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

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 biog enesis. 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 obt ained six peroxisome-deficient mutants by visual screening of the mislocali zations of the peroxisomal GFPs. Mutual cell fusion experiments indicated t hat the six mutants isolated were divided into four complementation groups. Several of the mutants obtained possessed defective genes: the PEX2 gene w as defective in SK24 and PT54; the PEX5 gene in SK32 and the PEX7 gene in P T13 and PT32. BE41, which belonged to the fourth complementation group, was not determined. When peroxisomal forms of BFP were transiently expressed i n mutant cells, the peroxisomal BFPs appending both PTS1 and PTS2 appeared to bypass either the PTS1 or PTS2 pathway for localization in SK32. This ob servation suggested that other important machinery, in addition to the PTS1 or PTS2 pathway, could be involved in peroxisome biogenesis. Thus, our app roach using peroxisomal fluorescent proteins could facilitate the isolation and analysis of peroxisome-deficient CHO mutants and benefit studies on th e identification and role of the genes responsible for peroxisome biogenesi s. (C) 2000 Elsevier Science B.V. All rights reserved.