Peroxisomal and mitochondrial carnitine acetyltransferase isozymes of the n-alkane-assimilating yeast, Candida tropicalis, occurred by alternative initiation of translation from the transcripts of a single gene.
Carnitine acetyltransferase (CAT; EC 2.3.1.7) is localized in two subcellular organelles, peroxisomes and mitochondria, in an n-alkane-assimilating yeast, Candida tropicalis. The isozymes are synthesized from the first and second ATG codon of the open reading frame of one gene, CtCAT. Primer extension analysis and RNase protection assay (RPA) revealed that multiple transcription initiation sites were found upstream of the first ATG codon. 5' ends could not be detected between the first and second ATG codons. These results suggested that the peroxisomal CAT of C. tropicalis, initiating at the second AUG codon of the transcripts, was synthesized by a translational readthrough of the first AUG codon of the open reading frame. When CtCAT was introduced into the other yeast, Saccharomyces cerevisiae, 5' ends of transcripts and the protein products were similar to those observed in C. tropicalis. This suggested that the transcripts harbored sufficient information to bring about alternative initiation of translation in both yeasts. Using S. cerevisiae as the host cell, introduction of mutations into the sequence near the first AUG codon or a deletion in the region between the first and second AUG codons resulted in an increased ratio of translation from the first AUG codon, although initiation sites of transcription did not change. Moreover, replacing the 5' leader sequence to that of C. tropicalis isocitrate lyase promoter (UPR-ICL) eliminated the product initiating at the second AUG codon. The transcript from these cells was shorter than those detected from the native CtCAT-harboring cells. From these results, it was strongly suggested that peroxisomal and mitochondrial CAT isozymes occurred by the alternative initiation of translation mainly dependent on the structure and sequence context of the region from the 5' end to the second AUG codon, and not the insufficient length of the 5' leader.
Ueda
M
M
Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
Kawachi
H
H
Atomi
H
H
Tanaka
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D002175:Candida / Q000201:enzymology*; D002332:Carnitine O-Acetyltransferase / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics*; D008830:Microbodies / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic
D007527:Isoenzymes; D012333:RNA, Messenger; D002332:Carnitine O-Acetyltransferase