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Aurelio Serrano - One of the best experts on this subject based on the ideXlab platform.
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the h translocating inorganic pyrophosphatase from arabidopsis thaliana is more sensitive to sodium than its na translocating counterpart from methanosarcina mazei
Frontiers in Plant Science, 2020Co-Authors: Jose R Perezcastineira, Aurelio SerranoAbstract:Overexpression of membrane-bound K+-dependent H+-translocating inorganic Pyrophosphatases (H+-PPases) from higher plants has been widely used to alleviate the sensitivity toward NaCl in these organisms, a strategy that had been previously tested in Saccharomyces cerevisiae. On the other hand, H+-PPases have been reported to functionally complement the yeast cytosolic soluble pyrophosphatase (IPP1). Here, the efficiency of the K+-dependent Na+-PPase from the archaeon Methanosarcina mazei (MVP) to functionally complement IPP1 has been compared to that of its H+-pumping counterpart from Arabidopsis thaliana (AVP1). Both membrane-bound integral PPases (mPPases) supported yeast growth equally well under normal conditions, however, cells expressing MVP grew significantly better than those expressing AVP1 under salt stress. The subcellular distribution of the heterologously-expressed mPPases was crucial in order to observe the phenotypes associated with the complementation. In vitro studies showed that the PPase activity of MVP was less sensitive to Na+ than that of AVP1. Consistently, when yeast cells expressing MVP were grown in the presence of NaCl only a marginal increase in their internal PPi levels was observed with respect to control cells. By contrast, yeast cells that expressed AVP1 had significantly higher levels of this metabolite under the same conditions. The H+-pumping activity of AVP1 was also markedly inhibited by Na+. Our results suggest that mPPases primarily act by hydrolysing the PPi generated in the cytosol when expressed in yeast, and that AVP1 is more susceptible to Na+ inhibition than MVP both in vivo and in vitro. Based on this experimental evidence, we propose Na+-PPases as biotechnological tools to generate salt-tolerant plants.
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inorganic pyrophosphatase defects lead to cell cycle arrest and autophagic cell death through nad depletion in fermenting yeast
Journal of Biological Chemistry, 2013Co-Authors: Gloria Serranobueno, Agustin Hernandez, Guillermo Lopezlluch, Jose R Perezcastineira, Placido Navas, Aurelio SerranoAbstract:Abstract Inorganic Pyrophosphatases are required for the anabolism to take place in all living organisms. Defects in genes encoding these hydrolytic enzymes are considered inviable, although its exact nature has not been studied at cellular and molecular physiology levels. Using a conditional mutant in IPP1, the Saccharomyces cerevisiae gene encoding the cytosolic soluble pyrophosphatase, we show that respiring cells arrest in S-phase upon Ipp1p deficiency but they remain viable and resume growth if accumulated pyrophosphate is removed. However, fermenting cells arrest in G1/G0 phase and suffer massive vacuolisation and eventual cell death by autophagy. Impaired NAD+ metabolism is a major determinant of cell death in this scenario since demise can be avoided under conditions favouring accumulation of the oxidised pyridine coenzyme. These results posit that the mechanisms related to excess pyrophosphate toxicity in eukaryotes are dependent on the cell's energy metabolism.
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inorganic pyrophosphatase defects lead to cell cycle arrest and autophagic cell death through nad depletion in fermenting yeast
Journal of Biological Chemistry, 2013Co-Authors: Gloria Serranobueno, Agustin Hernandez, Guillermo Lopezlluch, Jose R Perezcastineira, Placido Navas, Aurelio SerranoAbstract:Inorganic Pyrophosphatases are required for anabolism to take place in all living organisms. Defects in genes encoding these hydrolytic enzymes are considered inviable, although their exact nature has not been studied at the cellular and molecular physiology levels. Using a conditional mutant in IPP1, the Saccharomyces cerevisiae gene encoding the cytosolic soluble pyrophosphatase, we show that respiring cells arrest in S phase upon Ipp1p deficiency, but they remain viable and resume growth if accumulated pyrophosphate is removed. However, fermenting cells arrest in G1/G0 phase and suffer massive vacuolization and eventual cell death by autophagy. Impaired NAD+ metabolism is a major determinant of cell death in this scenario because demise can be avoided under conditions favoring accumulation of the oxidized pyridine coenzyme. These results posit that the mechanisms related to excess pyrophosphate toxicity in eukaryotes are dependent on the energy metabolism of the cell.
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functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant h translocating Pyrophosphatases
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Jose R Perezcastineira, Rosa L Lopezmarques, Jose M Villalba, Manuel Losada, Aurelio SerranoAbstract:Abstract Two types of proteins that hydrolyze inorganic pyrophosphate (PPi), very different in both amino acid sequence and structure, have been characterized to date: soluble and membrane-bound proton-pumping Pyrophosphatases (sPPases and H+-PPases, respectively). sPPases are ubiquitous proteins that hydrolyze PPi releasing heat, whereas H+-PPases, so far unidentified in animal and fungal cells, couple the energy of PPi hydrolysis to proton movement across biological membranes. The budding yeast Saccharomyces cerevisiae has two sPPases that are located in the cytosol and in the mitochondria. Previous attempts to knock out the gene coding for a cytosolic sPPase (IPP1) have been unsuccessful, thus suggesting that this protein is essential for growth. Here, we describe the generation of a conditional S. cerevisiae mutant (named YPC-1) whose functional IPP1 gene is under the control of a galactose-dependent promoter. Thus, YPC-1 cells become growth arrested in glucose but they regain the ability to grow on this carbon source when transformed with autonomous plasmids bearing diverse foreign H+-PPase genes under the control of a yeast constitutive promoter. The heterologously expressed H+-PPases are distributed among different yeast membranes, including the plasma membrane, functional complementation by these integral membrane proteins being consistently sensitive to external pH. These results demonstrate that hydrolysis of cytosolic PPi is essential for yeast growth and that this function is not substantially affected by the intrinsic characteristics of the PPase protein that accomplishes it. Moreover, this is, to our knowledge, the first direct evidence that H+-PPases can mediate net hydrolysis of PPi in vivo. YPC-1 mutant strain constitutes a convenient expression system to perform studies aimed at the elucidation of the structure–function relationships of this type of proton pumps.
Jose R Perezcastineira - One of the best experts on this subject based on the ideXlab platform.
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the h translocating inorganic pyrophosphatase from arabidopsis thaliana is more sensitive to sodium than its na translocating counterpart from methanosarcina mazei
Frontiers in Plant Science, 2020Co-Authors: Jose R Perezcastineira, Aurelio SerranoAbstract:Overexpression of membrane-bound K+-dependent H+-translocating inorganic Pyrophosphatases (H+-PPases) from higher plants has been widely used to alleviate the sensitivity toward NaCl in these organisms, a strategy that had been previously tested in Saccharomyces cerevisiae. On the other hand, H+-PPases have been reported to functionally complement the yeast cytosolic soluble pyrophosphatase (IPP1). Here, the efficiency of the K+-dependent Na+-PPase from the archaeon Methanosarcina mazei (MVP) to functionally complement IPP1 has been compared to that of its H+-pumping counterpart from Arabidopsis thaliana (AVP1). Both membrane-bound integral PPases (mPPases) supported yeast growth equally well under normal conditions, however, cells expressing MVP grew significantly better than those expressing AVP1 under salt stress. The subcellular distribution of the heterologously-expressed mPPases was crucial in order to observe the phenotypes associated with the complementation. In vitro studies showed that the PPase activity of MVP was less sensitive to Na+ than that of AVP1. Consistently, when yeast cells expressing MVP were grown in the presence of NaCl only a marginal increase in their internal PPi levels was observed with respect to control cells. By contrast, yeast cells that expressed AVP1 had significantly higher levels of this metabolite under the same conditions. The H+-pumping activity of AVP1 was also markedly inhibited by Na+. Our results suggest that mPPases primarily act by hydrolysing the PPi generated in the cytosol when expressed in yeast, and that AVP1 is more susceptible to Na+ inhibition than MVP both in vivo and in vitro. Based on this experimental evidence, we propose Na+-PPases as biotechnological tools to generate salt-tolerant plants.
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inorganic pyrophosphatase defects lead to cell cycle arrest and autophagic cell death through nad depletion in fermenting yeast
Journal of Biological Chemistry, 2013Co-Authors: Gloria Serranobueno, Agustin Hernandez, Guillermo Lopezlluch, Jose R Perezcastineira, Placido Navas, Aurelio SerranoAbstract:Abstract Inorganic Pyrophosphatases are required for the anabolism to take place in all living organisms. Defects in genes encoding these hydrolytic enzymes are considered inviable, although its exact nature has not been studied at cellular and molecular physiology levels. Using a conditional mutant in IPP1, the Saccharomyces cerevisiae gene encoding the cytosolic soluble pyrophosphatase, we show that respiring cells arrest in S-phase upon Ipp1p deficiency but they remain viable and resume growth if accumulated pyrophosphate is removed. However, fermenting cells arrest in G1/G0 phase and suffer massive vacuolisation and eventual cell death by autophagy. Impaired NAD+ metabolism is a major determinant of cell death in this scenario since demise can be avoided under conditions favouring accumulation of the oxidised pyridine coenzyme. These results posit that the mechanisms related to excess pyrophosphate toxicity in eukaryotes are dependent on the cell's energy metabolism.
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inorganic pyrophosphatase defects lead to cell cycle arrest and autophagic cell death through nad depletion in fermenting yeast
Journal of Biological Chemistry, 2013Co-Authors: Gloria Serranobueno, Agustin Hernandez, Guillermo Lopezlluch, Jose R Perezcastineira, Placido Navas, Aurelio SerranoAbstract:Inorganic Pyrophosphatases are required for anabolism to take place in all living organisms. Defects in genes encoding these hydrolytic enzymes are considered inviable, although their exact nature has not been studied at the cellular and molecular physiology levels. Using a conditional mutant in IPP1, the Saccharomyces cerevisiae gene encoding the cytosolic soluble pyrophosphatase, we show that respiring cells arrest in S phase upon Ipp1p deficiency, but they remain viable and resume growth if accumulated pyrophosphate is removed. However, fermenting cells arrest in G1/G0 phase and suffer massive vacuolization and eventual cell death by autophagy. Impaired NAD+ metabolism is a major determinant of cell death in this scenario because demise can be avoided under conditions favoring accumulation of the oxidized pyridine coenzyme. These results posit that the mechanisms related to excess pyrophosphate toxicity in eukaryotes are dependent on the energy metabolism of the cell.
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functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant h translocating Pyrophosphatases
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Jose R Perezcastineira, Rosa L Lopezmarques, Jose M Villalba, Manuel Losada, Aurelio SerranoAbstract:Abstract Two types of proteins that hydrolyze inorganic pyrophosphate (PPi), very different in both amino acid sequence and structure, have been characterized to date: soluble and membrane-bound proton-pumping Pyrophosphatases (sPPases and H+-PPases, respectively). sPPases are ubiquitous proteins that hydrolyze PPi releasing heat, whereas H+-PPases, so far unidentified in animal and fungal cells, couple the energy of PPi hydrolysis to proton movement across biological membranes. The budding yeast Saccharomyces cerevisiae has two sPPases that are located in the cytosol and in the mitochondria. Previous attempts to knock out the gene coding for a cytosolic sPPase (IPP1) have been unsuccessful, thus suggesting that this protein is essential for growth. Here, we describe the generation of a conditional S. cerevisiae mutant (named YPC-1) whose functional IPP1 gene is under the control of a galactose-dependent promoter. Thus, YPC-1 cells become growth arrested in glucose but they regain the ability to grow on this carbon source when transformed with autonomous plasmids bearing diverse foreign H+-PPase genes under the control of a yeast constitutive promoter. The heterologously expressed H+-PPases are distributed among different yeast membranes, including the plasma membrane, functional complementation by these integral membrane proteins being consistently sensitive to external pH. These results demonstrate that hydrolysis of cytosolic PPi is essential for yeast growth and that this function is not substantially affected by the intrinsic characteristics of the PPase protein that accomplishes it. Moreover, this is, to our knowledge, the first direct evidence that H+-PPases can mediate net hydrolysis of PPi in vivo. YPC-1 mutant strain constitutes a convenient expression system to perform studies aimed at the elucidation of the structure–function relationships of this type of proton pumps.
Jose Luis Millan - One of the best experts on this subject based on the ideXlab platform.
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novel inhibitors of alkaline phosphatase suppress vascular smooth muscle cell calcification
Journal of Bone and Mineral Research, 2007Co-Authors: Sonoko Narisawa, Dympna Harmey, Manisha C Yadav, Charles W Oneill, Marc Hoylaerts, Jose Luis MillanAbstract:UNLABELLED: We report three novel inhibitors of the physiological pyrophosphatase activity of alkaline phosphatase and show that these compounds are capable of reducing calcification in two models of vascular calcification (i.e., they suppress in vitro calcification by cultured Enpp1(-/-) VSMCs and they inhibit the increased pyrophosphatase activity in a rat aortic model). INTRODUCTION: Genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) leads to accumulation of the calcification inhibitor inorganic pyrophosphate (PP(i)). TNALP deficiency ameliorates the hypermineralization phenotype in Enpp1(-/-) and ank/ank mice, two models of osteoarthritis and soft tissue calcification. We surmised that the pharmacological inhibition of TNALP pyrophosphatase activity could be used to prevent/suppress vascular calcification. MATERIALS AND METHODS: Comprehensive chemical libraries were screened to identify novel drug-like compounds that could inhibit TNALP pyrophosphatase function at physiological pH. We used these novel compounds to block calcification by cultured vascular smooth muscle cells (VSMCs) and to inhibit the upregulated pyrophosphatase activity in a rat aortic calcification model. RESULTS: Using VSMC cultures, we determined that Enpp1(-/-) and ank/ank VSMCs express higher TNALP levels and enhanced in vitro calcification compared with wildtype cells. By high-throughput screening, three novel compounds, 5,361,418, 5,923,412, and 5,804,079, were identified that inhibit TNALP pyrophosphatase function through an uncompetitive mechanism, with high affinity and specificity when measured at both pH 9.8 and 7.5. These compounds were shown to reduce the calcification by Enpp1(-/-) VSMCs. Furthermore, using an ex vivo rat whole aorta PP(i) hydrolysis assay, we showed that pyrophosphatase activity was inhibited by all three lead compounds, with compound 5,804,079 being the most potent at pH 7.5. CONCLUSIONS: We conclude that TNALP is a druggable target for the treatment and/or prevention of ectopic calcification. The lead compounds identified in this study will serve as scaffolds for medicinal chemistry efforts to develop drugs for the treatment of soft tissue calcification.
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novel inhibitors of alkaline phosphatase suppress vascular smooth muscle cell calcification
Journal of Bone and Mineral Research, 2007Co-Authors: Sonoko Narisawa, Dympna Harmey, Manisha C Yadav, Charles W Oneill, Marc Hoylaerts, Jose Luis MillanAbstract:We report three novel inhibitors of the physiological pyrophosphatase activity of alkaline phosphatase and show that these compounds are capable of reducing calcification in two models of vascular calcification (i.e., they suppress in vitro calcification by cultured Enpp1−/− VSMCs and they inhibit the increased pyrophosphatase activity in a rat aortic model). Introduction: Genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) leads to accumulation of the calcification inhibitor inorganic pyrophosphate (PPi). TNALP deficiency ameliorates the hypermineralization phenotype in Enpp1−/− and ank/ank mice, two models of osteoarthritis and soft tissue calcification. We surmised that the pharmacological inhibition of TNALP pyrophosphatase activity could be used to prevent/suppress vascular calcification. Materials and Methods: Comprehensive chemical libraries were screened to identify novel drug-like compounds that could inhibit TNALP pyrophosphatase function at physiological pH. We used these novel compounds to block calcification by cultured vascular smooth muscle cells (VSMCs) and to inhibit the upregulated pyrophosphatase activity in a rat aortic calcification model. Results: Using VSMC cultures, we determined that Enpp1−/− and ank/ank VSMCs express higher TNALP levels and enhanced in vitro calcification compared with wildtype cells. By high-throughput screening, three novel compounds, 5361418, 5923412, and 5804079, were identified that inhibit TNALP pyrophosphatase function through an uncompetitive mechanism, with high affinity and specificity when measured at both pH 9.8 and 7.5. These compounds were shown to reduce the calcification by Enpp1−/− VSMCs. Furthermore, using an ex vivo rat whole aorta PPi hydrolysis assay, we showed that pyrophosphatase activity was inhibited by all three lead compounds, with compound 5804079 being the most potent at pH 7.5. Conclusions: We conclude that TNALP is a druggable target for the treatment and/or prevention of ectopic calcification. The lead compounds identified in this study will serve as scaffolds for medicinal chemistry efforts to develop drugs for the treatment of soft tissue calcification.
Adrian Goldman - One of the best experts on this subject based on the ideXlab platform.
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sulfolobus acidocaldarius inorganic pyrophosphatase structure thermostability and effect of metal ion in an archael pyrophosphatase
Protein Science, 1999Co-Authors: Velimatti Leppanen, Adrian Goldman, Reijo Lahti, Gunter Schafer, Heli Nummelin, Thomas HansenAbstract:The first crystal structure of an inorganic pyrophosphatase (S-PPase) from an archaebacterium, the thermophile Sulfolobus acidocaldarius, has been solved by molecular replacement and refined to an R-factor of 19.7% at 2.7 A. S-PPase is a D3 homohexameric protein with one Mg2+ per active site in a position similar to, but not identical with, the first activating metal in mesophilic Pyrophosphatases (PPase). In mesophilic PPases, Asp65, Asp70, and Asp102 coordinate the Mg2+, while only Asp65 and Asp102 do in S-PPase, and the Mg2+ moves by 0.7 A. S-PPase may therefore be deactivated at low temperature by mispositioning a key metal ion. The monomer S-PPase structure is very similar to that of Thermus thermophilus (T-PPase) and Escherichia coli (E-PPase), root-mean-square deviations around 1 A/Calpha. But the hexamer structures of S- and T-PPase are more tightly packed and more similar to each other than they are to that of E-PPase, as shown by the increase in surface area buried upon oligomerization. In T-PPase, Arg116 creates an interlocking ionic network to both twofold and threefold related monomers; S-PPase has hydrophilic interactions to threefold related monomers absent in both E- and T-PPase. In addition, the thermostable PPases have about 7% more hydrogen bonds per monomer than E-PPase, and, especially in S-PPase, additional ionic interactions anchor the C-terminus to the rest of the protein. Thermostability in PPases is thus due to subtle improvements in both monomer and oligomer interactions.
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A site-directed mutagenesis study of Saccharomyces cerevisiae pyrophosphatase. Functional conservation of the active site of soluble inorganic Pyrophosphatases.
European journal of biochemistry / FEBS, 1996Co-Authors: Pirkko Heikinheimo, Pekka Pohjanjoki, Mikko Tasanen, Atte Helminen, Barry S Cooperman, Adrian GoldmanAbstract:We report the expression and initial characterization of 19 active-site variants of Saccharomyces cerevisiae inorganic pyrophosphatase (PPase), including measurements of thermostability, oligomeric structure and specific activity at pH 7.2. 13 of the 19 conservative substitutions resulted in at least a fivefold decrease in activity, indicating that these residues are important for yeast PPase catalysis. The E58D, D117E, D120E and D152E variants had no activity under the conditions tested, suggesting that Glu58, Asp117, Asp120 and Asp152 may have crucial roles in catalysis. The effects of the mutations on catalytic activity were very similar to those observed with the corresponding variants of Escherichia coli PPase, proving conclusively that the active site and mechanism of soluble PPases are conserved. The D71E variant was more thermostable and the K56R, R78K, D115E and K154R variants were more thermolabile than the wild-type enzyme, whereas subunit:subunit interactions were somewhat weakened by the K56R, R78K, Y89F and K154R substitutions. These results suggest that Lys56, Asp71, Arg78, Tyr89, Asp115 and Lys154 are structurally important for yeast PPase.
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The structure of E.coli soluble inorganic pyrophosphatase at 2.7 A resolution.
Protein engineering, 1994Co-Authors: Jussi Kankare, Barry S Cooperman, Genevieve S. Neal, Tiina A. Salminen, Tuomo Glumoff, Adrian GoldmanAbstract:The structure of E.coli soluble inorganic pyrophosphatase has been refined at 2.7 A resolution to an R-factor of 20.9%. The overall fold of the molecule is essentially the same as yeast pyrophosphatase, except that yeast pyrophosphatase is longer at both the N- and C-termini. Escherichia coli pyrophosphatase is a mixed alpha + beta protein with a complicated topology. The active site cavity, which is also very similar to the yeast enzyme, is formed by seven beta-strands and an alpha-helix and has a rather asymmetric distribution of charged residues. Our structure-based alignment extends and improves upon earlier sequence alignment studies; it shows that probably no more than 14, not 15-17 charged and polar residues are part of the conserved enzyme mechanism of Pyrophosphatases. Six of these conserved residues, at the bottom of the active site cavity, form a tight group centred on Asp70 and probably bind the two essential Mg2+ ions. The others, more spreadout and more positively charged, presumably bind substrate. Escherichia coli pyrophosphatase has an extra aspartate residue in the active site cavity, which may explain why the two enzymes bind divalent cation differently. Based on the structure, we have identified a sequence motif that seems to occur only in soluble inorganic Pyrophosphatases.
Agustin Hernandez - One of the best experts on this subject based on the ideXlab platform.
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inorganic pyrophosphatase defects lead to cell cycle arrest and autophagic cell death through nad depletion in fermenting yeast
Journal of Biological Chemistry, 2013Co-Authors: Gloria Serranobueno, Agustin Hernandez, Guillermo Lopezlluch, Jose R Perezcastineira, Placido Navas, Aurelio SerranoAbstract:Abstract Inorganic Pyrophosphatases are required for the anabolism to take place in all living organisms. Defects in genes encoding these hydrolytic enzymes are considered inviable, although its exact nature has not been studied at cellular and molecular physiology levels. Using a conditional mutant in IPP1, the Saccharomyces cerevisiae gene encoding the cytosolic soluble pyrophosphatase, we show that respiring cells arrest in S-phase upon Ipp1p deficiency but they remain viable and resume growth if accumulated pyrophosphate is removed. However, fermenting cells arrest in G1/G0 phase and suffer massive vacuolisation and eventual cell death by autophagy. Impaired NAD+ metabolism is a major determinant of cell death in this scenario since demise can be avoided under conditions favouring accumulation of the oxidised pyridine coenzyme. These results posit that the mechanisms related to excess pyrophosphate toxicity in eukaryotes are dependent on the cell's energy metabolism.
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inorganic pyrophosphatase defects lead to cell cycle arrest and autophagic cell death through nad depletion in fermenting yeast
Journal of Biological Chemistry, 2013Co-Authors: Gloria Serranobueno, Agustin Hernandez, Guillermo Lopezlluch, Jose R Perezcastineira, Placido Navas, Aurelio SerranoAbstract:Inorganic Pyrophosphatases are required for anabolism to take place in all living organisms. Defects in genes encoding these hydrolytic enzymes are considered inviable, although their exact nature has not been studied at the cellular and molecular physiology levels. Using a conditional mutant in IPP1, the Saccharomyces cerevisiae gene encoding the cytosolic soluble pyrophosphatase, we show that respiring cells arrest in S phase upon Ipp1p deficiency, but they remain viable and resume growth if accumulated pyrophosphate is removed. However, fermenting cells arrest in G1/G0 phase and suffer massive vacuolization and eventual cell death by autophagy. Impaired NAD+ metabolism is a major determinant of cell death in this scenario because demise can be avoided under conditions favoring accumulation of the oxidized pyridine coenzyme. These results posit that the mechanisms related to excess pyrophosphate toxicity in eukaryotes are dependent on the energy metabolism of the cell.
