A single gene produces mitochondrial, cytoplasmic, and peroxisomal NADP-dependent isocitrate dehydrogenase in Aspergillus nidulans.
NADP-dependent isocitrate dehydrogenase enzymes catalyze the decarboxylation of isocitrate to 2-oxoglutarate accompanied by the production of NADPH. In mammals two different genes encode mitochondrial and cytoplasmic/peroxisomal located enzymes, whereas in Saccharomyces cerevisiae three separate genes specify compartment specific enzymes. We have identified a single gene, idpA, in the filamentous fungus Aspergillus nidulans that specifies a protein with a high degree of identity to mammalian and S. cerevisiae enzymes. Northern blot analysis and reverse transcription-polymerase chain reaction revealed the presence of two idpA transcripts and two transcription start points were identified by sequencing cDNA clones and by 5'-rapid amplification 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 amounts during growth in glucose containing media. The longer transcript is predicted to encode a polypeptide containing an N-terminal mitochondrial targeting sequence as well as a C-terminal tripeptide (ARL) as a potential peroxisomal targeting signal. The shorter transcript is predicted to encode a polypeptide lacking the mitochondrial targeting signal but retaining the C-terminal sequence. Immunoblotting using antibody raised against S. cerevisiae Idp1p detected two polypeptides consistent with these predictions. The functions of the predicted targeting sequences were confirmed by microscopic analysis of transformants containing fluorescent protein fusion constructs. Using anti-Idp1p antibodies, protein localization to mitochondria and peroxisomes was observed during growth on glucose whereas cytoplasmic and peroxisomal localization was found upon acetate or fatty acid induction. Therefore, we have established that by the use of two transcription start points a single gene is sufficient to specify localization of NADP-dependent isocitrate dehydrogenase to three different cellular compartments in A. nidulans.
Szewczyk
E
E
Department of Genetics, University of Melbourne, Parkville, Victoria 3010, Australia.
Andrianopoulos
A
A
Davis
M A
MA
Hynes
M J
MJ
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D020224:Expressed Sequence Tags; D007521:Isocitrate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D020675:Peroxisomes / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013347:Subcellular Fractions
D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)