The gluconate shunt is an alternative route for directing glucose into the pentose phosphate pathway in fission yeast
2017; Elsevier BV; Volume: 292; Issue: 33 Linguagem: Inglês
10.1074/jbc.m117.798488
ISSN1083-351X
AutoresMark E. Corkins, Stevin Wilson, Jean‐Christophe Cocuron, Ana Paula Alonso, Amanda Bird,
Tópico(s)Genetic Neurodegenerative Diseases
ResumoGlycolysis and the pentose phosphate pathway both play a central role in the degradation of glucose in all domains of life. Another metabolic route that can facilitate glucose breakdown is the gluconate shunt. In this shunt glucose dehydrogenase and gluconate kinase catalyze the two-step conversion of glucose into the pentose phosphate pathway intermediate 6-phosphogluconate. Despite the presence of these enzymes in many organisms, their only established role is in the production of 6-phosphogluconate for the Entner-Doudoroff pathway. In this report we performed metabolic profiling on a strain of Schizosaccharomyces pombe lacking the zinc-responsive transcriptional repressor Loz1 with the goal of identifying metabolic pathways that were altered by cellular zinc status. This profiling revealed that loz1Δ cells accumulate higher levels of gluconate. We show that the altered gluconate levels in loz1Δ cells result from increased expression of gcd1. By analyzing the activity of recombinant Gcd1 in vitro and by measuring gluconate levels in strains lacking enzymes of the gluconate shunt we demonstrate that Gcd1 encodes a novel NADP+-dependent glucose dehydrogenase that acts in a pathway with the Idn1 gluconate kinase. We also find that cells lacking gcd1 and zwf1, which encode the first enzyme in the pentose phosphate pathway, have a more severe growth phenotype than cells lacking zwf1. We propose that in S. pombe Gcd1 and Idn1 act together to shunt glucose into the pentose phosphate pathway, creating an alternative route for directing glucose into the pentose phosphate pathway that bypasses hexokinase and the rate-limiting enzyme glucose-6-phosphate dehydrogenase. Glycolysis and the pentose phosphate pathway both play a central role in the degradation of glucose in all domains of life. Another metabolic route that can facilitate glucose breakdown is the gluconate shunt. In this shunt glucose dehydrogenase and gluconate kinase catalyze the two-step conversion of glucose into the pentose phosphate pathway intermediate 6-phosphogluconate. Despite the presence of these enzymes in many organisms, their only established role is in the production of 6-phosphogluconate for the Entner-Doudoroff pathway. In this report we performed metabolic profiling on a strain of Schizosaccharomyces pombe lacking the zinc-responsive transcriptional repressor Loz1 with the goal of identifying metabolic pathways that were altered by cellular zinc status. This profiling revealed that loz1Δ cells accumulate higher levels of gluconate. We show that the altered gluconate levels in loz1Δ cells result from increased expression of gcd1. By analyzing the activity of recombinant Gcd1 in vitro and by measuring gluconate levels in strains lacking enzymes of the gluconate shunt we demonstrate that Gcd1 encodes a novel NADP+-dependent glucose dehydrogenase that acts in a pathway with the Idn1 gluconate kinase. We also find that cells lacking gcd1 and zwf1, which encode the first enzyme in the pentose phosphate pathway, have a more severe growth phenotype than cells lacking zwf1. We propose that in S. pombe Gcd1 and Idn1 act together to shunt glucose into the pentose phosphate pathway, creating an alternative route for directing glucose into the pentose phosphate pathway that bypasses hexokinase and the rate-limiting enzyme glucose-6-phosphate dehydrogenase. The Embden-Meyerhof-Parnas pathway (glycolysis) and the pentose phosphate pathway play a central role in the degradation of glucose in organisms from all domains of life. In glycolysis, glucose is broken down to pyruvate to provide energy in the form of ATP, metabolic intermediates, and reduced nicotinamide adenine dinucleotide. In the pentose phosphate pathway, glucose is broken down to provide reducing equivalents in the form of NADPH and pentose sugars that are biosynthetic precursors of nucleic acids and amino acids (1.Stanton R.C. Glucose-6-phosphate dehydrogenase, NADPH, and cell survival.IUBMB Life. 2012; 64: 362-369Crossref PubMed Scopus (461) Google Scholar, 2.Stincone A. Prigione A. Cramer T. Wamelink M.M. Campbell K. Cheung E. Olin-Sandoval V. Grüning N.M. Krüger A. Tauqeer Alam M. Keller M.A. Breitenbach M. Brindle K.M. Rabinowitz J.D. Ralser M. The return of metabolism: biochemistry and physiology of the pentose phosphate pathway.Biol. Rev. Camb. Philos. Soc. 2015; 90: 927-963Crossref PubMed Scopus (764) Google Scholar, 3.Lunt S.Y. Vander Heiden M.G. Aerobic glycolysis: meeting the metabolic requirements of cell proliferation.Annu. Rev. Cell Dev. Biol. 2011; 27: 441-464Crossref PubMed Scopus (2044) Google Scholar). As the processes of glycolysis and the pentose phosphate pathway run in parallel, all cells have mechanisms to tightly regulate the flow of glucose into each pathway. On entering cells, the majority of glucose is phosphorylated to glucose 6-phosphate (Glu-6-P) in the first step of glycolysis. The intermediate Glu-6-P can then be further broken down in the process of glycolysis, or shunted into the pentose phosphate pathway. In the first committed step of the pentose phosphate pathway, Glu-6-P dehydrogenase catalyzes the dehydrogenation of Glu-6-P. This irreversible, rate-limiting step of the pentose phosphate pathway is typically highly regulated within cells, and therefore holds a prominent position in determining the overall flow of glucose into the pentose phosphate pathway (1.Stanton R.C. Glucose-6-phosphate dehydrogenase, NADPH, and cell survival.IUBMB Life. 2012; 64: 362-369Crossref PubMed Scopus (461) Google Scholar, 2.Stincone A. Prigione A. Cramer T. Wamelink M.M. Campbell K. Cheung E. Olin-Sandoval V. Grüning N.M. Krüger A. Tauqeer Alam M. Keller M.A. Breitenbach M. Brindle K.M. Rabinowitz J.D. Ralser M. The return of metabolism: biochemistry and physiology of the pentose phosphate pathway.Biol. Rev. Camb. Philos. Soc. 2015; 90: 927-963Crossref PubMed Scopus (764) Google Scholar). The gluconate shunt is a less studied metabolic route that can facilitate the breakdown of glucose. In the gluconate shunt, glucose is oxidized by glucose dehydrogenase to gluconate, which is then phosphorylated by gluconate kinase to produce 6-phosphogluconate (4.Peekhaus N. Conway T. What's for dinner?: Entner-Doudoroff metabolism in Escherichia coli.J. Bacteriol. 1998; 180: 3495-3502Crossref PubMed Google Scholar, 5.Tsai C.S. Ye H.G. Shi J.L. Carbon-13 NMR studies and purification of gluconate pathway enzymes from Schizosaccharomyces pombe.Arch. Biochem. Biophys. 1995; 316: 155-162Crossref PubMed Scopus (24) Google Scholar). As 6-phosphogluconate is the second intermediate of the pentose phosphate pathway, the gluconate shunt potentially creates a route for directing glucose into the pentose phosphate pathway that bypasses the rate-limiting enzyme Glu-6-P dehydrogenase. However, the only established role for the gluconate shunt is found in plants, algae, cyanobacteria, and some bacteria, which all use the Entner-Doudoroff (ED) 2The abbreviations used are: ED, Entner-Doudoroff; EV, empty vector; GC-MS, gas chromatography-mass spectrometry; LC-MS, liquid chromatography-mass spectrometry; YES, yeast extract plus supplements; Adh1, alcohol dehydrogenase 1; ZL-EMM, zinc-limited minimal medium. pathway to degrade glucose or gluconate (4.Peekhaus N. Conway T. What's for dinner?: Entner-Doudoroff metabolism in Escherichia coli.J. Bacteriol. 1998; 180: 3495-3502Crossref PubMed Google Scholar, 6.Chen X. Schreiber K. Appel J. Makowka A. Fähnrich B. Roettger M. Hajirezaei M.R. Sönnichsen F.D. Schönheit P. Martin W.F. Gutekunst K. The Entner-Doudoroff pathway is an overlooked glycolytic route in cyanobacteria and plants.Proc. Natl. Acad. Sci. U.S.A. 2016; 113: 5441-5446Crossref PubMed Scopus (122) Google Scholar, 7.Patra T. Koley H. Ramamurthy T. Ghose A.C. Nandy R.K. The Entner-Doudoroff pathway is obligatory for gluconate utilization and contributes to the pathogenicity of Vibrio cholerae.J. Bacteriol. 2012; 194: 3377-3385Crossref PubMed Scopus (50) Google Scholar). In the ED pathway 6-phosphogluconate is dehydrated to generate 2-keto-3-deoxygluconate-6-phosphate, which is then cleaved to generate pyruvate and glyceraldehyde 3-phosphate. The gluconate shunt is therefore a metabolic route that can be used to direct glucose and gluconate to the ED pathway (6.Chen X. Schreiber K. Appel J. Makowka A. Fähnrich B. Roettger M. Hajirezaei M.R. Sönnichsen F.D. Schönheit P. Martin W.F. Gutekunst K. The Entner-Doudoroff pathway is an overlooked glycolytic route in cyanobacteria and plants.Proc. Natl. Acad. Sci. U.S.A. 2016; 113: 5441-5446Crossref PubMed Scopus (122) Google Scholar, 8.Fliege R. Tong S. Shibata A. Nickerson K.W. Conway T. The Entner-Doudoroff pathway in Escherichia coli is induced for oxidative glucose metabolism via pyrroloquinoline quinone-dependent glucose dehydrogenase.Appl. Environ. Microbiol. 1992; 58: 3826-3829Crossref PubMed Google Scholar). Despite this established role for the gluconate pathway, glucose dehydrogenase and gluconate kinase activities have been detected in mammals, fission yeast, and flies, which all lack the key ED pathway enzymes (5.Tsai C.S. Ye H.G. Shi J.L. Carbon-13 NMR studies and purification of gluconate pathway enzymes from Schizosaccharomyces pombe.Arch. Biochem. Biophys. 1995; 316: 155-162Crossref PubMed Scopus (24) Google Scholar, 9.Campbell D.P. 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Biochemical characterization of human gluconokinase and the proposed metabolic impact of gluconic acid as determined by constraint based metabolic network analysis.PLoS ONE. 2014; 9: e98760Crossref PubMed Scopus (27) Google Scholar). In this report we used metabolomics profiling to identify metabolites that accumulate in a fission yeast strain lacking the transcriptional repressor Loz1. In Schizosaccharomyces pombe Loz1 plays a central role in zinc homeostasis by regulating the expression of genes required for zinc uptake and zinc storage (14.Corkins M.E. May M. Ehrensberger K.M. Hu Y.M. Liu Y.H. Bloor S.D. Jenkins B. Runge K.W. Bird A.J. Zinc finger protein Loz1 is required for zinc-responsive regulation of gene expression in fission yeast.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 15371-15376Crossref PubMed Scopus (34) Google Scholar, 15.Wilson S. Bird A.J. Zinc sensing and regulation in yeast model systems.Arch. Biochem. Biophys. 2016; 611: 30-36Crossref PubMed Scopus (33) Google Scholar). Loz1 also regulates the availability of zinc in cells by controlling the levels of the non-essential, abundant zinc-binding enzyme alcohol dehydrogenase 1 (Adh1). Specifically, under zinc-limiting conditions, inactivation of Loz1 3The descriptive names of genes or proteins discussed in the text are as follows: loz1, loss of zinc sensing 1; S. pombe, adh4, alcohol dehydrogenase 4; ght3, homology to Glut- and Hxt-transporter 3; gnd1, 6-phosphogluconate dehydrogenase 1; idn1, l-IDoNate catabolism 1; pgk1, phosphoglycerate kinase 1; zrt1, zinc-regulated transporter 1; zwf1, ZWischenferment 1; S. cerevisiae, MET30, METhionine requiring 30; MET4, METhionine requiring 4; Drosophila melanogaster-Zw, ZWischenferment; H. sapiens Glu-6-PD, glucose-6-phosphate dehydrogenase 1. results in the derepression of an adh1 antisense transcript, and the strong antisense transcription in turn inhibits the expression of adh1 (16.Ehrensberger K.M. Corkins M.E. Choi S. Bird A.J. The double zinc finger domain and adjacent accessory domain from the transcription factor loss of zinc sensing 1 (loz1) are necessary for DNA binding and zinc sensing.J. Biol. Chem. 2014; 289: 18087-18096Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 17.Ehrensberger K.M. Mason C. Corkins M.E. Anderson C. Dutrow N. Cairns B.R. Dalley B. Milash B. Bird A.J. Zinc-dependent regulation of the Adh1 antisense transcript in fission yeast.J. Biol. Chem. 2013; 288: 759-769Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Although the regulation of adh1 gene expression has been well characterized at a transcriptional level, it is largely unclear if the lowered levels of Adh1 in zinc-deficient cells also affects cellular metabolism. As Loz1 regulates the expression of adh1, and potentially other abundant zinc-binding proteins, the goal of this study was to determine whether the changes in transcription that are mediated by Loz1 also affect cell metabolism. By using metabolomic analyses to screen for metabolites whose levels were altered in fission yeast mutants with impaired Loz1 function, we found that the metabolite that showed the highest fold-increase in loz1Δ cells was d-gluconate. Here we show that the higher levels of d-gluconate in loz1Δ cells results from increased expression of gcd1, a gene encoding a novel NADP+-dependent glucose dehydrogenase. We also find that the function of gcd1 overlaps with zwf1, the gene encoding Glu-6-P dehydrogenase. We propose that in fission yeast the gluconate shunt creates an alternative route for directing glucose into the pentose phosphate pathway that bypasses the rate-limiting enzyme Glu-6-P dehydrogenase. Our previous studies revealed that cells lacking the transcriptional repressor Loz1, or cells expressing the hypomorphic allele loz1-1, have impaired zinc homeostasis and reduced levels of the enzyme Adh1 (14.Corkins M.E. May M. Ehrensberger K.M. Hu Y.M. Liu Y.H. Bloor S.D. Jenkins B. Runge K.W. Bird A.J. Zinc finger protein Loz1 is required for zinc-responsive regulation of gene expression in fission yeast.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 15371-15376Crossref PubMed Scopus (34) Google Scholar). To determine whether impaired Loz1 activity affected cell metabolism, wild-type, loz1Δ, and loz1-1 cells were grown to exponential phase in the nutrient-rich YES medium. Cells were then harvested and metabolites identified using both GC-MS and LC-MS. 314 unique metabolites were detected. 11 of these accumulated >2-fold in both loz1-1 and loz1Δ cells relative to the wild-type control, including a variety of lipids and hydrolyzed phospholipids, the amino acid ergothioneine, the organic acids citrate, cis-aconitate, and gluconate, and the alcohol 2,3-butanediol (Table 1). Of these metabolites, gluconate, a naturally occurring derivative of glucose, showed the highest fold-increase in loz1Δ. As little is known about the biological function of gluconate in eukaryotes, we chose to further investigate how and why gluconate levels were regulated by Loz1.Table 1Metabolites that accumulate to significantly higher levels (p ≤ 0.05) in loz1 mutantsFold increaseBiochemical nameloz1–1 /WTloz1Δ/WTGluconate3.494.24Mevalonate2.713.53Phytosphingosine3.773.342-Palmitoleoylglycerophosphoinositol3.193.07Ergothioneine2.092.87Citrate2.692.852,3-Butanediol2.542.811-Stearoylglycerophosphocholine (18:0)2.242.79cis-Aconitate2.402.222-Myristoylglycerophosphocholine2.412.051-Eicosenoylglycerophosphocholine (20:1n9)2.892.03 Open table in a new tab Studies of glucose metabolism in cell-free extracts of S. pombe have demonstrated the presence of two enzymes involved in gluconate metabolism, an NADP+-dependent glucose dehydrogenase, which catalyzes the oxidation of d-glucose to d-gluconate, and gluconate kinase, which phosphorylates d-gluconate to produce 6-phosphogluconate (Fig. 1) (5.Tsai C.S. Ye H.G. Shi J.L. Carbon-13 NMR studies and purification of gluconate pathway enzymes from Schizosaccharomyces pombe.Arch. Biochem. Biophys. 1995; 316: 155-162Crossref PubMed Scopus (24) Google Scholar, 18.Tsai C.S. Shi J.L. Ye H.G. Kinetic studies of gluconate pathway enzymes from Schizosaccharomyces pombe.Arch. Biochem. Biophys. 1995; 316: 163-168Crossref PubMed Scopus (21) Google Scholar). A single hexose transporter named Ght3 has also been identified that facilitates uptake of gluconate when glucose is limiting (19.Heiland S. Radovanovic N. Höfer M. Winderickx J. Lichtenberg H. Multiple hexose transporters of Schizosaccharomyces pombe.J. Bacteriol. 2000; 182: 2153-2162Crossref PubMed Scopus (63) Google Scholar). As the growth medium used for the metabolic profiling contained 3% glucose, which results in the strong repression of the Ght3 gluconate uptake system (supplemental Fig. S1), we predicted that the increased levels of gluconate in loz1Δ cells were likely to be a result of altered expression of one or both of the enzymes involved in gluconate metabolism. The simplest explanation for the increase in gluconate levels in the loz1 mutants was that the expression or activity of the gluconate kinase was reduced in these strains (Fig. 2A). In S. pombe a single gene named idn1 encodes a protein with high sequence similarity to characterized gluconate kinases (supplemental Fig. S2). To test whether Idn1 was required for the breakdown of gluconate levels in vivo, wild-type and idn1Δ cells were grown to exponential phase in YES medium and gluconate levels were measured by LC-MS/MS. As shown in Fig. 2B, idn1Δ cells accumulated ∼300-fold higher levels of gluconate relative to the wild-type control, consistent with Idn1 catalyzing the phosphorylation of gluconate to 6-phosphogluconate. As a complementary approach to examine Idn1 activity, we employed an enzymatic assay to measure gluconate levels in cell extracts. In these assays, intracellular gluconate levels are coupled to the consumption of 6-phosphogluconate (see "Experimental procedures"). When gluconate levels were measured by this method, idn1Δ cells accumulated ∼28-fold higher levels of gluconate relative to the wild-type control (Fig. 2C). This phenotype was also rescued by the introduction of a plasmid expressing an Idn1-GFP fusion protein from the constitutive pgk1 promoter (idn1Δ Idn1-GFP). These results are consistent with the predicted role of Idn1 in phosphorylating gluconate in vivo. As decreased expression of gluconate kinase would potentially lead to gluconate accumulating within cells, we next tested whether idn1 expression was altered in loz1Δ cells. To determine whether the transcription of idn1 was controlled by Loz1, a construct was generated in which ∼1200 bp of the idn1 promoter, extending from the open reading frame, was fused to the lacZ reporter gene. In wild-type and loz1Δ cells expressing this reporter, no significant changes in β-galactosidase activity were apparent following growth overnight in a zinc-limited minimal medium (ZL-EMM) supplemented with 0–200 μm zinc (Fig. 2D). The presence of a fully functional Loz1 also had little effect on idn1 mRNA levels, as similar levels of idn1 transcripts accumulated in wild-type, loz1-1, and loz1Δ cells grown to exponential phase in the zinc-rich YES medium (Fig. 2E). This was in contrast to the Loz1-regulated zrt1 and adh4 mRNA controls, which accumulated to higher levels in loz1 mutants grown in the zinc-replete YES medium. Idn1 protein levels were also not regulated by Loz1 or zinc as similar levels of the functional Idn1-GFP protein were detected in idn1Δ loz1Δ and idn1Δ cells (Fig. 2F), and in idn1Δ cells grown over a range of zinc levels (Fig. 2G). Although the above experiments do not eliminate a model in which Loz1 influences the activity of Idn1, the results indicate that the ability of loz1Δ and loz1-1 cells to accumulate gluconate is not a result of altered idn1 gene expression. An alternative explanation for the increased gluconate in the loz1 mutants is that the unknown glucose dehydrogenase is expressed at higher levels in the loz1-1 and loz1Δ cells. To gain insight into whether the first step of the gluconate shunt was regulated by Loz1, gluconate levels were compared in loz1Δ idn1Δ and idn1Δ cells. As shown in Fig. 2, B and C, ∼2-fold higher levels of gluconate accumulated in loz1Δ idn1Δ relative to idn1Δ. These results reveal that the increase in gluconate in loz1Δ cells is independent of Idn1, and are consistent with gluconate synthesis being higher in loz1Δ. As Loz1 represses its target gene expression when zinc is in excess, we predicted that the gene encoding the unknown glucose dehydrogenase might be expressed at higher levels in zinc-deficient cells. When published microarray data were searched for putative NADP+-dependent oxidoreductases that were regulated by zinc, we noted that SPCC794.01c, a gene encoding a putative NADP+-dependent Glu-6-P dehydrogenase, was induced in response to zinc deficiency in multiple analyses (17.Ehrensberger K.M. Mason C. Corkins M.E. Anderson C. Dutrow N. Cairns B.R. Dalley B. Milash B. Bird A.J. Zinc-dependent regulation of the Adh1 antisense transcript in fission yeast.J. Biol. Chem. 2013; 288: 759-769Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 20.Dainty S.J. Kennedy C.A. Watt S. Bähler J. Whitehall S.K. Response of Schizosaccharomyces pombe to zinc deficiency.Eukaryot. Cell. 2008; 7: 454-464Crossref PubMed Scopus (33) Google Scholar). In S. pombe, two additional genes (zwf1 and SPAC3C7.13c) are also predicted to encode NADP+-dependent Glc-6-P dehydrogenase, which catalyzes the first committed step of the pentose phosphate pathway (Fig. 1). Although all of the three putative Glc-6-P dehydrogenases from S. pombe have multiple conserved domains that are common to this family of proteins (supplemental Fig. S3), a notable exception was that the residues predicted to be involved in coordinating the phosphate moiety of Glc-6-P (21.Cosgrove M.S. Gover S. Naylor C.E. Vandeputte-Rutten L. Adams M.J. Levy H.R. An examination of the role of Asp-177 in the His-Asp catalytic dyad of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: X-ray structure and pH dependence of kinetic parameters of the D177N mutant enzyme.Biochemistry. 2000; 39: 15002-15011Crossref PubMed Scopus (47) Google Scholar, 22.Bautista J.M. Mason P.J. Luzzatto L. Human glucose-6-phosphate dehydrogenase: lysine 205 is dispensable for substrate binding but essential for catalysis.FEBS Lett. 1995; 366: 61-64Crossref PubMed Scopus (29) Google Scholar) were not conserved in SPCC794.01c (Fig. 3A). This lack of conservation suggested that SPCC794.01c might differ in its substrate specificity relative to other Glc-6-P dehydrogenases. To examine the substrate specificity of SPCC794.01c, His-tagged recombinant SPCC794.01c was purified from Escherichia coli (Fig. 3B). The activities of His-SPCC794.01c, the His tag alone (EV), were then assayed by measuring NADPH production in the presence of a given substrate (Fig. 3C). The activity of Glc-6-P dehydrogenase from Saccharomyces cerevisiae (Sc.Zwf1) was also measured as a control. As expected, incubation of the control enzyme (Sc.Zwf1) with each of the substrates led to an ∼8.5-fold increase in NADPH levels in the presence of Glc-6-P relative to the His tag control (EV). This activity was specific to Glc-6-P as no increase in NADPH was observed in the presence of glucose or galactose substrates. In contrast, incubation of SPCC794.01c in the presence of glucose resulted in an ∼12-fold increase in NADPH levels relative to the His tag control, and in the presence of Glc-6-P, a 3-fold increase in NADPH. No increase in NADPH was observed in the presence of a galactose substrate. Together these experiments reveal that SPCC794.01c has different substrate specificity than other Glc-6-P dehydrogenases and that it is able to use glucose as a substrate in vitro. As SPCC794.01c functions as a glucose dehydrogenase, it was named Gcd1 for Glucose dehydrogenase 1. To test whether Gcd1 is necessary for the increased gluconate in loz1Δ cells in vivo, wild-type, loz1Δ, and loz1Δ gcd1Δ cells were grown to exponential phase in YES medium and gluconate levels were measured by LC-MS/MS (Fig. 3D). As expected, higher levels of gluconate accumulated in loz1Δ cells relative to the wild-type control. This increase was not apparent in loz1Δ gcd1Δ indicating that Gcd1 is required for the increased levels of gluconate in loz1Δ cells. As Gcd1 was necessary for the Loz1-dependent increase in gluconate we next tested whether gcd1 was a Loz1 target gene. To determine whether the expression of gcd1 was dependent upon Loz1, a reporter construct containing ∼1450 bp of the gcd1 promoter fused to the lacZ gene was integrated into the genome of wild-type and loz1Δ cells. As Loz1 represses gene expression when zinc is in excess, β-galactosidase activity was examined following growth in ZL-EMM or ZL-EMM supplemented with 1–200 μm Zn2+. An ∼10-fold increase in β-galactosidase activity was observed in zinc-limited wild-type cells, whereas high levels of β-galactosidase activity were observed under all conditions in loz1Δ (Fig. 4A). Cellular gcd1 mRNA levels were also dependent upon zinc and Loz1 (Fig. 4, B and C). To examine the effects of zinc on Gcd1 protein levels, a strain was generated that expressed the endogenous Gcd1 protein fused to 13 myc epitope tags. As shown in Fig. 4D, there was an ∼2–3-fold increase in levels of Gcd1-Myc protein in cells grown overnight under zinc-limiting conditions relative to the levels of Gcd1-Myc that accumulated in zinc-replete cells. Thus, Gcd1 accumulates in both zinc-limited and zinc-replete cells, yet higher levels accumulate in zinc-starved cells consistent with the increased expression of gcd1 under this condition. To determine whether increased expression of gcd1 in zinc-limited cells also resulted in gluconate accumulation, gluconate levels were measured in strains grown in ZL-EMM with or without a 200 μm zinc supplement. Zinc-limited wild-type cells accumulated ∼3-fold higher levels of gluconate relative to zinc-replete cells (Fig. 4E). This fold-change was reduced in loz1Δ mutants, whereas deletion of gcd1 resulted in lower levels of gluconate accumulating under zinc-limiting conditions. Although these results are consistent with the Loz1-dependent derepression of gcd1 leading to increased gluconate accumulation in zinc-limited cells, the levels of gluconate that accumulated were small relative to the large increase in gluconate seen in idn1Δ (Fig. 4F). The increases in gluconate in idn1Δ cells were largely dependent upon Gcd1 as lower levels of gluconate were detected in gcd1Δ idn1Δ cells compared with idn1Δ. Taken together these results are consistent with Gcd1 and Idn1 acting in the same pathway. They also suggest that most of the gluconate generated within cells is rapidly phosphorylated by Idn1. Why would the expression of gcd1 be increased in response to zinc limitation? As Gcd1 is a NADP+-dependent enzyme and the phosphorylation of gluconate by Idn1 requires 1 ATP, there is no obvious energetic advantage of using the gluconate shunt to generate 6-phosphogluconate instead of hexokinase and Glc-6-P dehydrogenase (Fig. 1). However, as these reactions could run in parallel, a potential explanation for the increased expression of gcd1 is that it allows increased carbon flux into the pentose phosphate pathway to increase NADPH regeneration and/or the levels of needed biosynthetic intermediates. In support of this hypothesis, there are precedents for the regulation of NADPH levels by zinc in other yeast (23.Wu C.Y. Roje S. Sandoval F.J. Bird A.J. Winge D.R. Eide D.J. Repression of sulfate assimilation is an adaptive response of yeast to the oxidative stress of zinc deficiency.J. Biol. Chem. 2009; 284: 27544-27556Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). loz1Δ cells also accumulate ergothioneine (Table 1), which is potentially generated from precursors supplied by the pentose phosphate pathway (24.Cheah I.K. Halliwell B. Ergothioneine: antioxidant potential, physiological function and role in disease.Biochim. Biophys. Acta. 2012; 1822: 784-793Crossref PubMed Scopus (306) Google Scholar). To test whether the enzymes of the gluconate shunt influenced total cellular NADPH regeneration, NADP+ and NADPH levels were measured in wild-type and gcd1Δ cells following growth to exponential phase in YES medium. No significant changes in the NADP+/NADPH ratio were observed in gcd1Δ compared with wild-type cells (supplemental Fig. S4). We also observed no obvious growth defect of gcd1Δ cells in zinc-limiting medium, zinc-replete medium, or in the presence of strong oxidants such as H2O2 (Fig. 5 and data not shown). As we were unable to find any phenotype that was a result of loss of gcd1, we next tested whether there was redundancy between the gluconate shunt and the first steps of the pentose phosphate pathway. As two genes (zwf1 and SPAC3C7.13c) encode Glc-6-P dehydrogenase in S. pombe, we initially performed RNA blot analysis to examine their expression in exponentially growing cells. We also examined the expression of gnd1, which encodes the pentose phosphate enzyme phosphogluconate dehydrogenase, as a control. The RNA blot analysis revealed that zwf1 and gnd1 mRNAs were abundantly expressed in wild-type and loz1Δ cells (Fig. 4C). In contrast, we were unable to detect SPAC3C7.13c using this method. Due to the low expression of SPAC3C7.13c, our further experiments focused on zwf1. Genome-wide deletion studies in S. pombe suggest that zwf1Δ cells are inviable (25.Kim D.U. Hayles J. Kim D. Wood V. Park H.O. Won M. Yoo H.S. D
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