The cytosolic isozyme of NADP-specific isocitrate dehydrogenase (IDP2) was purified from a Saccharomyces cerevisiae mutant containing a chromosomal disruption in the gene encoding the mitochondrial isozyme (IDP1). IDP2 was shown to be a homodimer with a subunit molecular weight of approximately 45,000 and an isoelectric point of 5.5. Amino acid sequences were obtained for tryptic peptides of IDP2 and used to plan polymerase chain reactions. A resulting 400 bp DNA fragment was used as a hybridization probe to isolate the IDP2 gene from a yeast genomic DNA library. The complete nucleotide sequence of the IDP2 coding region was determined and translated into a 412-residue amino acid sequence. IDP2 and IDP1 were found to be identical in 71% of the aligned residue positions. The identity of the IDP2 gene was confirmed by genomic replacement with a disrupted IDP2 coding region. Haploid yeast strains lacking either or both IDP2 and IDP1 were constructed by genetic crosses of mutant strains containing disruptions in chromosomal IDP2 and IDP1 loci. No dramatic differences in growth rates with common carbon sources could be attributed to these disruptions.

Cellular and mitochondrial metabolite levels were measured in yeast TCA cycle mutants (sdh2⌬ or fum1⌬) lacking succinate dehydrogenase or fumarase activities. Cellular levels of succinate relative to parental strain levels were found to be elevated ϳ8-fold in the sdh2⌬ mutant and ϳ4-fold in the fum1⌬ mutant, and there was a preferential increase in mitochondrial levels in these mutant strains. The sdh2⌬ and fum1⌬ strains also exhibited 3-4-fold increases in expression of Cit2, the cytosolic form of citrate synthase that functions in the glyoxylate pathway. Codisruption of the SFC1 gene encoding the mitochondrial succinate/fumarate transporter resulted in higher relative mitochondrial levels of succinate and in substantial reductions of Cit2 expression in sdh2⌬sfc1⌬ and fum1⌬sfc1⌬ strains as compared with sdh2⌬ and fum1⌬ strains, suggesting that aberrant transport of succinate out of mitochondria mediated by Sfc1 is related to the increased expression of Cit2 in sdh2⌬ and fum1⌬ strains. A defect (rtg1⌬) in the yeast retrograde response pathway, which controls expression of several mitochondrial proteins and Cit2, eliminated expression of Cit2 and reduced expression of NAD-specific isocitrate dehydrogenase (Idh) and aconitase (Aco1) in parental, sdh2⌬, and fum1⌬ strains. Concomitantly, co-disruption of the RTG1 gene reduced the cellular levels of succinate in the sdh2⌬ and fum1⌬ strains, of fumarate in the fum1⌬ strain, and citrate in an idh⌬ strain. Thus, the retrograde response is necessary for maintenance of normal flux through the TCA and glyoxylate cycles in the parental strain and for metabolite accumulation in TCA cycle mutants.Intermediates of the tricarboxylic (TCA) 2 cycle are believed to be present in relatively low concentrations in mitochondria of actively respiring eukaryotic cells due to rapid flux through the cycle and to alternative biosynthetic uses for many of the intermediates. Flux through the cycle thus requires anaplerotic enzymes (e.g. pyruvate carboxylase to produce oxaloacetate) or pathways (e.g. the glyoxylate pathway in yeast that produces succinate) to replenish these intermediates. Defects in a TCA cycle enzyme would be expected to result in increased mitochondrial and/or cellular concentrations of the substrate of that enzyme.We and others (1-4) have been analyzing effects of disruptions of genes encoding TCA cycle enzymes in the yeast Saccharomyces cerevisiae. These gene disruptions are not lethal because yeast cells can survive without mitochondrial functions. However, the disruptions have quite distinct effects depending on loss of particular enzymes (5). We initiated studies of changes in metabolite profiles associated with loss of yeast TCA cycle enzymes. In a previous report (2), we described that citrate, a relatively abundant metabolite in the parental strain, accumulates to very high cellular levels in a mutant (idh⌬) lacking both Idh1 and Idh2 subunits of NAD-specific isocitrate dehydrogenase and in a mutant (aco1⌬) lacking aconitase. In addition to sharing similar gene and...

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.

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.Differentially compartmentalized isozymes of malate dehydrogenase in eucaryotic cells catalyze the NAD(H)-dependent interconversion of oxaloacetate and malate. In mammalian cells this reaction, catalyzed by mitochondrial and cytosolic isozymes, respectively, is a critical step in the tricarboxylic acid cycle and in gluconeogenesis. The two isozymes also participate in the malate/aspartate shuttle cycle, a mechanism for exchange of reducing equivalents between cellular compartments. In yeast and plant cells, a third isozyme localized in peroxisomes catalyzes a step in the glyoxylate pathway. This pathway allows formation of C 4 metabolites from C 2 precursors. The malate dehydrogenase isozyme family is therefore an ideal focus for analysis of structural features responsible for differential compartmentation and metabolic function.To initiate molecular genetic studies, the three isozymes of malate dehydrogenase have been purified from Saccharomyces cerevisiae, and the corresponding genes have been cloned, sequenced, and disrupted (1-3). The isozymes are all homodimers, and they exhibit similar kinetic properties (3). The aligned amino acid sequences have residue identities ranging from 43 to 50%. Among conserved residues are those with catalytic functions or those that participate in cofactor binding (4, 5). Salient differences include regions with putative functions in organellar targeting. The yeast mitochondrial isozyme (MDH1, subunit molecular weight ϭ 33,500), for example, has a 17-residue amino-terminal extension not present on the other isozymes; this extension is removed upon mitochondrial import (6). The peroxisomal isozyme (MDH3, subunit molecular weight ϭ 37,200) has a unique carboxyl-terminal tripeptide sequence, Ser-Lys-Leu. Similar SKL termini on other peroxisomal proteins have been found to be necessary and sufficient for organellar lo...

Yeast NAD+-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four heterodimers of regulatory IDH1 and catalytic IDH2 subunits. The crystal structure suggested that the interactions between tetramers in the octamer are restricted to defined regions in IDH1 subunits from each tetramer. Using truncation and mutagenesis, we constructed three tetrameric forms of IDH. Truncation of five residues from the amino-terminus of IDH1 did not alter the octameric form of the enzyme, but this truncation plus IDH1 G15D or IDH1 D168K residue substitutions produced tetrameric enzymes as assessed by sedimentation velocity ultracentrifugation. The IDH1 G15D substitution in the absence of any truncation of IDH1 was subsequently found to be sufficient for production of a tetrameric enzyme. The tetrameric forms of IDH exhibited ~50% reductions in Vmax and in cooperativity with respect to isocitrate relative to the wild-type enzyme, but they retained the property of allosteric activation by AMP. The truncated -5IDH1/IDH2 and tetrameric enzymes were much more sensitive than the wild-type enzyme to inhibition by the oxidant diamide and concomitant formation of a disulfide bond between IDH2 Cys-150 residues. Binding of ligands reduced the sensitivity of the wild-type enzyme to diamide but had no effect on inhibition of the truncated or tetrameric enzymes. These results suggest that the octameric structure of IDH has in part evolved for regulation of disulfide-bond formation and activity by ensuring the proximity of the amino terminus of an IDH1 subunit from one tetramer to the IDH2 Cys-150 residues in the other tetramer.