Rat pristanoyl-CoA oxidase. cDNA cloning and recognition of its C-terminal (SQL) by the peroxisomal-targeting signal 1 receptor.

The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5'-RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS-gel electrophoresis. The amino acid identity with rat palmitoyl-CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl-CoA oxidases (20%), suggesting that the palmitoyl-CoA oxidase/pristanoyl-CoA oxidase duplication occurred early in evolution. The carboxy-terminal tripeptide of pristanoyl-CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal-targeting signal-1 import receptor indicated that SQL functions as a peroxisome-targeting signal. Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl-CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl-CoA oxidase gene, if present, is only poorly or not transcribed.

The 80 kDa cytosolic protein that binds the C-terminal part of rat acyl-CoA oxidase is not a peroxisomal import receptor but a prolyl-endopeptidase.

In an attempt to identify putative peroxisomal import receptors, we investigated the cross-linking of a radioiodinated peptide consisting of the 13 last amino acids of acyl-CoA oxidase and comprising the carboxy-terminal SKL-peroxisomal targeting motive, to proteins present in different subcellular fractions from rat liver. The radiolabeled peptide could be cross-linked to an 80 kDa protein present in the cytosol but not to proteins present in other subcellular fractions including highly purified peroxisomes. Binding was reversible, saturable and dependent on the presence of Mg2+ and ATP or GTP but hydrolysis of the nucleotides was not required. Binding was abolished by pretreatment of the cytosol--but not of the peptide--with N-ethylmaleimide. Binding was not specific for peptides containing the carboxy-terminal SKL-motive, since binding was competed for by the SKL-peptide from which the SKL-motive had been deleted, by the SKL-peptide with reversed sequence and by the SV40 T-antigen nuclear localisation signal peptide, but not by other peptides tested. The 80 kDa binding protein cross-reacted with a monoclonal antibody against hsp90. Purification and internal peptide sequencing of the binding protein revealed its identity as prolyl-endopeptidase. In retrospect, we realized that the SKL-peptide and all competing peptides contained a proline residue, which was not present in the non-competing peptides. In recent experiments in yeast McNew et al. (McNew, J.A., Sykes, K. and Goodman, J.M. (1993) Mol. Biol. Cell 4, 223-232) cross-linked a peroxisomal targeting peptide to a 20 kDa cytosolic protein that was identified as proline isomerase despite the fact that the peptide did not contain proline. The experiments by McNew et al. in yeast and our experiments in the rat suggest that the (peroxisomal) targeting sequence cross-linking approach may not be suited for the identification of (peroxisomal) import receptors.

Large-scale purification and further characterization of rat pristanoyl-CoA oxidase.

The elution of pristanoyl-CoA oxidase from butyl-Sepharose required unusually high concentrations of ethylene glycol, enabling the large-scale purification of this oxidase in a single chromatographic step. The enzyme, the native molecular mass of which was estimated previously at 415 kDa by gel filtration (Van Veldhoven, P.P., Vanhove, G., Vanhoutte, F., Dacremont, G., Eyssen, H. J. & Mannaerts, G. P. (1991) J. Biol. Chem. 266, 24676-24683), migrated as a 513-kDa protein during native gel electrophoresis. It showed a typical flavoprotein spectrum and probably binds 4 mol FAD/mol enzyme. Its amino acid composition was different from those of other known acyl-CoA oxidases. Screening of different rat tissues, either for enzyme activity or by immunoblotting, revealed the highest level of pristanoyl-CoA oxidase in liver, followed by kidney, intestinal mucosa, spleen and lung. The oxidase activities, measured with 2-methylpalmitoyl-CoA as the substrate, in livers from other vertebrates including man were low compared to rat. This was also confirmed by immunoblotting which provided a clear signal only in rat liver, possibly indicating that pristanoyl-CoA oxidase might be a rat-specific oxidase.