Isolation of Chinese hamster ovary cell pex mutants: two PEX7-defective mutants.
We searched for novel Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis by an improved method using peroxisome targeting signal 2 (PTS2)-tagged enhanced green fluorescent protein (EGFP). From mutagenized TKaEG2 cells, the wild-type CHO-K1 stably expressing rat Pex2p and PTS2-EGFP, cell colonies resistant to the 9-(1(')-pyrene)nonanol/ultraviolet treatment were examined for intracellular location of PTS2-EGFP. Of six mutant cell clones two, ZPEG227 and ZPEG231, showed cytosolic PTS2-EGFP, indicative of impaired PTS2 import, and numerous PTS1-positive particles. PEX7 expression restored the impaired PTS2 import in both mutants. Cell fusion with fibroblasts from a patient with PEX7-defective rhizomelic chondrodysplasia punctata did not complement PTS2 import defect of ZPEG227 and ZPEG231, confirming that these two are pex7 mutants. Mutation analysis of PEX7 by reverse transriptase (RT)-PCR indicated that ZPEG227-allele carried an inactivating nonsense mutation, Trp158Ter. Therefore, ZPEG227 is a pex7 mutant possessing a newly identified mutation in mammalian pex7 cell lines.
Yanago
Eiko
E
Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Fukuoka 812-8581, Japan.
Hiromasa
Takahide
T
Matsumura
Tsuyoshi
T
Kinoshita
Naohiko
N
Fujiki
Yukio
Y
D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002459:Cell Fusion; D002470:Cell Survival / Q000528:radiation effects; D002806:Chondrodysplasia Punctata / Q000235:genetics; D006224:Cricetinae; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis; D008830:Microbodies / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation; D020125:Mutation, Missense; D053402:PHEX Phosphate Regulating Neutral Endopeptidase; D000074437:Peroxisomal Targeting Signal 2 Receptor; D011506:Proteins / Q000235:genetics*; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection; D014466:Ultraviolet Rays; D015211:Zellweger Syndrome / Q000235:genetics
D008164:Luminescent Proteins; C000614269:PEX7 protein, human; D000074437:Peroxisomal Targeting Signal 2 Receptor; D011506:Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D053402:PHEX Phosphate Regulating Neutral Endopeptidase; C506231:PHEX protein, human; C506229:Phex protein, rat