© 2005 The Japanese Biochemical Society.
Regular Paper |
Identification of Pex5pM, and Retarded Maturation of 3-Ketoacyl-CoA Thiolase and Acyl-CoA Oxidase in CHO Cells Expressing Mutant Pex5p Isoforms
1 Division of Molecular Cell Biology, Saga University Faculty of Medicine, Nabeshima 5-1-1, Saga 849-8501; 2 Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-0194; 3 Division of Genomic Research, Life Science Research Center, Gifu University, Yanagido 1-1, Gifu 500-8076; and 4 Department of Anatomy, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192
* To whom correspondence should be addressed. Tel/Fax: +81-76-434-7545, E-mail: imanaka{at}ms.toyama-mpu.ac.jp
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°C and only slightly discernible at 30°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°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°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.