A single gene produces mitochondrial, cytoplasmic, and peroxisomal NADP-dependent isocitrate dehydrogenase in Aspergillus nidulans

NADP-dependent isocitrate dehydrogenase enzymes catalyze the decarboxylatio n of isocitrate to 2-oxoglutarate accompanied by the production of NADPH. I n mammals two different genes encode mitochondrial and cytoplasmic/peroxiso mal located enzymes, whereas in Saccharomyces cerevisiae three separate gen es specify compartment specific enzymes. We have identified a single gene, idpA, in the filamentous fungus Aspergillus nidulans that specifies a prote in with a high degree of identity to mammalian and S. cerevisiae enzymes. N orthern blot analysis and reverse transcription-polymerase chain reaction r evealed the presence of two idpA transcripts and two transcription start po ints were identified by sequencing cDNA clones and by 5'-rapid amplificatio n of cDNA ends. The shorter transcript was found to be inducible by acetate and by fatty acids while the longer transcript was present in higher amoun ts during growth in glucose containing media. The longer transcript is pred icted to encode a polypeptide containing an N-terminal mitochondrial target ing sequence as well as a C-terminal tripeptide (ARL) as a potential peroxi somal targeting signal. The shorter transcript is predicted to encode a pol ypeptide lacking the mitochondrial targeting signal but retaining the C-ter minal sequence. Immunoblotting using antibody raised against S. cerevisiae Idp1p detected two polypeptides consistent with these predictions. The func tions of the predicted targeting sequences were confirmed by microscopic an alysis of transformants containing fluorescent protein fusion constructs. U sing anti-Idp1p antibodies, protein localization to mitochondria and peroxi somes was observed during growth on glucose whereas cytoplasmic and peroxis omal localization was found upon acetate or fatty acid induction. Therefore , we have established that by the use of two transcription start points a s ingle gene is sufficient to specify localization of NADP-dependent isocitra te dehydrogenase to three different cellular compartments in A. nidulans.