Identification and functional characterization of a novel, tissue- specific NAD+-dependent isocitrate dehydrogenase β subunit isoform

Abstract

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 (β1 and β2) and the γ subunit. Analysis of deduced amino acid sequences revealed that β1 and β2 encode 349 and 354 amino acids, respectively, and the two isoforms only differ in the most carboxyl 28 amino acids. The cDNA encodes 354 amino acids and is almost identical to monkey IDHγ. Northern analyses revealed that the smaller β2 transcript (1.3 kilobases) is primarily expressed in heart and skeletal muscle, whereas the larger β1 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 β1 and β2 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 β1 with β2 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 (β1 prevalent; optimal pH 8.0) versus heart (β2 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 β2 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.