Yeast PPA2 gene encodes a mitochondrial inorganic pyrophosphatase that is essential for mitochondrial function.

We have cloned a gene encoding a mitochondrial inorganic pyrophosphatase (PPase) in the yeast Saccharomyces cerevisiae by low stringency hybridization to PPA1, the yeast gene for cytoplasmic PPase. The new gene, PPA2, is located on chromosome 13 and encodes a protein whose sequence is 49% identical to the cytoplasmic enzyme. The protein differs from cytoplasmic PPase in that it has a leader sequence enriched in basic and hydroxylated residues, which is typically found in mitochondrial proteins. Yeast cells overproducing PPA2 had a 47-fold increase in mitochondrial PPase activity. This activity was further stimulated 3-fold by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which suggests that PPA2 is part of an energy-linked enzyme. Using gene disruptions, we found that PPA1 is required for cell growth. In contrast, cells disrupted for PPA2 are viable, but unable to grow on respiratory carbon sources. Fluorescence microscopy revealed that these cells have lost their mitochondrial DNA. We conclude that the mitochondrial PPase encoded by PPA2 is essential for mitochondrial function and maintenance of the mitochondrial genome.     

Characterization of a mitochondrial inorganic pyrophosphatase in Saccharomyces cerevisiae.

We have studied a mitochondrial inorganic pyrophosphatase (PPase) in the yeast Saccharomyces cerevisiae. The uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and the ionophores valinomycin and nigericin stimulate the PPase activity of repeatedly washed yeast mitochondria 2-3-fold. We have previously cloned a yeast gene, PPA2, encoding the catalytic subunit of a mitochondrial PPase. Uncouplers stimulate the PPase activity several-fold in mitochondria from both cells that overexpress PPA2 from a high copy number plasmid and cells with normal expression. These results indicate that the PPA2 polypeptide functions as an energy linked and membrane associated PPase. The stimulation of mitochondrial PPase activity by FCCP, but not by valinomycin and nigericin, was greatly enhanced by the presence of DTT. The antibiotics Dio-9, equisetin and the F0F1-ATPase inhibitor oligomycin also increase mitochondrial PPase activity several fold. This stimulation is much higher, whereas basal PPase activity is lower, in isotonic than in hypotonic solution, which indicates that intact membranes are a prerequisite for maximal effects.     

Global Genetic Determinants of Mitochondrial DNA Copy Number

Many human diseases including development of cancer is associated with depletion of mitochondrial DNA (mtDNA) content. These diseases are collectively described as mitochondrial DNA depletion syndrome (MDS). High similarity between yeast and human mitochondria allows genomic study of the budding yeast to be used to identify human disease genes. In this study, we systematically screened the pre-existing respiratory-deficient Saccharomyces cerevisiae yeast strains using fluorescent microscopy and identified 102 nuclear genes whose deletions result in a complete mtDNA loss, of which 52 are not reported previously. Strikingly, these genes mainly encode protein products involved in mitochondrial protein biosynthesis process (54.9%). The rest of these genes either encode protein products associated with nucleic acid metabolism (14.7%), oxidative phosphorylation (3.9%), or other protein products (13.7%) responsible for bud-site selection, mitochondrial intermembrane space protein import, assembly of cytochrome-c oxidase, vacuolar protein sorting, protein-nucleus import, calcium-mediated signaling, heme biosynthesis and iron homeostasis. Thirteen (12.7%) of the genes encode proteins of unknown function. We identified human orthologs of these genes, conducted the interaction between the gene products and linked them to human mitochondrial disorders and other pathologies. In addition, we screened for genes whose defects affect the nuclear genome integrity. Our data provide a systematic view of the nuclear genes involved in maintenance of mitochondrial DNA. Together, our studies i) provide a global view of the genes regulating mtDNA content; ii) provide compelling new evidence toward understanding novel mechanism involved in mitochondrial genome maintenance and iii) provide useful clues in understanding human diseases in which mitochondrial defect and in particular depletion of mitochondrial genome plays a critical role.     

Some features of methylpyrophosphate hydrolysis by yeast inorganic pyrophosphatase

The interaction of yeast inorganic pyrophosphatase with methylpyrophosphate was studied. In the presence of Mg2+ the rate of hydrolysis of the methylpyrophosphate-Zn2+ complex by the enzyme was shown to decrease. This was accompanied by competition of Zn2+ and Mg2+ for one site of Me2+ binding on the enzyme. The kinetics of combined hydrolysis of zinc methylpyrophosphate and zinc pyrophosphate were studied. It was found that both substrates are hydrolyzed at the same active site of the enzyme. Free methylpyrophosphate when bound to a specific phosphorylation site on the enzyme surface accelerated magnesium pyrophosphate hydrolysis. Some kinetic parameters of this hydrolysis were determined.     

Kinetics and thermodynamics of catalysis by the inorganic pyrophosphatase of Escherichia coli in both directions

Combined evidence obtained from the measurements of pyrophosphate hydrolysis and synthesis, oxygen exchange between phosphate and water, enzyme-bound pyrophosphate formation and Mg2+ binding enabled us to deduce the overall scheme of catalysis by Escherichia coli inorganic pyrophosphatase in the presence of Mg2+. We determined the equilibrium constants for Mg2+ binding to various enzyme species and forward and reverse rate constants for the four steps of the catalytic reaction, namely, binding/release of PPi, hydrolysis/synthesis of PPi and successive binding/release of two Pi molecules. Catalysis by the E. coli enzyme in both directions, in contrast to baker's yeast pyrophosphatase, occurs via a single pathway, which requires the binding of Mg2+ to the sites of four types. Three of them can be filled in the absence of the substrates, and the affinity of one of them to Mg2+ is increased by two orders of magnitude in the enzyme-substrate complexes. The distribution of 18O-labelled phosphate isotopomers during the exchange indicated that hydrolysis of pyrophosphate in the active site is appreciably reversible. The equilibrium constant for this process estimated from direct measurements is 5.0. The ratio of the maximal velocities of pyrophosphate hydrolysis and synthesis is 69. The rate of the synthesis is almost entirely determined by the rate of the release of pyrophosphate from the enzyme. In the hydrolytic reaction, enzyme-bound pyrophosphate hydrolysis and successive release of two phosphate molecules proceed with nearly equal rate constants.     

Spectral and kinetic studies of phosphate and magnesium ion binding to yeast inorganic pyrophosphatase

Inorganic pyrophosphatase must bind two phosphate molecules in order to catalyze pyrophosphate synthesis. In this report it is shown that Pi causes marked effect on the absorption spectrum of baker's yeast inorganic pyrophosphatase and this effect can be used to analyze Pi binding to this enzyme. A series of absorbance versus Pi concentration curves in the presence of 0.5-20 mM free Mg2+ were obtained at pH 7.2 and computer-fitted to 19 models. The dissociation constant of magnesium phosphate (8.5 +/- 0.4 mM) used in this analysis was measured with a Mg2+-sensitive electrode. The best model implies successive binding of two magnesium phosphate molecules or random-order binding of magnesium phosphate and free phosphate molecules. The first route predominates at physiological concentrations of Mg2+. The Pi-inhibition pattern of pyrophosphate hydrolysis confirmed that Pi adds to the active site and provided further evidence for the existence of an activating Pi-binding site. The possibility is raised that the pathways of pyrophosphate synthesis and hydrolysis by inorganic pyrophosphatase may differ in the sense that the binding of the fourth metal ion/subunit may facilitate the synthesis and inhibit the hydrolysis.     

Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean

Inorganic pyrophosphatase was purified from the vacuolar membrane of mung bean hypocotyl tissue by solubilization with lysophosphatidylcholine and QAE-Toyopearl chromatography. The molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 73,000 daltons. Among the amino-terminal first 30 amino acids are 25 nonpolar hydrophobic residues. For maximum activity, the purified pyrophosphatase required 1 mM Mg2+ and 50 mM K+. The enzyme reaction was stimulated by exogenous phospholipid in the presence of detergent. Excess pyrophosphate as well as excess magnesium inhibited the pyrophosphatase. The enzyme reaction was strongly inhibited by ATP, GTP, and CTP at 2 mM, and the inhibition was reversed by increasing the Mg2+ concentration. An antibody preparation raised in a rabbit against the purified enzyme inhibited both the reactions of pyrophosphate hydrolysis of the purified preparation and the pyrophosphate-dependent H+ translocation in the tonoplast vesicles. N,N'-Dicyclohexylcarbodiimide became bound to the purified pyrophosphatase and inhibited the reaction of pyrophosphate hydrolysis. It is concluded that the 73-kDa protein in vacuolar membrane functions as an H+-translocating inorganic pyrophosphatase.     

The evidence that the DNC (SLC25A19) is not the mitochondrial deoxyribonucleotide carrier

We review the evidence that the function of the SLC25A19 gene product, previously identified as the mitochondrial deoxyribonucleotide carrier (DNC), is actually the transport of thiamine pyrophosphate. This evidence comes from enzyme kinetics, homologous yeast protein alignments, gene knockout studies, and clinical samples from Amish Microcephaly patients. This diverse body of evidence consistently points to the conclusion that SLC25A19 is not the true mitochondrial DNC gene. The identification of the correct mitochondrial DNC is important for research on the genetic diseases of mitochondrial DNA maintenance and the toxicity experienced by many HIV patients undergoing antiretroviral therapy involving nucleoside analogs.     

Kinetic properties of mutant deoxyguanosine kinase in a case of reversible hepatic mtDNA depletion

DGUOK [dG (deoxyguanosine) kinase] is one of the two mitochondrial deoxynucleoside salvage pathway enzymes involved in precursor synthesis for mtDNA (mitochondrial DNA) replication. DGUOK is responsible for the initial rate-limiting phosphorylation of the purine deoxynucleosides, using a nucleoside triphosphate as phosphate donor. Mutations in the DGUOK gene are associated with the hepato-specific and hepatocerebral forms of MDS (mtDNA depletion syndrome). We identified two missense mutations (N46S and L266R) in the DGUOK gene of a previously reported child, now 10 years old, who presented with an unusual revertant phenotype of liver MDS. The kinetic properties of normal and mutant DGUOK were studied in mitochondrial preparations from cultured skin fibroblasts, using an optimized methodology. The N46S/L266R DGUOK showed 14 and 10% residual activity as compared with controls with dG and deoxyadenosine as phosphate acceptors respectively. Similar apparent negative co-operativity in the binding of the phosphate acceptors to the wild-type enzyme was found for the mutant. In contrast, abnormal bimodal kinetics were shown with ATP as the phosphate donor, suggesting an impairment of the ATP binding mode at the phosphate donor site. No kinetic behaviours were found for two other patients with splicing defects or premature stop codon. The present study represents the first characterization of the enzymatic kinetic properties of normal and mutant DGUOK in organello and our optimized protocol allowed us to demonstrate a residual activity in skin fibroblast mitochondria from a patient with a revertant phenotype of MDS. The residual DGUOK activity may play a crucial role in the phenotype reversal.     

A novel inorganic pyrophosphatase in Thermococcus onnurineus NA1

The TON_0002 gene, which is in close proximity to the DNA polymerase locus in Thermococcus onnurineus NA1, has been shown to encode an inorganic pyrophosphatase. Its genomic position and function suggest a role for pyrophosphate hydrolysis during DNA polymerization. This is the first report of an inorganic pyrophosphatase belonging to the haloacid dehalogenase superfamily, in which unique residues in motif I and II have been replaced with Trp and Gly, respectively. The optimum pyrophosphatase activity of the recombinant enzyme occurred at pH 6, and it displayed an absolute dependence on divalent metal ions, among which Ni²⁺ was the most efficient. The site-specific mutation of the Gly residue in motif II to Ala or Ser residue exhibited only a slight change in the enzymatic activity and the K _m value.     

Crystalline inorganic pyrophosphatase isolated from baker's yeast

Crystalline inorganic pyrophosphatase has been isolated from baker's yeast. The crystalline enzyme is a protein of the albumin type with an isoelectric point near pH 4.8. Its molecular weight is of the order of 100,000. It contains about 5 per cent tyrosine and 3.5 per cent tryptophane. It is most stable at pH 6.8. The new crystalline protein acts as a specific catalyst for the hydrolysis of inorganic pyrophosphate into orthophosphate ions. It does not catalyze the hydrolysis of the pyrophosphate radical of such organic esters as adenosine di- and triphosphate, or thiamine pyrophosphate. Crystalline pyrophosphatase requires the presence of Mg, Co, or Mn ions as activators. These ions are antagonized by calcium ions. Mg is also antagonized by Co or Mn ions. The rate of the enzymatic hydrolysis of inorganic pyrophosphate is proportional to the concentration of enzyme and is a function of pH, temperature, concentration of substrate, and concentration of activating ion. The approximate conditions for optimum rate are: 40 degrees C. and pH 7.0 at a concentration of 3 to 4 x 10(-3)M Na(4)P(2)O(7) and an equivalent concentration of magnesium salt. The enzymatic hydrolysis of Na(4)P(2)O(7) or K(4)P(2)O(7) proceeds to completion and is irreversible under the conditions at which hydrolysis is occurring. Details are given of the method of isolation of the crystalline enzyme.     

Inhibition of the orthophosphatase and pyrophosphatase activities of human alkaline-phosphatase preparations

1. Purified human liver and small-intestinal alkaline orthophosphatases release inorganic phosphate at appreciable rates from a variety of organic pyrophosphate substrates. 2. The pyrophosphatase action is inhibited by Mg²⁺ ions at concentrations that activate the hydrolysis of orthophosphate substrates by these enzymes. 3. The results of mixed-substrate experiments, denaturation studies with heat or urea and starch-gel electrophoresis suggest that both orthophosphatase and pyrophosphatase activities are, in each preparation, properties of a single enzyme. 4. Intestinal phosphatase shows greater pyrophosphatase activity relative to orthophosphatase than the liver enzyme.     

Organic pyrophosphates as substrates for human alkaline phosphatases

1. Purified human liver and small-intestinal alkaline orthophosphatases release inorganic phosphate at appreciable rates from a variety of organic pyrophosphate substrates. 2. The pyrophosphatase action is inhibited by Mg²⁺ ions at concentrations that activate the hydrolysis of orthophosphate substrates by these enzymes. 3. The results of mixed-substrate experiments, denaturation studies with heat or urea and starch-gel electrophoresis suggest that both orthophosphatase and pyrophosphatase activities are, in each preparation, properties of a single enzyme. 4. Intestinal phosphatase shows greater pyrophosphatase activity relative to orthophosphatase than the liver enzyme.     

Properties of mutated Rhodospirillum rubrum H+-pyrophosphatase expressed in Escherichia coli

The membrane-bound proton pumping inorganic pyrophosphate synthase/pyrophosphatase (H(+)-PPi synthase/H(+)-PPase) from the photosynthetic bacterium Rhodospirillum rubrum was functionally expressed in Escherichia coli C43(DE3) cells. Based on a new topology model of the enzyme, charged residues predicted to be located near or within the membrane were selected for site-directed mutagenesis. Several of these mutations resulted in an almost complete inactivation of the enzyme. Four mutated residues appear to show a selective impairment of proton translocation and are thus likely to be involved in coupling pyrophosphate hydrolysis with electrogenic proton pumping. Two of these mutations, R176K and E584D, caused increased tolerance to salt. In addition, the former mutation caused an increased K(m) of one order of magnitude for the hydrolysis reaction. These results and their possible implications for the enzyme function are discussed.     

pdf1, a palmitoyl protein thioesterase 1 Ortholog in Schizosaccharomyces pombe: a yeast model of infantile Batten disease

Infantile Batten disease is a severe neurodegenerative storage disorder caused by mutations in the human PPT1 (palmitoyl protein thioesterase 1) gene, which encodes a lysosomal hydrolase that removes fatty acids from lipid-modified proteins. PPT1 has orthologs in many species, including lower organisms and plants, but not in Saccharomyces cerevisiae. The fission yeast Schizosaccharomyces pombe contains a previously uncharacterized open reading frame (SPBC530.12c) that encodes the S. pombe Ppt1p ortholog fused in frame to a second enzyme that is highly similar to a previously cloned mouse dolichol pyrophosphatase (Dolpp1p). In the present study, we characterized this interesting gene (designated here as pdf1, for palmitoyl protein thioesterase-dolichol pyrophosphate phosphatase fusion 1) through deletion of the open reading frame and complementation by plasmids bearing mutations in various regions of the pdf1 sequence. Strains bearing a deletion of the entire pdf1 open reading frame are nonviable and are rescued by a pdf1 expression plasmid. Inactivating mutations in the Dolpp1p domain do not rescue the lethality, whereas mutations in the Ppt1p domain result in cells that are viable but abnormally sensitive to sodium orthovanadate and elevated extracellular pH. The latter phenotypes have been previously associated with class C and class D vacuolar protein sorting (vps) mutants and vacuolar membrane H(+)-ATPase (vma) mutants in S. cerevisiae. Importantly, the Ppt1p-deficient phenotype is complemented by the human PPT1 gene. These results indicate that the function of PPT1 has been widely conserved throughout evolution and that S. pombe may serve as a genetically tractable model for the study of human infantile Batten disease.     

Chloroplast inorganic pyrophosphatase

An alkaline, Mg²⁺-dependent inorganic pyrophosphatase has been isolated from previously isolated spinach chloroplast. The activity of the enzyme was increased 100-fold, with a 42% yield, upon purification from the total soluble chloroplast enzymes. The pH optimum for the enzyme shifts from 9.0 at 5 mM Mg²⁺ to 7.0 at 40 mM Mg²⁺. The substrate for the reaction appears to be magnesium pyrophosphate, and anionic pyrophosphate is an effective inhibitor. There seems to be also an activating effect of Mg²⁺ on the enzyme at pH 7. No other cation substitutes for Mg²⁺ in activating the hydrolysis of pyrophosphate. Among anions tested, only F⁻ caused severe inhibition. The enzyme is inactive towards fructose 1,6-diphosphate, thiamine pyrophosphate, ATP, and ADP. The possibility that this enzyme is subject to metabolic regulation is discussed in relation to an indicated role of pyrophosphate in the regulation of photosynthetic carbon reduction.     

MtDNA mutations are a common cause of severe disease phenotypes in children with Leigh syndrome

Leigh syndrome is a common clinical manifestation in children with mitochondrial disease and other types of inborn errors of metabolism. We characterised clinical symptoms, prognosis, respiratory chain function and performed extensive genetic analysis of 25 Swedish children suffering from Leigh syndrome with the aim to obtain insights into the molecular pathophysiology and to provide a rationale for genetic counselling. We reviewed the clinical history of all patients and used muscle biopsies in order to perform molecular, biochemical and genetic investigations, including sequencing the entire mitochondrial DNA (mtDNA), the mitochondrial DNA polymerase (POLGA) gene and the surfeit locus protein 1 (SURF1) gene. Respiratory chain enzyme activity measurements identified five patients with isolated complex I deficiency and five with combined enzyme deficiencies. No patient presented with isolated complex IV deficiency. Seven patients had a decreased ATP production rate. Extensive sequence analysis identified eight patients with pathogenic mtDNA mutations and one patient with mutations in POLGA. Mutations of mtDNA are a common cause of LS and mtDNA analysis should always be included in the diagnosis of LS patients, whereas SURF1 mutations are not a common cause of LS in Sweden. Unexpectedly, age of onset, clinical symptoms and prognosis did not reveal any clear differences in LS patients with mtDNA or nuclear DNA mutations.     

Identification of cytosolic Mg2+-dependent soluble inorganic pyrophosphatases in potato and phylogenetic analysis

Using polyclonal antibodies raised against a previously cloned potato Mg2+-dependent soluble inorganic pyrophosphatase (ppa1 gene) [8], a second gene, called ppa2, could be isolated. A single locus homologous to ppa2 was mapped on potato chromosomes, unlinked to the two loci identified for ppa1. From a phylogenetic and structural point of view, the PPA1 and PPA2 polypeptides are more closely related to prokaryotic than to eukaryotic Mg2+-dependent soluble inorganic pyrophosphatases (soluble PPases). Subcellular localization by immunogold electron microscopy, using sections from leaf parenchyma cells, showed that PPA1 and PPA2 are localized to the cytosol. Based on these observations, the likely phylogenetic origin and the physiological significance of the cytosolic soluble pyrophosphatases are discussed.     

The influence of metal ions on the orthophosphatase and inorganic pyrophosphatase activities of human alkaline phosphatase

1. The differential effects of adding Zn(2+) and Mg(2+) on the orthophosphatase and inorganic pyrophosphatase activities of human intestinal alkaline phosphatase were studied. 2. In the presence of excess of Zn(2+), inorganic pyrophosphatase activity is inhibited. At higher concentrations of pyrophosphate, hydrolysis of this substrate takes place, but is inhibited competitively by the Zn(2+)-pyrophosphate complex. This complex also acts as a competitive inhibitor of orthophosphate hydrolysis. 3. Excess of Mg(2+) also inhibits pyrophosphatase action by removal of substrate; at low concentrations, this ion activates pyrophosphatase, as is the case with orthophosphatase. 4. It is concluded that, when interactions between metal ions and pyrophosphate are taken into account, the effects of these ions are consistent with the view that alkaline phosphatases possess both orthophosphatase and inorganic pyrophosphatase activities.