Kinetic properties and metabolic contributions of yeast mitochondrial and cytosolic NADP+-specific isocitrate dehydrogenases

To compare kinetic properties of homologous isozymes of NADP+-specific isocitrate dehydrogenase, histidine-tagged forms of yeast mitochondrial (IDP1) and cytosolic (IDP2) enzymes were expressed and purified. The isozymes were found to share similar apparent affinities for cofactors. However, with respect to isocitrate, IDP1 had an apparent Km value approximately 7-fold lower than that of IDP2, whereas, with respect to alpha-ketoglutarate, IDP2 had an apparent Km value approximately 10-fold lower than that of IDP1. Similar Km values for substrates and cofactors in decarboxylation and carboxylation reactions were obtained for IDP2, suggesting a capacity for bidirectional catalysis in vivo. Concentrations of isocitrate and alpha-ketoglutarate measured in extracts from the parental strain were found to be similar with growth on different carbon sources. For mutant strains lacking IDP1, IDP2, and/or the mitochondrial NAD+-specific isocitrate dehydrogenase (IDH), metabolite measurements indicated that major cellular flux is through the IDH-catalyzed reaction in glucose-grown cells and through the IDP2-catalyzed reaction in cells grown with a nonfermentable carbon source (glycerol and lactate). A substantial cellular pool of alpha-ketoglutarate is attributed to IDH function during glucose growth, and to both IDP1 and IDH function during growth on glycerol/lactate. Complementation experiments using a strain lacking IDH demonstrated that overexpression of IDP1 partially compensated for the glutamate auxotrophy associated with loss of IDH. Collectively, these results suggest an ancillary role for IDP1 in cellular glutamate synthesis and a role for IDP2 in equilibrating and maintaining cellular levels of isocitrate and alpha-ketoglutarate.     

Dual compartmental localization and function of mammalian NADP+-specific isocitrate dehydrogenase in yeast

Isozymes of NADP⁺-specific isocitrate dehydrogenase (IDP) provide NADPH in cytosolic, mitochondrial, and peroxisomal compartments of eukaryotic cells. Analyses of purified IDP isozymes from yeast and from mouse suggest a general correspondence of pH optima for catalysis and pI values with pH values reported for resident cellular compartments. However, mouse IDP2, which partitions between cytosolic and peroxisomal compartments in mammalian cells, exhibits a broad pH optimum and an intermediate pI value. Mouse IDP2 was found to similarly colocalize in both cellular compartments when expressed in yeast at levels equivalent to those of endogenous yeast isozymes. The mouse enzyme can compensate for loss of yeast cytosolic IDP2 and of peroxisomal IDP3. Removal of the peroxisomal targeting signal of the mouse enzyme precludes both localization in peroxisomes and compensation for loss of yeast IDP3.     

Peroxisomal localization and function of NADP-specific isocitrate dehydrogenases in yeast

Yeast peroxisomal NADP⁺-specific isocitrate dehydrogenase (IDP3) contains a canonical type I peroxisomal targeting sequence (a carboxyl-terminal Cys-Lys-Leu tripeptide), and provides the NADPH required for β-oxidation of some fatty acids in that organelle. Cytosolic yeast IDP2 carrying a PTS1 (IDP2^+CKL) was only partially localized to peroxisomes, and the enzyme was able to function in lieu of either peroxisomal IDP3 or cytosolic IDP2. The analogous isocitrate dehydrogenase enzyme (IDPA) from Aspergillus nidulans, irrespective of the presence or absence of a putative PTS1, was found to exhibit patterns of dual compartmental distribution and of dual function in yeast similar to those observed for IDP2^+CKL. To test a potential cellular limit on peroxisomal levels, authentic yeast IDP3, which is normally strictly peroxisomal, was over-expressed. This also resulted in dual distribution and function of the enzyme in both the cytosol and in peroxisomes, supporting the possibility of a restriction on organellar amounts of IDP.     

Isolation, nucleotide sequence, and disruption of the Saccharomyces cerevisiae gene encoding mitochondrial NADP(H)-specific isocitrate dehydrogenase

Mitochondrial NADP(H)-specific isocitrate dehydrogenase (IDP1) was purified from yeast cells grown with acetate as a carbon source. IDP1 was shown to be a dimer with a subunit molecular weight of approximately 45,000. Immunochemical levels of IDP1 were found to vary in inverse proportion with those of mitochondrial NAD(H)-specific isocitrate dehydrogenase in cells grown with glucose or with acetate as a carbon source. A 20-residue amino-terminal sequence was obtained for IDP1, and degenerate oligonucleotides were used to synthesize a 50-base pair polymerase chain reaction product corresponding to the coding region for a portion of the amino terminus. The 50-base pair DNA fragment was used as a hybridization probe to identify plasmids containing the IDP1 gene in a yeast genomic DNA library. The complete nucleotide sequence of the IDP1 coding region was determined and translated into a 412-residue amino acid sequence for the mature protein which is preceded by a putative 16-residue mitochondrial targeting presequence. A haploid yeast strain containing a chromosomal disruption of the IDP1 locus was constructed and found to be capable of growth with glucose but not with other carbon sources, suggesting that IDP1 provides a critical function and may be the primary source of NADPH in yeast mitochondria.     

A multilocus system for studying tissue and subcellular specialization. The pH and temperature dependence of the two major NADP-dependent isocitrate dehydrogenase isozymes of the fish Fundulus heteroclitus

In the teleost fish Fundulus heteroclitus, there are three NADP-dependent isocitrate dehydrogenase isozymes. IDH-B2 is the only cytoplasmic isozyme, and IDH-C2 dominates the mitochondria of all tissues other than liver, where IDH-A2 is expressed. Since fish are ectotherms, their intracellular temperature and pH change directly with environmental temperature. In order to evaluate the influence of these environmental parameters on a model fish NADP-isocitrate dehydrogenase system, the major cytoplasmic (IDH-B2) and mitochondrial (IDH-C2) isozymes were kinetically evaluated as a function of pH and temperature. Whereas Vfmax and KmISOCm (where ISOC is isocitrate) were pH-independent, the Km for NADP was pH-dependent for both isozymes. The cytoplasmic isozyme (IDH-B2) had smaller KmNADP values between pH 7.0 and pH 8.0 than the mitochondrial form (IDH-C2). Vfmax and Km for substrate and coenzyme were temperature-dependent. Energy of activation for IDH-B2 and IDH-C2 was 10.6 and 12.8 kcal/mol, respectively. Both proteins had delta G not equal to values of about 15.8 kcal/mol, with significantly different distributions between delta H not equal to and delta S not equal to. The cytoplasmic isozyme (IDH-B2) appears to have a greater rate of catalysis than the mitochondrial enzyme (IDH-C2) at temperatures less than 30 degrees C. Moreover, the IDH-B2 isozyme had lower KmNADP values than the IDH-C2 isozyme at all temperatures, whereas the KmISOC values for the two isozymes were indistinguishable. Our data suggest that the two major NADP-dependent isocitrate dehydrogenase isozymes have unique physiological and metabolic functions that are adapted to the tissues and cellular compartments in which they are expressed.     

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of mitochondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the 'mitochondrial' isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The 'cytosolic' isozyme accounted for only 1-2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.     

Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.     

The soluble citric acid cycle enzymes ofDrosophila melanogaster. II. Tissue and intracellular distribution of aconitase and NADP-dependent isocitrate dehydrogenase

Aconitase (aconitate hydratase) (AH) and NADP-dependent isocitrate dehydrogenase (IDH-NADP) are found in every larval and adultDrosophila tissue. Their specific activities as well as the ratios of their absolute activities differ significantly from tissue to tissue. There are tissue-specific differences in the pattern of IDH-NADP isozymes in adults and in larvae. No clear-cut tissuespecific patterns exist for AH isozymes. Most of the activity of both enzymes is found in the supernatant fraction of whole fly homogenates. Only 35% of the AH activity and 16% of the IDH-NADP activity are associated with mitochondria. The patterns of supernatant and mitochondrial IDH-NADP isozymes are the same. On the other hand, the supernatant possesses AH isozymes not found in the mitochondria.     

A multilocus system for studying tissue and subcellular specialization. The three NADP-dependent isocitrate dehydrogenase isozymes of the fish Fundulus heteroclitus

Three NADP-dependent isocitrate dehydrogenase isozymes in the teleost, Fundulus heteroclitus (L.), exhibit differences in tissue and subcellular distribution. These three proteins were purified and characterized as to native and subunit molecular weight, isoelectric pH, susceptibility to thermal denaturation, and certain kinetic parameters (Km and Vmax) for the oxidative decarboxylation of isocitrate at 25 degrees C and pH 7.4. The enzymes are dimers of 90 +/- 4 kDa with subunit molecular masses of 45 +/- 3 kDa. Isoelectric pH values were 7.00, 5.19, and 5.29 for IDH-A2, IDH-B2 and IDH-C2 (where IDH represents isocitrate dehydrogenase), respectively. While the monomer-dimer equilibrium is not influenced by substrates, the equilibrium appears to respond to buffer concentration and temperature. Enzyme activity is not affected upon dilution in the presence of buffer containing bovine serum albumin, however, its activity declines rapidly in the absence of bovine serum albumin. Thermal stability varies among the isozymes, and they do not denature by a simple first-order process. The presence of substrates, metal, and coenzymes independently provided enzyme stability, suggesting a random mechanism of substrate and cofactor binding. While IDH-A2 and IDH-B2 have identical KISOCm, IDH-B2 has a lower KNADPm. The most common mitochondrial isozyme (IDH-C2) has a greater KISOCm than either the less common mitochondrial isozyme (IDH-A2) or the cytoplasmic enzyme (IDH-B2). The KNADPm for IDH-C2 was the same as that of IDH-A2 but greater than that of IDH-B2. These Km differences are consistent with the cytoplasmic-mitochondrial shuttling of NADPH-reducing equivalents into the cytoplasm.     

Antioxidant function of cytosolic sources of NADPH in yeast

The relative antioxidant functions of thiol-dependent mechanisms and of direct catalytic inactivation of H2O2 were examined using a collection of yeast mutants containing disruptions in single or multiple genes encoding two major enzymatic sources of NADPH [glucose-6-phosphate dehydrogenase (ZWF1) and cytosolic NADP+-specific isocitrate dehydrogenase (IDP2)] and in genes encoding two major cellular peroxidases [mitochondrial cytochrome c peroxidase (CCP1) and cytosolic catalase (CTT1)]. Both types of mechanisms were found to be important for growth in the presence of exogenous H2O2. In the absence of exogenous oxidants, however, loss of ZWF1 and IDP2, but not loss of CTT1 and CCP1, was found to be detrimental not only to growth but also to viability of cells shifted to rich medium containing oleate or acetate. The loss in viability correlates with increased levels of intracellular oxidants apparently produced during normal metabolism of these carbon sources. Acute effects in DeltaZWF1DeltaIDP2 mutants following shifts to these nonpermissive media include an increase in the number of cells demonstrating a transient decrease in growth rate and in cells containing apparent nuclear DNA strand breaks. Cumulative effects are reflected in phenotypes, including sensitivity to acetate medium and a reduction in mating efficiency, that become more pronounced with time following disruption of the ZWF1 and IDP2 genes. These results suggest that cellular mechanisms dependent on NADPH are crucial metabolic antioxidants.     

Characterization of mouse endoplasmic reticulum methionine-R-sulfoxide reductase

Methionine-R-sulfoxide reductases (MsrBs) catalyze a stereospecific reduction of methionine-R-sulfoxides to methionines in proteins. Mammals possess three MsrB genes. MsrB1 (SelR) is a selenoprotein located in the cytosol and nucleus, MsrB2 (CBS-1) is a mitochondrial protein, and MsrB3 is a recently identified protein with an unusual localization pattern. Human MsrB3 occurs in two protein forms, MsrB3A and MsrB3B, which can be targeted to the endoplasmic reticulum (ER) and mitochondria, respectively. These forms are generated by alternative first exon splicing that introduces contrasting N-terminal signal peptides. Herein, we characterized mouse MsrB3 and found no evidence of alternative splicing of its gene. The ER signal was located upstream of the predicted mitochondrial signal sequence in a single coding region, whose product was targeted to the ER. Although the mitochondrial signal could function if placed at the N-terminus, it did not target MsrB3 to mitochondria as part of the entire coding region. In addition, immunoblot assays detected no mitochondrial MsrB3 in examined mouse tissues. The data suggest that, in mice, MsrB3 is largely or exclusively an ER-resident protein, and that the reduction of methionine-R-sulfoxides in different cellular compartments is provided by individual MsrB isozymes.     

Mitochondrial and cytosolic NADPH systems and isocitrate dehydrogenase indicator metabolites during ureogensis from ammonia in isolated rat hepatocytes.

1. Citrate isocitrate and 2-oxoglutarate levels were determined in isolated rat hepatocytes and in particulate and soluble fractions, thereof, obtained by the digitonin and silicone oil fractionation technique. 2. Caculated from isocitrate/2-oxoglutarate ratios ("indicator metabolite method"), the redox potential of mitochondrial free NADPH is -402 mV, whereas that of the extramitochondrial (cytosolic) space is about 10 mV more positive, -392 mV. 3; Addition of ammonia (either as ammonium chloride or from urea plus urease) to isolated hepatocytes causes preferential oxidation of mitochondrial NADPH, is demonstrated by spectrophotometry of the dihydro band and by the changes in the isocitrate/2-oxoglutarate ratios. The redox potential difference of free NADPH between mitochondria and cytosol is abolished or even reserved. 4. It is concluded that during urogenesis from ammonia mitochondrial isocitrate oxidation is shifted largely in favor of the NADP-linked as opposed to the NAD-linked enzyme; isocitrate concentration under these conditions is less than 10 muM, below the Km (isocitrate) of the NAD-linked enzyme but in the range of that for the NADP-linked enzyme. 5. Both in the absence and in the presence of ammonia there is a concentration gradient across the mitochondrial inner membrane (from mitochondria to cytosol) for citrate, isocitrate, and also, to a smaller extent, for 2-oxoglutarate. 6. These results and data in the literature on enzyme activity are in agreement with the assumption of near-equilibrium of NADP-dependent isocitrate dehydrogenases in the mitochondrial matrix and cytosolic spaces in the absence of ammonia; accordingly, during urea formation from added ammonia the redox potential of mitochondrial free NADPH is increased to -391 mV or possibly even higher if there exists an indicator error under this condition.     

The control of isocitrate oxidation by rat liver mitochondria

1. The factors capable of affecting the rate of isocitrate oxidation in intact mitochondria include the rate of isocitrate penetration, the activity of the NAD-specific and NADP-specific isocitrate dehydrogenases, the activity of the transhydrogenase acting from NADPH to NAD(+), the rate of NADPH oxidation by the reductive synthesis of glutamate and the activity of the respiratory chain. A quantitative assessment of these factors was made in intact mitochondria. 2. The kinetic properties of the NAD-specific and NADP-specific isocitrate dehydrogenases extracted from rat liver mitochondria were examined. 3. The rate of isocitrate oxidation through the respiratory chain in mitochondria with coupled phosphorylation is approximately equal to the maximal of the NAD-specific isocitrate dehydrogenase but at least ten times as great as the transhydrogenase activity from NADPH to NAD(+). 4. It is concluded that the energy-dependent inhibition of isocitrate oxidation by palmitoylcarnitine oxidation is due to an inhibition of the NAD-specific isocitrate dehydrogenase. 5. Kinetic studies of NAD-specific isocitrate dehydrogenase demonstrated that its activity could be inhibited by one or more of the following: an increased reduction of mitochondrial NAD, an increased phosphorylation of mitochondrial adenine nucleotides or a fall in the mitochondrial isocitrate concentration. 6. Uncoupling agents stimulate isocitrate oxidation by an extent equal to the associated stimulation of transhydrogenation from NADPH to NAD(+). 7. A technique is described for continuously measuring with a carbon dioxide electrode the synthesis of glutamate from isocitrate and ammonia.     

Ca(2+) modulates respiratory and steroidogenic activities of human term placental mitochondria

We investigated the effects of calcium on the oxidative metabolism and steroidogenic activity of human term placental mitochondria. Submicromolar Ca(2+) concentrations stimulated state 3 oxygen consumption with 2-oxoglutarate and isocitrate and activated the 2-oxoglutarate and the NAD-isocitrate dehydrogenases by diminishing their Michaelis-Menten constants. Ca(2+) inhibited NADP-isocitrate dehydrogenase (NADP-ICDH) and the synthesis of progesterone. The NADP-ICDH maximal velocity was threefold higher than that of NAD-ICDH and had a threefold lower K(m) for isocitrate than NAD-ICDH. Isocitrate but not malate or 2-oxoglutarate supported progesterone synthesis. Calcium inhibition of progesterone synthesis was observed with isocitrate but not with malate or 2-oxoglutarate. Tight regulation of NADP-isocitrate dehydrogenase by calcium ions suggests that this enzyme plays an important role in placental mitochondrial metabolism.     

Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios.

1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system.     

Synthesis of phosphoenolpyruvate from propionate in sheep liver

1. Utilization of propionate by sheep liver mitochondria was stimulated equally by pyruvate or alpha-oxoglutarate, with formation predominantly of malate. Pyruvate increased conversion of propionate carbon into citrate, whereas alpha-oxoglutarate increased formation of phosphoenolpyruvate. The fraction of metabolized propionate converted into phosphoenolpyruvate was about 17% in the presence or absence of alpha-oxoglutarate and about 7% in the presence of pyruvate. Pyruvate consumption was inhibited by 80% by 5mm-propionate. 2. Compared with rat liver, sheep liver was characterized by very high activities of phosphoenolpyruvate carboxykinase and moderately high activities of aconitase in the mitochondria and by low activities of ;malic' enzyme, pyruvate kinase and lactate dehydrogenase in the cytosol. Activities of phosphoenolpyruvate carboxy-kinase were similar in liver cytosol from rats and sheep. Activities of malate dehydrogenase and NADP-linked isocitrate dehydrogenase in sheep liver were about half those in rat liver. 3. The phosphate-dicarboxylate antiport was active in sheep liver mitochondria, but compared with rat liver mitochondria the citrate-malate antiport showed only low activity and mitochondrial aconitase was relatively inaccessible to external citrate. The rate of swelling of mitochondria induced by phosphate in solutions of ammonium malate was inversely related to the concentration of malate. 4. The results are discussed in relation to gluconeogenesis from propionate in sheep liver. It is proposed that propionate is converted into malate by the mitochondria and the malate is converted into phosphoenolpyruvate by enzymes in the cytosol. In this way sufficient NADH would be generated in the cytosol to convert the phosphoenolpyruvate into glucose.     

Identification of Enzymes and Quantification of Metabolic Fluxes in the Wild Type and in a Recombinant Aspergillus oryzae Strain

Two α-amylase-producing strains of Aspergillus oryzae, a wild-type strain and a recombinant containing additional copies of the α-amylase gene, were characterized with respect to enzyme activities, localization of enzymes to the mitochondria or cytosol, macromolecular composition, and metabolic fluxes through the central metabolism during glucose-limited chemostat cultivations. Citrate synthase and isocitrate dehydrogenase (NAD) activities were found only in the mitochondria, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase (NADP) activities were found only in the cytosol, and isocitrate dehydrogenase (NADP), glutamate oxaloacetate transaminase, malate dehydrogenase, and glutamate dehydrogenase (NAD) activities were found in both the mitochondria and the cytosol. The measured biomass components and ash could account for 95% (wt/wt) of the biomass. The protein and RNA contents increased linearly with increasing specific growth rate, but the carbohydrate and chitin contents decreased. A metabolic model consisting of 69 fluxes and 59 intracellular metabolites was used to calculate the metabolic fluxes through the central metabolism at several specific growth rates, with ammonia or nitrate as the nitrogen source. The flux through the pentose phosphate pathway increased with increasing specific growth rate. The fluxes through the pentose phosphate pathway were 15 to 26% higher for the recombinant strain than for the wild-type strain.     

NAD(+)-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 (Mr approximately 40,000) and IDH2 (Mr approximately 39,000). We have isolated and characterized a yeast genomic clone containing the IDH2 gene. The amino acid sequence deduced from the gene indicates that IDH2 is synthesized as a precursor of 369 amino acids (Mr 39,694) and is processed upon mitochondrial import to yield a mature protein of 354 amino acids (Mr 37,755). Amino acid sequence comparison between S. cerevisiae IDH2 and S. cerevisiae NADP(+)-dependent isocitrate dehydrogenase shows no significant sequence identity, whereas comparison of IDH2 and Escherichia coli NADP(+)-dependent isocitrate dehydrogenase reveals a 33% sequence identity. To confirm the identity of the IDH2 gene and examine the relationship between IDH1 and IDH2, the IDH2 gene was disrupted by genomic replacement in a haploid yeast strain. The disruption strain expressed no detectable IDH2, as determined by Western blot analysis, and was found to lack NAD(+)-dependent isocitrate dehydrogenase activity, indicating that IDH2 is essential for a functional enzyme. Overexpression of IDH2, however, did not result in increased NAD(+)-dependent isocitrate dehydrogenase activity, suggesting that both IDH1 and IDH2 subunits are required for catalytic activity. The disruption strain was unable to utilize acetate as a carbon source and exhibited a 2-fold slower growth rate than wild type strains on glycerol or lactate. This growth phenotype is consistent with NAD(+)-dependent isocitrate dehydrogenase performing an essential role in the oxidative function of the citric acid cycle.     

A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.