Two acyl-coenzyme A oxidases in peroxisomes of the yeast Candida tropicalis: primary structures deduced from genomic DNA sequence.

Abstract

We report the complete nucleotide sequence of two genes encoding major peroxisomal polypeptides (PXPs) of Candida tropicalis. One, POX4, encodes PXP-4, which is the most abundant polypeptide in cells grown on oleic acid, and the other, POX5, is the gene for PXP-5. Each of the two polypeptides was found to be the subunit of a distinct long-chain acyl-coenzyme A oxidase: acyl-CoA oxidase II (PXP-4) or acyl-CoA oxidase I (PXP-5). Both the genes had no intron and gave a single open reading frame. The NH2-terminal sequences, except the initiator methionine, and the calculated molecular weights of the deduced polypeptides were consistent with those of the respective PXPs. Well-conserved sequences of 12 and 16 hydrophobic amino acids were present in the middle of the polypeptide, instead of at the NH2 terminus, and may be internal signal sequences for the peroxisomal location of PXPs. Although the two polypeptides were significantly homologous throughout their sequences, the local homologies in two regions out offive were markedly diverged from the average (63%); the homology in the second region was 93%, whereas that in the fourth one was only 24%. The implications of this finding are discussed in respect to the multiplicity of peroxisomal enzymes and the presence of multifunctional proteins in peroxisomes.

A novel peroxisomal nonspecific lipid‐transfer protein from Candida tropicalis

“…Procedures for thc manipulation [5] and sequencing [6] of DNA, DNA [5] and KNA [6] blot hybridization experiments, SDSjPAGE and the staining of proteins [24], and the assay of the protein [lo] with bovine serum albumin as the standard were as described previously. Acyl-CoA oxidase activity was assayed at 25°C as described by Shimizu et al [26].…”

“…Poly(A)-rich RNA was prepared as described previously [5] from cells grown in YPBO medium, composed of 0.3% yeast extract, 0.5% peptone, 0.5% K2HP04, 0.5% KH2P04, 1% Brij 58 and 1% oleic acid, or in YYBG medium, which is similar to YPBO but contains 2% glucose instead of oleic acid. Acyl-CoA oxidase was obtained as the major fraction from Mono Q column chromatography during the isolation of PXP-4 [6]. This fraction was about 20% PXP-5, but it contained no PXP-18.…”

“…Egg yolk phosphatidylethanolamine and phosphatidylcholine were purchased from Green Cross Corp. (Osaka) and acyl-CoAs from Sigma (St. Louis, MO, USA). The sources of the other enzymes and biological materials have been listed elsewhere [5,6,241.…”

“…2) identical to that of the purified PXP-18 (see below) found by Edman degradation : Ser-Val-Glu-Val-Asp-Gly-Phe-Asn-Ala-Ser-ProLeu-Phe-Lys-(Glu/Gln)-. The initiator methionine had been removed from the mature protein as in PXP-2 [7], PXP-4 [6], PXP-5 [6] and PXP-9 [8]. The deduced amino acid sequence of PXP-18 had considerable similarity with that of nsLTP (SCP-2) purified from rat [17] and bovine [19] livers.…”

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.…”