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 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.
Ito
M
M
Department of Biology, Saga Medical School, Saga, Japan. itohm1@post.saga-med.ac.jp
Ito
R
R
Huang
Y
Y
Miura
S
S
Imamura
A
A
Suzuki
Y
Y
Shimozawa
N
N
D000818:Animals; D016466:CHO Cells; D002374:Catalase / Q000032:analysis; D002459:Cell Fusion; D002469:Cell Separation / Q000379:methods; D006224:Cricetinae; D005347:Fibroblasts; D005456:Fluorescent Dyes; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D018901:Peroxisomal Disorders / Q000145:classification; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011956:Receptors, Cell Surface / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D014162:Transfection
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D008565:Membrane Proteins; C000614264:PEX5 protein, human; C000614269:PEX7 protein, human; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; C409504:blue fluorescent protein, Aequorea victoria; C085380:peroxisomal targeting sequence receptor; D049452:Green Fluorescent Proteins; D002374:Catalase