P NMR spectroscopy. Surprisingly, we found that, under the high phosphate conditions, the Δplc1Δpho3 and Δarg82Δpho3 strains had significantly reduced intracellular polyphosphate levels, viz. 14 and 24%, respectively, of the level in the wild-type strain (Δpho3). Moreover, intracellular polyphosphate was reduced to an undetectable level in the Δkcs1Δpho3 strain. In contrast to the polyphosphate levels, intracellular orthophosphate levels in these double disruptants were ∼1.6-fold higher than that in the wild-type strain (Fig. 3A). These observations indicate that, despite the increase in intracellular orthophosphate levels, expression of PHO5 in the Δplc1Δpho3, Δarg82Δpho3, and Δkcs1Δpho3 strains is constitutive; this differs from its expression in the wild-type strain, where it is repressed (Fig. 1B). Therefore, these findings further suggest that Plc1p, Arg82p, and Kcs1p regulate the PHO pathway independently of intracellular orthophosphate levels. On the other hand, the levels of both intracellular orthophosphate and polyphosphate in the Δadk1Δpho3 strain were similar to those in the wild-type strain (Fig. 3B). Previous studies showed that the constitutive expression of PHO5 in the Δpho84 strain, a disruptant for a high affinity phosphate transporter, could be suppressed by increasing intracellular orthophosphate levels through the deletion of PHM3, PHM4, or both PHM1 and PHM2 (5Auesukaree C. Homma T. Tochio H. Shirakawa M. Kaneko Y. Harashima S. J. Biol. Chem. 2004; 279: 17289-17294Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 31Bun-ya M. Nishimura M. Harashima S. Oshima Y. Mol. Cell. Biol. 1991; 11: 3229-3238Crossref PubMed Scopus (340) Google Scholar). We therefore knocked out PHM3 in the Δadk1Δpho3 strain and found that the resulting triple mutant showed defects in accumulating intracellular polyphosphate and thus contained higher levels of intracellular orthophosphate compared with the wild-type strain (Fig. 3B). Under the high pho