Temperature-Sensitive Phenotype of Chinese Hamster Ovary Cells Defective in PEX5 Gene

Abstract

SK32 mutant cells, which were isolated as peroxisome-deficient Chinese hamster ovary (CHO) cells by an advantage of a visible peroxisome form of green fluorescent protein (GFP), were found to suffer from a functional loss of PEX5 gene encoding for PTS1R. The sequence analysis of cDNA indicated that PEX5 gene encoded for the two isoforms composed of 603 amino acids (PTS1RS) and 640 amino acids (PTS1RL). The mutation changed glycine to arginine at amino acid position 343 of PTS1RL (corresponding to the position 306 of PTS1RS) in SK32 cells. The mutant cells exhibited a temperature-sensitive (TS) phenotype on the peroxisomal localizations of the recombinant GFP and urate oxidase appending a genuine peroxisome targeting signal 1 (PTS1), a tripeptide of Ser-Lys-Leu (SKL) at the C-terminus, but did not on that of catalase harboring a divergent PTS1, Lys-Ala-Asn-Leu (KANL) sequence. 3-ketoacyl-CoA thiolase (hereafter referred to as thiolase), which harbors an extension sequence (PTS2) at the N-terminus, never appeared to be affected on the peroxisomal localization in the mutant cells. When thiolase was examined on the molecular size in the mutant cells, the enzyme existed as the larger precursor form in the peroxisomes at 37°C and a considerable part (almost half) was converted to the mature size at 30°C. These results indicate that the amino acid substitution, Gly306Arg in PTS1RS and/or Gly343Arg in PTSRL, gives rise to TS phenotype on the peroxisomal translocation of PTS1 proteins and the maturation of PTS2 protein.