Redox responses in yeast to acetate as the carbon source.

Following a shift to medium with acetate as the carbon source, a parental yeast strain exhibited a transient moderate 20% reduction in total cellular [NAD(+)+NADH] but showed a approximately 10-fold increase in the ratio of [NAD(+)]:[NADH] after 36h. A mutant strain (idhDelta) lacking the tricarboxylic acid cycle enzyme isocitrate dehydrogenase had 50% higher cellular levels of [NAD(+)+NADH] relative to the parental strain but exhibited similar changes in cofactor concentrations following a shift to acetate medium, despite an inability to grow on that carbon source; essentially all of the cofactor was in the oxidized form within 36h. The salvage pathway for NAD(H) biosynthesis was found to be particularly important for viability during early transition of the parental strain to stationary phase in acetate medium. However, oxygen consumption was not affected, suggesting that the NAD(H) produced during this time may support other cellular functions. The idhDelta mutant exhibited increased flux through the salvage pathway in acetate medium but was dependent on the de novo pathway for viability. Long-term chronological lifespans of the parental and idhDelta strains were similar, but viability of the mutant strain was dependent on both pathways for NAD(H) biosynthesis.

Effect of AMP on mRNA binding by yeast NAD+-specific isocitrate dehydrogenase.

Yeast mitochondrial NAD+-specific isocitrate dehydrogenase (IDH) has previously been shown to bind specifically to 5'-untranslated regions of yeast mitochondrial mRNAs, and transcripts containing these regions have been found to allosterically inhibit activity of the enzyme. This inhibition is relieved by AMP, an allosteric activator of this regulatory enzyme of the tricarboxylic acid cycle. We further investigated these enzyme/ligand interactions to determine if binding of RNA and AMP by IDH is competitive or independent. Gel mobility shift experiments indicated no effect of AMP on formation of an IDH/RNA complex. Similarly, sedimentation velocity ultracentrifugation experiments used to analyze interactions in solution indicated that AMP alone had little effect on the formation or stability of an RNA/IDH complex. However, when these sedimentation experiments were conducted in the presence of isocitrate, which has been shown to be essential for binding of AMP by IDH, the proportion of RNA sedimenting in a complex with IDH was significantly reduced by AMP. These results suggest that AMP can affect the binding of RNA by IDH but that this effect is apparent only in the presence of substrate. They also suggest that the catalytic activity of IDH in vivo may be subject to complex allosteric control determined by relative mitochondrial concentrations of mRNA, isocitrate, and AMP. We also found evidence for binding of 5'-untranslated regions of mitochondrial mRNAs by yeast mitochondrial NADP+-specific isocitrate dehydrogenase (IDP1) but not by the corresponding cytosolic isozyme (IDP2). However, this appears to be a nonspecific interaction since no evidence was obtained for any effect on the catalytic activity of IDP1.

Kinetic and physiological effects of alterations in homologous isocitrate-binding sites of yeast NAD(+)-specific isocitrate dehydrogenase.

Yeast NAD(+)-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four each of two homologous but nonidentical subunits designated IDH1 and IDH2. Models based on the crystallographic structure of Escherichia coli isocitrate dehydrogenase suggest that both yeast subunits contain isocitrate-binding sites. Identities in nine residue positions are predicted for the IDH2 site whereas four of the nine positions differ between the IDH1 and bacterial enzyme sites. Thus, we speculate that the IDH2 site is catalytic and that the IDH1 site may bind but not catalytically alter isocitrate. This was examined by kinetic analyses of enzymes with independent and concerted replacement of residues in each yeast IDH subunit site with the residues that differ in the other subunit site. Mutant enzymes were expressed in a yeast strain containing disrupted IDH1 and IDH2 loci and affinity-purified for kinetic analyses. The primary effects of various residue replacements in IDH2 were reductions of 30->300-fold in V(max) values, consistent with the catalytic function of this subunit. In contrast, replacement of all four residues in IDH1 produced a 17-fold reduction in V(max) under the same assay conditions, suggesting that the IDH1 site is not the primary catalytic site. However, single or multiple residue replacements in IDH1 uniformly increased half-saturation concentrations for isocitrate, implying that isocitrate can be bound at this site. Both subunits appear to contribute to cooperativity with respect to isocitrate, but AMP activation is lost only with residue replacements in IDH1. Overall, results are consistent with isocitrate binding by IDH2 for catalysis and with isocitrate binding by IDH1 being a prerequisite for allosteric activation by AMP. The effects of residue substitutions on enzyme function in vivo were assessed by analysis of various growth phenotypes. Results indicate a positive correlation between the level of IDH catalytic activity and the ability of cells to grow with acetate or glycerol as carbon sources. In addition, lower levels of activity are associated with increased production of respiratory-deficient (petite) segregants.

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.

Subunit interactions of yeast NAD+-specific isocitrate dehydrogenase.

Yeast mitochondrial NAD(+)-specific isocitrate dehydrogenase is an octamer composed of four each of two nonidentical but related subunits designated IDH1 and IDH2. IDH2 was previously shown to contain the catalytic site, whereas IDH1 contributes regulatory properties including cooperativity with respect to isocitrate and allosteric activation by AMP. In this study, interactions between IDH1 and IDH2 were detected using the yeast two-hybrid system, but interactions between identical subunit polypeptides were not detected with this or other methods. A model for heterodimeric interactions between the subunits is therefore proposed for this enzyme. A corollary of this model, based on the three-dimensional structure of the homologous enzyme from Escherichia coli, is that some interactions between subunits occur at isocitrate binding sites. Based on this model, two residues (Lys-183 and Asp-217) in the regulatory IDH1 subunit were predicted to be important in the catalytic site of IDH2. We found that individually replacing these residues with alanine results in mutant enzymes that exhibit a drastic reduction in catalysis both in vitro and in vivo. Also based on this model, the two analogous residues (Lys-189 and Asp-222) of the catalytic IDH2 subunit were predicted to contribute to the regulatory site of IDH1. A K189A substitution in IDH2 was found to produce a decrease in activation of the enzyme by AMP and a loss of cooperativity with respect to isocitrate. A D222A substitution in IDH2 produces similar regulatory defects and a substantial reduction in V(max) in the absence of AMP. Collectively, these results suggest that the basic structural/functional unit of yeast isocitrate dehydrogenase is a heterodimer of IDH1 and IDH2 subunits and that each subunit contributes to the isocitrate binding site of the other.

Allosteric inhibition of NAD+-specific isocitrate dehydrogenase by a mitochondrial mRNA.

NAD+-specific isocitrate dehydrogenase (IDH) has been reported to bind sequences in 5'-untranslated regions of yeast mitochondrial mRNAs. In the current study, an RNA transcript containing the 5'-untranslated region of the mRNA from the yeast mitochondrial COX2 gene is shown to be an allosteric inhibitor of the affinity-purified yeast enzyme. At 0.1 microM concentrations of the transcript, velocity of the IDH reaction is reduced to 20% of the value obtained in the absence of the RNA transcript. This inhibition is due to a 2. 5-fold increase in the S0.5 value for isocitrate. Significant inhibition of IDH activity is also obtained with a transcript containing a portion of the 5'-untranslated region of the yeast mitochondrial ATP9 gene and with an antisense form of the COX2 transcript, both of which contain potential stem-loop secondary structures implicated in binding of IDH. In contrast, much higher concentrations of yeast tRNA or poly(A)mRNA, respectively, 33- and 60-fold greater than that required for the COX2 transcript, are required to produce a 50% decrease in velocity. These results suggest that inhibition of activity is relatively specific for the 5'-untranslated regions of mitochondrial mRNAs. All measurable inhibition of IDH activity by RNA is eliminated by addition of 100 microM concentrations of the allosteric activator AMP. At equivalent concentrations, dAMP is less efficient than AMP as an allosteric activator of IDH and is proportionally less effective in protecting against inhibition of activity by the COX2 transcript. Other nucleotides that are not allosteric activators fail to protect IDH activity from inhibitory effects of RNA. Thus, alleviation of catalytic inhibition of IDH by mitochondrial mRNA correlates with the property of allosteric activation.

Applications of the yeast two-hybrid system.

In recent years, the yeast two-hybrid system has become the method of choice for detection and analysis of protein-protein interactions in an in vivo context. This system, which capitalizes on the significant genetic history and ease of protocols for manipulation of Saccharomyces cerevisiae, is accessible to most laboratories and is applicable to the pursuit of a large variety of experimental goals. To date, the two-hybrid system has seen widespread application for identification of interaction partners by screening methods using a particular protein of interest as a "bait." Large-scale ventures are also in progress, for example, a cataloging of interactions among the cellular proteins in yeast. However, this method also has tremendous potential for more focused analyses of specific proteins and should become more routine as an alternative or adjunct approach for many structure-function investigations.

Dependence of peroxisomal beta-oxidation on cytosolic sources of NADPH.

Growth of Saccharomyces cerevisiae with a fatty acid as carbon source was shown previously to require function of either glucose-6-phosphate dehydrogenase (ZWF1) or cytosolic NADP+-specific isocitrate dehydrogenase (IDP2), suggesting dependence of beta-oxidation on a cytosolic source of NADPH. In this study, we find that DeltaIDP2DeltaZWF1 strains containing disruptions in genes encoding both enzymes exhibit a rapid loss of viability when transferred to medium containing oleate as the carbon source. This loss of viability is not observed following transfer of a DeltaIDP3 strain lacking peroxisomal isocitrate dehydrogenase to medium with docosahexaenoate, a nonpermissive carbon source that requires function of IDP3 for beta-oxidation. This suggests that the fatty acid- phenotype of DeltaIDP2DeltaZWF1 strains is not a simple defect in utilization. Instead, we propose that the common function shared by IDP2 and ZWF1 is maintenance of significant levels of NADPH for enzymatic removal of the hydrogen peroxide generated in the first step of peroxisomal beta-oxidation in yeast and that inadequate levels of the reduced form of the cofactor can produce lethality. This proposal is supported by the finding that the sensitivity to exogenous hydrogen peroxide previously reported for DeltaZWF1 mutant strains is less pronounced when analyses are conducted with a nonfermentable carbon source, a condition associated with elevated expression of IDP2. Under those conditions, similar slow growth phenotypes are observed for DeltaZWF1 and DeltaIDP2 strains, and co-disruption of both genes dramatically exacerbates the H2O2s phenotype. Collectively, these results suggest that IDP2, when expressed, and ZWF1 have critical overlapping functions in provision of reducing equivalents for defense against endogenous or exogenous sources of H2O2.

Sources of NADPH and expression of mammalian NADP+-specific isocitrate dehydrogenases in Saccharomyces cerevisiae.

To compare roles of specific enzymes in supply of NADPH for cellular biosynthesis, collections of yeast mutants were constructed by gene disruptions and matings. These mutants include haploid strains containing all possible combinations of deletions in yeast genes encoding three differentially compartmentalized isozymes of NADP+-specific isocitrate dehydrogenase and in the gene encoding glucose-6-phosphate dehydrogenase (Zwf1p). Growth phenotype analyses of the mutants indicate that either cytosolic NADP+-specific isocitrate dehydrogenase (Idp2p) or the hexose monophosphate shunt is essential for growth with fatty acids as carbon sources and for sporulation of diploid strains, a condition associated with high levels of fatty acid synthesis. No new biosynthetic roles were identified for mitochondrial (Idp1p) or peroxisomal (Idp3p) NADP+-specific isocitrate dehydrogenase isozymes. These and other results suggest that several major presumed sources of biosynthetic reducing equivalents are non-essential in yeast cells grown under many cultivation conditions. To develop an in vivo system for analysis of metabolic function, mammalian mitochondrial and cytosolic isozymes of NADP+-specific isocitrate dehydrogenase were expressed in yeast using promoters from the cognate yeast genes. The mammalian mitochondrial isozyme was found to be imported efficiently into yeast mitochondria when fused to the Idp1p targeting sequence and to substitute functionally for Idp1p for production of alpha-ketoglutarate. The mammalian cytosolic isozyme was found to partition between cytosolic and organellar compartments and to replace functionally Idp2p for production of alpha-ketoglutarate or for growth on fatty acids in a mutant lacking Zwf1p. The mammalian cytosolic isozyme also functionally substitutes for Idp3p allowing growth on petroselinic acid as a carbon source, suggesting partial localization to peroxisomes and provision of NADPH for beta-oxidation of that fatty acid.

Identification of a cytosolically directed NADH dehydrogenase in mitochondria of Saccharomyces cerevisiae.

The reoxidation of NADH generated in reactions within the mitochondrial matrix of Saccharomyces cerevisiae is catalyzed by an NADH dehydrogenase designated Ndi1p (C. A. M. Marres, S. de Vries, and L. A. Grivell, Eur. J. Biochem. 195:857-862, 1991). Gene disruption analysis was used to examine possible metabolic functions of two proteins encoded by open reading frames having significant primary sequence similarity to Ndi1p. Disruption of the gene designated NDH1 results in a threefold reduction in total mitochondrial NADH dehydrogenase activity in cells cultivated with glucose and in a fourfold reduction in the respiration of isolated mitochondria with NADH as the substrate. Thus, Ndh1p appears to be a mitochondrial dehydrogenase capable of using exogenous NADH. Disruption of a closely related gene designated NDH2 has no effect on these properties. Growth phenotype analyses suggest that the external NADH dehydrogenase activity of Ndh1p is important for optimum cellular growth with a number of nonfermentable carbon sources, including ethanol. Codisruption of NDH1 and genes encoding malate dehydrogenases essentially eliminates growth on nonfermentable carbon sources, suggesting that the external mitochondrial NADH dehydrogenase and the malate-aspartate shuttle may both contribute to reoxidation of cytosolic NADH under these growth conditions.

Expression and mutagenesis of mammalian cytosolic NADP+-specific isocitrate dehydrogenase.

Rat liver cytosolic NADP+-specific isocitrate dehydrogenase (IDP2) was expressed in bacteria as a fusion protein with maltose binding protein (MBP). High levels of expression were obtained. The fusion protein was purified from bacterial lysates by affinity chromatography with an amylose resin and found to be catalytically active. IDP2 was separated from MBP by cleavage with protease Xa and purified to homogeneity by FPLC anion-exchange chromatography. A specific activity of 56.3 units/mg and respective apparent Km values for dl-isocitrate and NADP+ of 9.7 +/- 2.9 microM and 11.5 +/- 0.2 microM were obtained for the purified enzyme. These values are similar to those previously reported for cytosolic isocitrate dehydrogenase isolated from a variety of tissues. Evolutionarily conserved arginine residues implicated in substrate binding were changed to glutamate residues using PCR based site-directed mutagenesis of the bacterial fusion plasmid. Mutant enzymes containing residue changes of R100E, R109E, R119E, or R132E were expressed, purified, and characterized by initial rate kinetic analyses. The R119E and R109E mutant enzymes exhibited respective 15- and 31-fold increases in Km values for dl-isocitrate relative to the wild-type enzyme. In contrast, Km values for NADP+ were, respectively, unchanged and increased 9-fold. The most significant reductions in kcat/Km values were obtained for the R100E, R109E, and R132E enzymes. These results suggest that substrate binding residues are highly conserved between bacterial and mammalian enzymes despite low overall homology.

Metabolic effects of altering redundant targeting signals for yeast mitochondrial malate dehydrogenase.

Eukaryotic cells contain highly homologous isozymes of malate dehydrogenase which catalyze the same reaction in different cellular compartments. To examine whether the metabolic functions of these isozymes are interchangeable, we have altered the cellular localization of mitochondrial malate dehydrogenase (MDH1) in yeast. Since a previous study showed that removal of the targeting presequence from MDH1 does not prevent mitochondrial import in vivo, we tested the role of a putative cryptic targeting sequence near the amino terminus of the mature polypeptide. Three residues in this region were changed to residues present in analogous positions in the other two yeast MDH isozymes. Alone, these replacements did not affect activity or localization of MDH1 but, in combination with deletion of the presequence, prevented mitochondrial import in vivo. Measurable levels of the resulting cytosolic form of MDH1 were low with expression from a centromere-based plasmid but were comparable to normal cellular levels with expression from a multicopy plasmid. The cytosolic form of MDH1 restored the ability of a deltaMDH1 disruption strain to grow on ethanol or acetate, suggesting that mitochondrial localization of MDH1 is not essential for its function in the TCA cycle. This TCA cycle function observed for the cytosolic form of MDH1 is unique to that isozyme since overexpression of MDH2 and of a cytosolic form of MDH3 in a deltaMDH1 strain failed to restore growth. Finally, only partial restoration of growth of a deltaMDH2 disruption mutant was attained with the cytosolic form of MDH1, suggesting that MDH2 may also have unique metabolic functions.

Affinity purification and kinetic analysis of mutant forms of yeast NAD+-specific isocitrate dehydrogenase.

Polyhistidine tags were added to the carboxyl termini of the two homologous subunits of yeast NAD+-specific isocitrate dehydrogenase (IDH). The tag in either the IDH1 or IDH2 subunit permits one-step affinity purification from yeast cellular extracts of catalytically active and allosterically responsive holoenzyme. This expression system was used to investigate subunit-specific contributions of residues with putative functions in adenine nucleotide binding. The primary effect of simultaneous replacement of the adjacent Asp-279 and Ile-280 residues in IDH1 with alanines is a dramatic loss of activation by AMP. In contrast, alanine replacement of the homologous Asp-286 and Ile-287 residues in IDH2 does not alter the allosteric response to AMP, but produces a 160-fold reduction in Vmax due to a 70-fold increase in the S0.5 value for NAD+. These results suggest that the targeted aspartate/isoleucine residues may contribute to regulator binding in IDH1 and to cofactor binding in IDH2, i.e. that these homologous residues are located in regions that have evolved for binding the adenine nucleotide components of different ligands. In other mutant enzymes, an alanine replacement of Asp-191 in IDH1 eliminates measurable catalytic activity, and a similar substitution of the homologous Asp-197 in IDH2 produces pleiotropic catalytic effects. A model is presented for the primary function of IDH2 in catalysis and of IDH1 in regulation, with crucial roles for these single aspartate residues in the communication and functional interdependence of the two subunits.

Expression of pig heart mitochondrial NADP-dependent isocitrate dehydrogenase in Escherichia coli.

Pig heart mitochondrial NADP-specific isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The 1.2-kbp cDNA encoding this porcine mitochondrial NADP-specific enzyme has now been inserted into an expression vector, pMAL-c2, to be expressed as a fusion protein with maltose binding protein. Initially, the vector was constructed with a cleavage site for protease Factor Xa between the maltose binding protein and isocitrate dehydrogenase; however, since Factor Xa was also found to digest isocitrate dehydrogenase, a thrombin recognition site was substituted. The fusion protein was expressed in Escherichia coli by IPTG induction at 25 degrees C, and was separated from the endogenous E. coli isocitrate dehydrogenase by affinity chromatography on an amylose resin which adsorbs maltose binding protein and its fusion products. Cleavage of the fusion protein with thrombin generated pig heart NADP-specific isocitrate dehydrogenase, which was purified to homogeneity by affinity chromatography on Matrex Gel Red-A resin and gel filtration by FPLC. A 41-fold increase in specific activity to 37 enzyme units/mg with an approximate yield of 34% for the expressed enzyme was achieved by this purification procedure. This enzyme exhibits a single band (M(r) = 46,600) on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, under standard assay conditions, has a Km for DL-isocitrate of 7.74 +/- 0.18 microM and a Km for NADP+ of 6.63 +/- 1.34 microM. These values are similar to the Kms measured for the enzyme purified from pig heart. The amino-terminal sequence of the expressed enzyme is identical with that of authentic porcine enzyme and distinguishable from the E. coli enzyme at 17 of the 18 residues determined. We conclude that this expression and purification system yields pure pig heart mitochondrial NADP-specific isocitrate dehydrogenase and should allow generation of wild-type and mutant enzymes in amounts suitable for their biochemical characterization and comparison.

Expression and gene disruption analysis of the isocitrate dehydrogenase family in yeast.

Mammalian and yeast cells contain three isozymes of isocitrate dehydrogenase: mitochondrial NAD- and NADP-specific enzymes and a cytosolic NADP-specific enzyme. Independent metabolic functions of these enzymes in Saccharomyces cerevisiae were examined by analyses of expression and of phenotypes displayed by mutants containing all possible combinations of isozyme gene disruptions. All three isocitrate dehydrogenases are expressed at high levels with growth on nonfermentable carbon sources, whereas the mitochondrial NADP-specific enzyme constitutes the major cellular activity with growth on glucose. Distinct growth phenotypes are observed for mutants expressing a single isozyme, and expression of at least one isozyme is necessary for glutamate-independent growth. The NADP-specific tricarboxylic acid cycle isocitrate dehydrogenase from Escherichia coli was expressed in mitochondrial and cytosolic compartments of the yeast disruption mutants using plasmids carrying gene fusions of yeast promoters and a mitochondrial targeting presequence with the bacterial coding sequence. The bacterial enzyme is competent for restoration of NADP-specific functions in either compartment but does not compensate for function of the yeast NAD-specific tricarboxylic acid cycle enzyme.

Assembly and function of a cytosolic form of NADH-specific isocitrate dehydrogenase in yeast.

Mitochondrial NAD-dependent isocitrate dehydrogenase catalyzes a rate-limiting step in the tricarboxylic acid cycle. Yeast isocitrate dehydrogenase is an octomer composed of two subunits (IDH1 and IDH2) encoded by different genes and possessing independent mitochondrial targeting presequences. Oligonucleotide-directed mutagenesis was used to remove the presequences from each gene and from both genes carried on centromere-based expression plasmids. Effects on cellular localization were examined in a yeast strain containing chromosomal disruptions of IDH1 and IDH2 loci. Each subunit was found to be dependent upon its presequence for mitochondrial localization, and the subunits are independently imported into mitochondria under most growth conditions. Furthermore, an active holoenzyme can be assembled in the cytosol and this ''cytosolic'' form of isocitrate dehydrogenase can reverse the acetate- growth phenotype characteristic of the DeltaIDH1/ DeltaIDH2 disruption strain, indicating functional replacement of the mitochondrial enzyme. However, transformants containing plasmids lacking either the IDH1 or IDH2 presequence coding regions were unexpectedly found to be capable of growth on acetate medium. Further investigation demonstrated that cellular localization of the IDH1 subunit can be biased by this stringent growth pressure.

Expression and function of a mislocalized form of peroxisomal malate dehydrogenase (MDH3) in yeast.

The malate dehydrogenase isozyme MDH3 of Saccharomyces cerevisiae was found to be localized to peroxisomes by cellular fractionation and density gradient centrifugation. However, unlike other yeast peroxisomal enzymes that function in the glyoxylate pathway, MDH3 was found to be refractory to catabolite inactivation, i.e. to rapid inactivation and degradation following glucose addition. To examine the structural requirements for organellar localization, the Ser-Lys-Leu carboxyl-terminal tripeptide, a common motif for localization of peroxisomal proteins, was removed by mutagenesis of the MDH3 gene. This resulted in cytosolic localization of MDH3 in yeast transformants. To examine structural requirements for catabolite inactivation, a 12-residue amino-terminal extension from the yeast cytosolic MDH2 isozyme was added to the amino termini of the peroxisomal and mislocalized "cytosolic" forms of MDH3. This extension was previously shown to be essential for catabolite inactivation of MDH2 but failed to confer this property to MDH3. The mislocalized cytosolic forms of MDH3 were found to be catalytically active and competent for metabolic functions normally provided by MDH2.

N-ethylmaleimide profiling of yeast NADP-dependent isocitrate dehydrogenase.

Yeast NADP-dependent isocitrate dehydrogenase is inactivated by N-ethyl-maleimide (NEM) at pH 7.7 and 30 degrees C. Reaction with cysteine382 occurs most rapidly and is accompanied by loss of about 50% of the enzymatic activity. A slower phase of inactivation ensues during which lysine343 is the major target of NEM, while minor products result from reaction at cysteine73 and cysteine354. Protection against the second phase of inactivation is provided by NADP, NAPH, or manganous-isocitrate. Comparison of the time-dependence of inactivation and the products of reaction with N-ethyl-ethylmaleimide (NEM profiling) of the pig heart (G. E. Smyth and R. F. Colman, 1991, J. Biol. Chem. 266, 14918-14925) and yeast NADP-specific isocitrate dehydrogenases have been coupled with an examination of the crystal structure of the Escherichia coli isocitrate dehydrogenase. The following conclusions have been reached: while no cysteine is essential for activity, yeast Cys382/pig Cys379 is close to the adenine portion of the NADP binding site, and pig Cys269 is located in the region of the metal-isocitrate binding site.

Glucose-induced phosphorylation of the MDH2 isozyme of malate dehydrogenase in Saccharomyces cerevisiae.

The cytosolic isozyme of malate dehydrogenase, MDH2, was previously shown to be subject to rapid inactivation and proteolysis following the addition of glucose to yeast cultures growing on nonfermentable carbon sources. In this report, we show that MDH2 is phosphorylated during the process of glucose-induced degradation. A truncated active form of MDH2 lacking the first 12 residues of the amino terminus was previously found to be resistant to glucose-induced degradation and, as shown in this study, is not subject to phosphorylation. Site-directed mutagenesis was conducted to change Ser-12 in the authentic enzyme to Ala-12 and to Asp-12. The S12A substitution has little effect on glucose-induced phosphorylation and degradation, whereas the enzyme with the S12D substitution is subject to phosphorylation and inactivation but not to rapid degradation. This provides clear evidence that inactivation is not simply a result of degradation. Additional mutagenesis was conducted to change His-214, a critical active site residue, to Leu-214. Analysis of expression of full-length and truncated forms of the H214L enzyme demonstrated that catalytic inactivity is not a prerequisite for degradation and confirmed an essential role for the amino terminus of the authentic enzyme in this phenomenon.