Rapid isolation and characterization of CHO mutants deficient in peroxisome biogenesis using the peroxisomal forms of fluorescent proteins

Abstract

We isolated and characterized CHO mutants deficient in peroxisome assembly using green fluorescent protein (GFP) and blue fluorescent protein (BFP) as the fluorescent probes to study the molecular mechanism of peroxisome biogenesis. We used stable transformants of CHO cells expressing GFP appending peroxisome targeting signal-1 (PTS1) and/or peroxisome targeting signal-2 (PTS2) as the parent strains for rapid isolation of the mutants. We have obtained six peroxisome-deficient mutants by visual screening of the mislocalizations of the peroxisomal GFPs. Mutual cell fusion experiments indicated that the six mutants isolated were divided into four complementation groups. Several of the mutants obtained possessed defective genes: the PEX2 gene was defective in SK24 and PT54; the PEX5 gene in SK32 and the PEX7 gene in PT13 and PT32. BE41, which belonged to the fourth complementation group, was not determined. When peroxisomal forms of BFP were transiently expressed in mutant cells, the peroxisomal BFPs appending both PTS1 and PTS2 appeared to bypass either the PTS1 or PTS2 pathway for localization in SK32. This observation suggested that other important machinery, in addition to the PTS1 or PTS2 pathway, could be involved in peroxisome biogenesis. Thus, our approach using peroxisomal fluorescent proteins could facilitate the isolation and analysis of peroxisome-deficient CHO mutants and benefit studies on the identification and role of the genes responsible for peroxisome biogenesis.

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

“…The two isoforms (PTS1RL and PTS1RS) are produced by an alternative splicing from PEX5 gene and the PTS1RL isoform is supposed to be involved in both PTS1 and PTS2 pathways (16,17). The cytosolic mislocalization of BFP appending PTS2 therefore seemed to be plausible in SK32 cells (14). In this study, we report that SK32 mutant cells show TS phenotype on the assembly pathway of both PTS1 and PTS2 proteins.…”

“…CHO-K1 expressing a peroxisomal form of GFP appending SKL sequence at the C-terminus was used as a wild-type cell line and cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and 0.4 mg/ml Hygromycin B. SK32 mutant cell line was isolated from the parent cell line as described (14).…”

“…Each plasmid was then transfected into SK32 mutant cells to examine the functional loss of PEX5 gene. The transfection was carried out by a liposome-mediated procedure as described (14). A recombinant cDNA encoding for rat urate oxidase, in which arginine was replaced by Lysine at the penultimate residue (19), was constructed into a eukaryotic expression vector pOPI3 (Stratagene).…”

“…A mutant strain SK32 exhibited a characteristic phenotype, cytosolic mislocalization of blue fluorescent protein (BFP) appending either PTS1 or PTS2, and peroxisomal localization of BFP appending both PTS1 and PTS2 (14). Complementation analysis with human PBD patient fibroblasts suggested that SK32 was defective in PEX5 gene (14).…”

“…Complementation analysis with human PBD patient fibroblasts suggested that SK32 was defective in PEX5 gene (14). PEX5 gene encodes a recep-tor for PTS1 proteins (referred to as PTS1R), with which the proteins are delivered to peroxisomes (15).…”