Isolation, Characterization, and Disruption of the Yeast Gene Encoding Cytosolic NADP-Specific Isocitrate Dehydrogenase

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Abstract
The cytosolic isozyme of NADP-specific isocitrate dehydrogenase (IDP2) was purified from a Saccharomyces cerevisiae mutant containing a chromosomal disruption in the gene encoding the mitochondrial isozyme (IDP1). IDP2 was shown to be a homodimer with a subunit molecular weight of approximately 45,000 and an isoelectric point of 5.5. Amino acid sequences were obtained for tryptic peptides of IDP2 and used to plan polymerase chain reactions. A resulting 400 bp DNA fragment was used as a hybridization probe to isolate the IDP2 gene from a yeast genomic DNA library. The complete nucleotide sequence of the IDP2 coding region was determined and translated into a 412-residue amino acid sequence. IDP2 and IDP1 were found to be identical in 71% of the aligned residue positions. The identity of the IDP2 gene was confirmed by genomic replacement with a disrupted IDP2 coding region. Haploid yeast strains lacking either or both IDP2 and IDP1 were constructed by genetic crosses of mutant strains containing disruptions in chromosomal IDP2 and IDP1 loci. No dramatic differences in growth rates with common carbon sources could be attributed to these disruptions.