High-level expression and molecular cloning of genes encoding Candida tropicalis peroxisomal proteins.

Abstract

The development of peroxisomes in the cells of Candida tropicalis grown on oleic acid was accompanied by a markedly high expression of peroxisomal proteins. On the basis of this finding, the nuclear DNA library of this yeast was screened by differential hybridization, and 102 clones of oleic acid-inducible sequences were isolated. Seven coding regions were found to form clusters in three stretches of the genomic DNA. Five of the regions were identified as genes for peroxisomal polypeptides (PXPs). The coding sequence for PXP-2 hybrid selected an additional mRNA for PXP-4, the subunit of long-chain acyl coenzyme A oxidase, which was the most abundant PXP. PXP-2 and PXP-4 were close in apparent molecular weight and generated similar peptides when digested with a protease. The gene for PXP-4 was adjacent to that for PXP-2 on the genome and also hybridized to the mRNA coding for PXP-5. These and other similar results suggest that the genes for the peroxisomal proteins of this organism arose by duplication of a few ancestral genes.The enzyme composition of peroxisomes, intracellular compartments generating and degrading hydrogen peroxide, varies widely in contrast to their ubiquitous distribution. The peroxisomes of methanol-assimilating yeasts contain only a few enzymes (10, 40), whereas the equivalents of fatty acidutilizing yeasts have, as in the case of hepatic peroxisomes, more than a dozen enzymes in addition to a n-oxidation system for long-chain fatty acids (1,6,12,20,28,39). In yeast cells, unlike in hepatocytes, fatty acids are oxidized solely in peroxisomes (20), which have a novel long-chain acyl coenzyme A synthetase whose product is exclusively utilized for 3-oxidation but not for lipid synthesis (17,18,28).Candida tropicalis pK233 is a typical yeast strain which assimilates fatty acids or alkanes as the sole source of carbon and energy. The cells grown on glucose contain few peroxisomes, whereas those grown on oleic acid or n-alkanes (C10-C13) develop large numbers of the organelles (30). The peroxisomes of this yeast strain have been purified to near homogeneity (16), and the induction of many associated enzymes has been reported (39). C. tropicalis is thus a suitable unicellular system for studying the biogenesis of peroxisomes and its regulation. In this regard, special attention was directed to reports (29, 31) suggesting the presence of DNA in the peroxisomes of this organism. Extensive searches for the proposed DNA, however, provided no evidence for this and led us to conclude that the biogenesis of peroxisomes must be entirely controlled by nuclear genes (16).We report here that the development of peroxisomes in cells grown on oleic acid is accompanied by a markedly high expression of peroxisomal proteins. On the basis of this finding, we isolated oleic acid-inducible DNA sequences by differential hybridization. At least five oleate-inducible sequences were identified as genes encoding peroxisomal polypeptides (PXPs) and were found to form clusters in the genomic DNA fragments. Genes for the per...

A novel peroxisomal nonspecific lipid‐transfer protein from Candida tropicalis

“…A 4.2-kb BglII-Hind111 fragment of genomic DNA carrying the POX18 gene of C. tropicalis pK233 (ATCC 20336) was obtained from plasmid pC7 [5] and subcloned in pBR322, giving pC7-5. The 1.4-kb BglII-SpeI fragment of pC7 was subcloned in pGEM3 (Promega, Madison, WI, USA), giving pG18.…”

“…Thus, the entire protein molecule must be systematically analysed if the yeast peroxisomal targeting sequence is to be identified. Since a small protein is more convenient for this purpose, we chose a 16-kDa protein, PXP-18, which is encoded by the gene POX18 and induced markedly by oleic acid [5]. Its function is not known.…”

“…The targeting sequences of five peroxisomal proteins from higher eucaryotes have been found at the carboxy-terminus and shown to contain a tripeptide, Ser-Lys/His-Leu, at or near the end of the sequence [2 -41. We have cloned genes encoding peroxisomal proteins of the yeast Candida tropicalis [5] and sequenced three genes for the subunits (PXP-2, PXP-4 and PXP-5) of acylCoA oxidase isozymes [6, 71 and the gene for catalase (PXP-9) [8]. None of the polypeptides encoded by these genes has the Ser-Lys/His-Leu sequence in their carboxy-terminal regions.…”

Catalase gene of the yeast Candida tropicalis

“…2, underlined). The molecular mass of 54937 Da, calculated from this amino acid sequence, coincided well with the value (54 kDa) on the sodium dodecylsulfateipolyacrylamide slab gel electrophore-RESULTS AND DISCUSSION Identification and sequencing of genomic DNA f o r catalase of C. tropicalis A genomic DNA recombinant library prepared from C. tropicatis was screened for the gene encoding PXP-9, which seemed to be a catalase protein, by the procedure applied to other PXPs [16]; that is, by means of cell-free translation of hybrid-selected mRNA and peptide mapping of the translation product. The gene, POX9, was confirmed as the catalase gene by blot-hybridization analysis of this genomic DNA and the cDNA probe for catalase obtained previously [17].…”

Interspecific complementation analysis by protoplast fusion of Candida tropicalis and Candida albicans adenine auxotrophs

“…The synthesis of peroxisomes in C. tropicalis is induced manyfold by growth on these substrates as sole carbon and energy sources (11). Recently, several genes encoding C. tropicalis peroxisomal proteins have been cloned into Escherichia coli (5,10,14,19). The development of genetic systems appropriate to C. tropicalis may be of value in elucidating mechanisms of pathogenesis, the basis of drug resistance, and the induction and targeting of peroxisomal proteins.…”