Identification of Pex5pM, and Retarded Maturation of 3-Ketoacyl-CoA Thiolase and Acyl-CoA Oxidase in CHO Cells Expressing Mutant Pex5p Isoforms

Abstract

Recently, we isolated CHO cells, termed SK32 cells, that express mutant Pex5p (G432R), and showed mislocalization of catalase in the cytosol, but peroxisomal localization of 3-ketoacyl-CoA thiolase (thiolase) in the mutant cells [Ito, R. et al. (2001) Biochem. Biophys. Res. Commun. 288, 321-327]. While analyzing the mutant cells, we found a novel Pex5p isoform (Pex5pM), which was shorter by seven amino acids than Pex5pL and longer by 30 amino acids than Pex5pS. Similar levels of mRNA syntheses for the PEX5 gene were observed in both the wild type and mutant cells, but the protein levels of Pex5p isoforms were markedly reduced in the mutant cells cultured at 37 degrees C and only slightly discernible at 30 degrees C, suggesting that they could be rapidly degraded. Furthermore, we characterized the peroxisomal localization of thiolase and acyl-CoA oxidase (Aox) in SK32 cells. The proteins in the organelle fraction were protected from proteinase K-digestion in the mutant cells, indicating that they were translocated inside peroxisomes. However, the conversion of Aox from component A to components B and C was completely prevented at both 30 and 37 degrees C, and the precursor form of thiolase was partially processed to the mature one in a temperature-sensitive manner. Transformed SK32 cells stably expressing one of the wild type Pex5p isoforms were isolated, and then the maturation steps for thiolase and Aox were examined. Pex5pM and S restored the processing of the two enzymes, but Pex5pL did not. In addition, Pex5pL prevented the maturation of thiolase observed at 30 degrees C. These results indicate that (i) the novel Pex5pM is functional and (ii) a seven amino acids-insertion, which is present in the L isoform but absent in the M isoform, plays some role in the process of maturation of thiolase and Aox.

Novel peroxisomal protease Tysnd1 processes PTS1- and PTS2-containing enzymes involved in β-oxidation of fatty acids

“…Of note is the observation that a large fraction of Acox1 and Scp2 was not processed in the peroxisomes (e.g. Otera et al , 2001), whereas thiolase was fully converted to its mature form inside the organelle (Ito et al , 2005). CHO cells, termed SK32, that express mutant PTS1 receptor Pex5p (G432R), were reported to differentially process thiolase and Acox1 (Ito et al , 2005).…”

“…Otera et al , 2001), whereas thiolase was fully converted to its mature form inside the organelle (Ito et al , 2005). CHO cells, termed SK32, that express mutant PTS1 receptor Pex5p (G432R), were reported to differentially process thiolase and Acox1 (Ito et al , 2005). Whereas at 37°C the peroxisomal processing of thiolase was completely prevented, it was partially restored at 30°C.…”

“…The conversion of Acox1 in the peroxisomes was completely prevented at both temperatures. Forced expression of wild‐type Pex5p in the mutant cells restored the normal processing of both peroxisomal proteins (Ito et al , 2005). Hence, it is likely that Acox1 becomes more susceptible to the cleavage by Tysnd1 when it forms a complex with Pex5p and/or other components of the translocation machinery.…”