Cloning and characterization of the gene encoding the IDH1 subunit of NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae.

J.R Cupp,L Mcalister-Henn

doi:10.1016/s0021-9258(18)42019-4

Abstract

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 and IDH2. The gene encoding IDH2 was previously cloned and sequenced (Cupp, J.R., and McAlister-Henn, L. (1991) J. Biol. Chem. 266, 22199-22205), and in this paper we describe the isolation of a yeast genomic clone containing the IDH1 gene. A fragment of the IDH1 gene was amplified by the polymerase chain reaction method utilizing degenerate oligonucleotides based on tryptic peptide sequences of the purified subunit; this fragment was used to isolate a full length IDH1 clone. The nucleotide sequence of the IDH1 coding region was determined and encodes a 360-residue polypeptide including an 11-residue mitochondrial targeting presequence. Amino acid sequence comparison between IDH1 and IDH2 reveals a 42% sequence identity, and both IDH1 and IDH2 show approximately 32% identity to Escherichia coli NAD(P)(+)-dependent isocitrate dehydrogenase. To examine the function of the IDH1 subunit and to determine the metabolic role of NAD(+)-dependent isocitrate dehydrogenase the IDH1 gene was disrupted in a wild type haploid yeast strain and in a haploid strain lacking IDH2. The IDH1 disruption strains expressed no detectable IDH1 as determined by Western blot analysis, and these strains were found to lack NAD(+)-dependent isocitrate dehydrogenase activity indicating that IDH1 is essential for a functional enzyme. Over-expression of IDH1 in a strain containing IDH2 restored wild type activity but did not result in increased levels of activity, suggesting that both IDH1 and IDH2 are required for a functional enzyme. Growth phenotype analysis of the IDH1 disruption strains revealed that they grew at a reduced rate on the nonfermentable carbon sources examined (glycerol, lactate, and acetate), consistent with NAD(+)-dependent isocitrate dehydrogenase performing a critical role in oxidative function of the citric acid cycle. In addition, the IDH1 disruption strains grew at wild type rates in the absence of glutamate, indicating that these strains are not glutamate auxotrophs.

Highlights

Saccharomyces cerevisiae is composed of two noni- rate to a-ketoglutaratien all cellular organisms
The nucleotide sequence of the I D H l coding region richia coli (I), Saccharomyces cereuisiae (2), and pig and bovine heart (3, 4)C. haracterization of the prokaryotic NAD(P)+-dependent IDH (NAD(P)+-IDH)f’rom E. coli has shown it tobe a homodimer with a subunit molecular weight of 45,000 (1) which is regulated by phosphorylation ( 5 )
Metabolic Role of NAD+-IDH-Because of the importance of the conversion of isocitrate to a-ketoglutarate in central metabolic pathways, much effort has been invested in understanding the kinetics and regulation of NAD+-IDH.TOfurther characterize this complex allosteric enzyme our work has focused on determination of the structure andfunction of the two individual subunits of the enzyme from S. cerevisiae

Summary

RESULTS

Cloning and Nucleotide Sequence-Yeast mutants lacking individual subunits of NAD+-IDH were previously described (9), and the ZDH2 gene was cloned by complementation of thesemutants (10). The nucleotide sequence of this fragment was determined, and the deduced amino acid sequence contained the tryptic peptides B and C obtainedfrom IDHl (Fig. 1B) andexhibited 43% identity with the aligned amino acid sequence of IDH2. These results strongly suggested that theamplified fragment encodes a portion of the yeast IDHl gene. A partial restriction map of the 1.8-kb BglII fragment and the strategy used for nucleotide sequencing of this region are shown in Fig. lA.An open reading frame was found to encode the amino terminus of the mature IDHl polypeptide (nucleotides 34-72, Fig. 1B).

GTG GCT AAC CCA ACT

Wild type

DISCUSSION