Identification and functional characterization of a novel, tissue-specific NAD(+)-dependent isocitrate dehydrogenase...

To understand the interactions and functional role of each of the three mitochondrial NAD(+)-dependent isocitrate dehydrogenase (IDH) subunits (alpha, beta, and gamma), we have characterized human cDNAs encoding two beta isoforms (beta(1) and beta(2)) and the gamma subunit. Analysis of deduced amino acid sequences revealed that beta(1) and beta(2) encode 349 and 354 amino acids, respectively, and the two isoforms only differ in the most carboxyl 28 amino acids. The gamma cDNA encodes 354 amino acids and is almost identical to monkey IDHgamma. Northern analyses revealed that the smaller beta(2) transcript (1.3 kilobases) is primarily expressed in heart and skeletal muscle, whereas the larger beta(1) mRNA (1.6 kilobases) is prevalent in nonmuscle tissues. Sequence analysis of the IDHbeta gene indicates that the difference in the C-terminal 28 amino acids between beta(1) and beta(2) proteins results from alternative splicing of a single transcript. Among the various combinations of human IDH subunits co-expressed in bacteria, alphabetagamma, alphabeta, and alphagamma combinations exhibited significant amounts of IDH activity, whereas subunits produced alone and betagamma showed no detectable activity. These data suggest that the alpha is the catalytic subunit and that at least one of the other two subunits plays an essential supporting role for activity. Substitution of beta(1) with beta(2) in the co-expression system lowered the pH optimum for IDH activity from 8.0 to 7.6. This difference in optimal pH was analogous to what was observed in mouse kidney and brain (beta(1) prevalent; optimal pH 8.0) versus heart (beta(2) prevalent; pH 7.6) mitochondria. Experiments with a specially designed splicing reporter construct stably transfected into HT1080 cells indicate that acidic conditions favor a splicing pattern responsible for the muscle- and heart-specific beta(2) isoform. Taken together, these data indicate a regulatory role of IDHbeta isoforms in determining the pH optimum for IDH activity through the tissue-specific alternative splicing.