Immunochemically distinct NADP-linked isocitrate dehydrogenase isozymes in mitochondria and peroxisomes of Candida tropicalis.
Although peroxisomal localization of NADP-linked isocitrate dehydrogenase (Idp) was first demonstrated in Candida tropicalis, the mitochondrial isozyme has not been found in this yeast. Here we report that the presence of mitochondrial Idp in the yeast was demonstrated by screening for its gene with a DNA probe containing conserved sequences of Idps from various organisms. The nucleotide sequence of the gene (CtIDP1) revealed a 1,290-bp open reading frame corresponding to a 430-amino-acid protein with a high similarity to previously reported Idps. Overexpression of CtIDP1 in Saccharomyces cerevisiae gave a high intracellular Idp activity, and the purified recombinant Idp was shown to be a homodimer with a subunit molecular mass of approximately 44 kDa, different from that of peroxisomal Idp (45 kDa) previously purified from C. tropicalis. Western blot analysis of the subcellular fractions from acetate-grown C. tropicalis with polyclonal antibodies raised against the recombinant CtIdp1 showed that the CtIdp1 in C. tropicalis was localized in mitochondria but not in peroxisomes. Similar levels of CtIDP1 mRNA and its protein product were detected in cells grown on glucose, acetate, and n-alkane, although a slight decrease was observed in n-alkane-grown cells. From these results, CtIdp1 was demonstrated to be mitochondrial Idp. The properties of mitochondrial Idp and peroxisomal Idp isozymes were proven to be similar, but they were immunochemically distinct, suggesting the presence of another gene responsible for peroxisomal Idp in C. tropicalis.
Imajo
T
T
Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-01, Japan.
Kawachi
H
H
Atomi
H
H
Sanuki
S
S
Yamamoto
S
S
Ueda
M
M
Tanaka
A
A
D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D019976:Cloning, Organism; D015870:Gene Expression; D005800:Genes, Fungal / Q000235:genetics; D007521:Isocitrate Dehydrogenase / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D007527:Isoenzymes / Q000032:analysis* / Q000235:genetics; D008830:Microbodies / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)