Isolation and characterization of novel phenotype CHO cell mutants defective in peroxisome assembly, using ICR191 as a potent mutagenic agent.
To elucidate molecular and cellular mechanisms of peroxisome biogenesis, we have isolated Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis by making use of enhanced green fluorescent protein (EGFP) and a frameshift-inducing mutagen ICR191. CHO-TKa cells stably expressing Pex2p were transformed with a cDNA encoding EGFP fused with peroxisomal targeting signal type 2 (PTS2-EGFP), termed Tka/EG2. TKa/EG2 cells were mutagenized with ICR191 and cultured in the presence of P9OH (9-(1'-pyrene) nonanol) followed by an exposure to UV. P9OH/UV-resistant and morphologically peroxisome-deficient mutant cells were isolated by directly observing cytosolic localization of EGFP, without cell staining. By a combination of cell-fusion and PEX transfection, we determined complementation groups (CGs) of 16 cell mutants isolated here. The mutants were classified into five CGs, including pex2, pex3, pex5, pex6, and pex7 cell mutants. In contrast to typical pex6 mutants with the impaired import of both PTS1- and PTS2-proteins, two clones, ZPEG236 and ZPEG244, showed a distinct, novel phenotype where PTS1-protein import was normal despite the abrogated PTS2 import. Dysfunction of Pex3p in pex3 ZPEG 238 was due to one base (G) insertion in the codon for Asn7 resulting in a frameshift, thereby inducing a distinct 31 amino-acid sequence and a termination. pex2 ZPEG239 showed a mutation in codon GAG for Glu(201) to a nonsense mutation, TAG. Thus, the method developed here using ICR191 could be useful for isolation of further novel cell mutants impaired in peroxisome biogenesis.
Ghaedi
Kamran
K
Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Fukuoka, Japan.
Fujiki
Yukio
Y
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000235:genetics; D000585:Aminacrine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000633:toxicity; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism; D004252:DNA Mutational Analysis; D016368:Frameshift Mutation; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D009153:Mutagens / Q000737:chemistry / Q000633:toxicity*; D009154:Mutation; D009588:Nitrogen Mustard Compounds / Q000737:chemistry / Q000633:toxicity*; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000187:drug effects* / Q000378:metabolism; D017354:Point Mutation; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D008565:Membrane Proteins; D009153:Mutagens; D009588:Nitrogen Mustard Compounds; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000585:Aminacrine; C004294:acridine half-mustard; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; C096998:Pex6 protein, rat