Analysis of Interactions with Mitochondrial mRNA Using Mutant Forms of Yeast NAD+-Specific Isocitrate Dehydrogenase†

The reaction catalyzed by mitochondrial NAD+-specific isocitrate dehydrogenase (IDH) is considered to be a rate limiting step in the tricarboxylic acid (TCA) cycle. The reaction is essentially irreversible under physiological conditions (1), and IDH is subject to multiple allosteric controls. The primary allosteric activator is ADP for the mammalian enzyme (2) or AMP for the Saccharomyces cerevisiae enzyme (3), while ATP is a negative allosteric regulator. These observations led to a hypothesis that rates of respiration are controlled at the level of IDH (4). These allosterically regulated eukaryotic IDHs are structurally complex. Yeast IDH is an octameric enzyme composed of four IDH1 and four IDH2 subunits (5). The IDH1 and IDH2 subunits are 42% identical in sequence (6, 7). Mutagenesis of analogous residues in these subunits has demonstrated that the catalytic isocitrate/Mg2+-binding site is primarily contained in IDH2 and shares structural similarity with the regulatory cooperative isocitrate-binding site primarily contained in IDH1 (8-10). Similarly, the cofactor NAD+-binding site in IDH2 shares structural similarity with the allosteric AMP-binding site in IDH1 (11, 12). In addition to allosteric regulation of energy metabolism, yeast IDH has also been found to be an mRNA-binding protein (13). This was revealed in a search for proteins that bind yeast mitochondrial mRNAs to identify possible regulators of translation (14), a major point for control of mitochondrial gene expression. IDH was shown to bind near conserved secondary structures in the 5'-untranslated regions (UTRs) of all eight major mRNAs encoded by mitochondrial genes in yeast (15). De Jong et al. (16) analyzed the importance of this interaction in vivo. They found that while the rate of mitochondrial translation was increased in a yeast strain lacking IDH, the newly synthesized proteins were rapidly degraded, resulting in an overall reduction in their steady-state levels. Since these mitochondrially-encoded proteins are primarily hydrophobic subunits of electron transport complexes, De Jong et al. (16) suggested that IDH binding may normally prevent premature translation in the matrix until the mRNAs are in close proximity of known translational activators in the inner mitochondrial membrane (17, 18). Therefore, IDH was proposed to act as a transient negative regulator of translation to ensure that hydrophobic subunits of respiratory complexes are synthesized only in close proximity of their cellular membrane destination. We have elucidated another important aspect of the IDH/mRNA interaction (19). We found that addition of short RNA transcripts containing yeast mitochondrial UTRs dramatically inhibits IDH activity. Inhibition was observed with the COX2 and ATP9 UTRs but not with addition of other unrelated RNAs (e.g. tRNAs) of similar size. The inhibition of IDH by mitochondrial mRNA transcripts is due to a decrease in the apparent affinity of IDH for isocitrate (19). Remarkably, all inhibitory effects of the mRNA are eliminated by the presence of the allosteric activator AMP. This relief from inhibition was shown to be specific for AMP, with little effect resulting from addition of ADP, ATP, or GMP. Thus, allosteric inhibition of IDH appears to be specific for mitochondrial mRNA transcripts containing IDH-binding sites, and alleviation of this inhibition correlates with the property of allosteric activation by AMP. Importantly, these kinetic effects were obtained with relative concentrations of enzyme, mRNA, isocitrate, and AMP that are similar to reported cellular levels of these molecules (15, 20, 21), suggesting that these interactions may be physiologically relevant. In particular, this macromolecular interaction could provide a mechanism for coordinate regulation of rates of flux through oxidative metabolic pathways and rates of synthesis of mitochondrially-encoded proteins. We previously used analytical ultracentrifugation techniques to examine the interaction of IDH and mitochondrial mRNAs (22). Complex formation was observed with near equimolar concentrations of the COX2 mRNA UTR transcript and IDH holoenzyme. This macromolecular interaction was not affected by the presence of isocitrate or of AMP alone; however, the combination of isocitrate and AMP produced a moderate increase in free mRNA. This requirement for both ligands is explained by the fact that binding of the allosteric activator requires isocitrate (10). To extend these studies, we have employed mutant forms of IDH with well characterized defects that alter specific kinetic and ligand-binding properties to determine structural properties important for catalytic inhibition and for binding of a mitochondrial mRNA transcript. Effects of expression of these mutant enzymes on mitochondrial translation were also examined.