Abstract
NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 (Mr approximately 40,000) and IDH2 (Mr approximately 39,000). We have isolated and characterized a yeast genomic clone containing the IDH2 gene. The amino acid sequence deduced from the gene indicates that IDH2 is synthesized as a precursor of 369 amino acids (Mr 39,694) and is processed upon mitochondrial import to yield a mature protein of 354 amino acids (Mr 37,755). Amino acid sequence comparison between S. cerevisiae IDH2 and S. cerevisiae NADP(+)-dependent isocitrate dehydrogenase shows no significant sequence identity, whereas comparison of IDH2 and Escherichia coli NADP(+)-dependent isocitrate dehydrogenase reveals a 33% sequence identity. To confirm the identity of the IDH2 gene and examine the relationship between IDH1 and IDH2, the IDH2 gene was disrupted by genomic replacement in a haploid yeast strain. The disruption strain expressed no detectable IDH2, as determined by Western blot analysis, and was found to lack NAD(+)-dependent isocitrate dehydrogenase activity, indicating that IDH2 is essential for a functional enzyme. Overexpression of IDH2, however, did not result in increased NAD(+)-dependent isocitrate dehydrogenase activity, suggesting that both IDH1 and IDH2 subunits are required for catalytic activity. The disruption strain was unable to utilize acetate as a carbon source and exhibited a 2-fold slower growth rate than wild type strains on glycerol or lactate. This growth phenotype is consistent with NAD(+)-dependent isocitrate dehydrogenase performing an essential role in the oxidative function of the citric acid cycle.
Highlights
Saccharomyces cerevisiae is composed of two noni- Characterization of purified NAD+-IDH hasshown it tobe a dentical subunits, designated IDHl (Mr-40,000) and IDHP (Mr -39,000)
The amino acid sequence deduced from the gene indicates that IDHP is synthesized as a precursor of 369 amino acids (MI39,694)and is processed upon mitochondrial import to yield a mature protein of 354 amino acids (Mr 37,755)
To confirm the identity of the IDH2 gene and examine the relationship between IDHl and IDHB, the IDH2 gene was disrupted by genomic replacement in complex oligomeric enzyme that is subject to extensive allosteric regulation
Summary
Western Blot Analysis-Samples of mitochondrial protein extracts (20 pg each) for Western blot analysis were electrophoresed on 10%. Yeast Strains and GrowthConditions-Yeast strains lacking a polyacrylamide-sodiumdodecyl sulfate gels [23]. Separated proteins functional NAD+-IDHwere previously identified [5] from screens of were transferred to polyvinylidenemembranes (Immobilon,Millipore mutants that failed to grow on acetate as a carbon source (provided Corp.) using a graphite dry blotting apparatus [24]. Complementation experiments were perfomed using albumin, and incubated in a 1:300 dilution of anti-IDH antiserum the NAD+-IDHmutants designated idhl-1 and idh2-I. Detection was performed using ‘2SI-labeledprotein-A followed by were previously characterized and shown to lack the 40,000 and 39,000 autoradiography. The AZDHZ strain was constructed using the wild type haploid yeast strain S173-6B
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