Identification and Functional Characterization of a Novel, Tissue-specific NAD+-dependent Isocitrate Dehydrogenase β Subunit Isoform

To understand the interactions and functional role of each of the three mitochondrial NAD + -dependent isocitrate dehydrogenase (IDH) subunits (α, β, and γ), we have characterized human cDNAs encoding two β isoforms (β 1 and β 2 ) and the γ subunit. Analysis of deduced amino acid sequences revealed that β 1 and β 2 encode 349 and 354 amino acids, respectively, and the two isoforms only differ in the most carboxyl 28 amino acids. The γ cDNA encodes 354 amino acids and is almost identical to monkey IDHγ. Northern analyses revealed that the smaller β 2 transcript (1.3 kilobases) is primarily expressed in heart and skeletal muscle, whereas the larger β 1 mRNA (1.6 kilobases) is prevalent in nonmuscle tissues. Sequence analysis of the IDHβ gene indicates that the difference in the C-terminal 28 amino acids between β 1 and β 2 proteins results from alternative splicing of a single transcript. Among the various combinations of human IDH subunits co-expressed in bacteria, αβγ, αβ, and αγ combinations exhibited significant amounts of IDH activity, whereas subunits produced alone and βγ showed no detectable activity. These data suggest that the α is the catalytic subunit and that at least one of the other two subunits plays an essential supporting role for activity. Substitution of β 1 with β 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 (β 1 prevalent; optimal pH 8.0) versus heart (β 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 β 2 isoform. Taken together, these data indicate a regulatory role of IDHβ isoforms in determining the pH optimum for IDH activity through the tissue-specific alternative splicing.