Peroxisomal and Mitochondrial Carnitine Acetyltransferases of the n‐Alkane‐Assimilating Yeast Candida tropicalis

Abstract

A genomic DNA clone encoding carnitine acetyltransferases (EC 2.3.1.7), localized in two subcellular organelles, peroxisomes and mitochondria of an n-alkane-assimilating yeast Candida tropicalis, was isolated from the yeast AEMBL library using a carnitine acetyltransferase cDNA probe. Nucleotide sequence analysis disclosed that the open reading frame was 1881 bp, corresponding to 627 amino acids with a molecular mass of 70760 Da. Comparison of the predicted amino acid sequence of the C. tropicalis enzyme with that of Saccharonzyces cerevisiae mitochondrial matrix carnitine acetyltransferase revealed 46.3 % identity. It was noticeable that the C. tropicalis enzymes had amino acid sequences similar to both proposed mitochondrial and peroxisomal targeting signals. When the C. tropicalis gene was expressed in S. cerevisiae using its own 5'-upstream region, a 12-fold increase in activity was observed. Western blot analysis revealed the presence of two major proteins whose sizes corresponded to the peroxisomal and mitochondrial proteins detected in C. tropicalis. This suggested that peroxisomal and mitochondrial carnitine acetyltransferases were encoded by one gene, as suggested for the S. cerevisiae enzyme. Furthermore, we have separated and purified these enzymes from peroxisomes and mitochondria of C. tropicalis, and analyzed the amino-terminal amino acid sequences of each. The amino-terminal sequence of the mitochondrial enzyme suggested that a signal sequence had been cleaved during translocation into mitochondria. Concerning the peroxisomal enzyme, the evidence obtained indicated that in vivo the translation was initiated at the second methionine of the open reading frame.

Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis : cloning and expression in Saccharomyces cerevisiae

“…Gaps have been inserted to achieve the maximum similarity the heterologous yeast S. cerevisiae, indicating that the complete splicing of the intron in CtCIT was performed. These results together with the facts that several promoters of C. tropicalis genes are functional in S. cerevisiae (Umemura et al 1995;Kawachi et al 1996a;Murai et al 1996) and that high expression of C. tropicalis genes can be observed in S. cerevisiae (Kanai et al 1996;Kawachi et al 1996b) reflect a genetic relationship between S. cerevisiae and C. tropicalis.…”

Candida albicans CTN gene family is induced during macrophage infection: homology, disruption and phenotypic analysis of CTN3 gene

“…Cat2p is either mitochondrial or peroxisomal, depending on the differential splicing of the CAT2 gene. Translational initiation at the second splicing site, leading to the loss of the mitochondrial targeting signal (MTS), results in peroxisomal targeting (Elgersma et al, 1995;Kawachi et al, 1996;Stemple et al, 1998). In addition to the MTS, the protein also contains, in its C-terminal sequence, a tripeptide motif (PTS1) responsible for its peroxisomal import via interaction with the Pex5p protein (see for review Hettema et al).…”

A Single Gene Produces Mitochondrial, Cytoplasmic, and Peroxisomal NADP-dependent Isocitrate Dehydrogenase inAspergillus nidulans

“…Two other genes encode cytoplasmic enzymes lacking either signal (67,68). In C. tropicalis, a single gene also determines two forms of carnitine acetyltransferase but in this case the peroxisomal enzyme results from alternative initiation of translation (69,70). A similar situation for genes encoding carnitine acetyltransferase enzymes is likely in A. nidulans (57).…”