PEROXISOMAL AND MITOCHONDRIAL CARNITINE ACETYLTRANSFERASE ISOZYMES OFTHE 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 subc ellular organelles, peroxisomes and mitochondria, in an n-alkane-assim ilating yeast, Candida tropicalis. The isozymes are synthesized from t he first and second ATG codon of the open reading frame of one gene, C tCAT. Primer extension analysis and RNase protection assay (RPA) revea led that multiple transcription initiation sites were found upstream o f the first ATG codon. 5' ends could not be detected between the first and second ATG codons. These results suggested that the peroxisomal C AT of C. tropicalis, initiating at the second AUG codon of the transcr ipts, was synthesized by a translational readthrough of the first AUG codon of the open reading frame. When CtCAT was introduced into the ot her yeast, Saccharomyces cerevisiae, 5' ends of transcripts and the pr otein products were similar to those observed in C. tropicalis. This s uggested 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 sequen ce near the first AUG codon or a deletion in the region between the fi rst and second AUG codons resulted in an increased ratio of translatio n 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 prod uct initiating at the second AUG codon. The transcript from these cell s was shorter than those detected from the native CtCAT-harboring cell s. From these results. it was strongly suggested that peroxisomal and mitochondrial CAT isozymes occurred by the alternative initiation of t ranslation mainly dependent on the structure and sequence context of t he region from the 5' end to the second AUG codon, and not the insuffi cient length of the 5' leader. (C) 1998 Elsevier Science B.V.