Isolation, nucleotide sequence, and disruption of the Saccharomyces cerevisiae gene encoding mitochondrial NADP(H)-specific isocitrate dehydrogenase

Abstract

Mitochondrial NADP(H)-specific isocitrate dehydrogenase (IDP1) was purified from yeast cells grown with acetate as a carbon source. IDP1 was shown to be a dimer with a subunit molecular weight of approximately 45,000. Immunochemical levels of IDP1 were found to vary in inverse proportion with those of mitochondrial NAD(H)-specific isocitrate dehydrogenase in cells grown with glucose or with acetate as a carbon source. A 20-residue amino-terminal sequence was obtained for IDP1, and degenerate oligonucleotides were used to synthesize a 50-base pair polymerase chain reaction product corresponding to the coding region for a portion of the amino terminus. The 50-base pair DNA fragment was used as a hybridization probe to identify plasmids containing the IDP1 gene in a yeast genomic DNA library. The complete nucleotide sequence of the IDP1 coding region was determined and translated into a 412-residue amino acid sequence for the mature protein which is preceded by a putative 16-residue mitochondrial targeting presequence. A haploid yeast strain containing a chromosomal disruption of the IDP1 locus was constructed and found to be capable of growth with glucose but not with other carbon sources, suggesting that IDP1 provides a critical function and may be the primary source of NADPH in yeast mitochondria.