RESUMEN
We made use of the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) method and isolated peroxisome-deficient mutant cells. TKa cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNA encoding Pex2p (formerly peroxisome assembly factor-1, PAF-1) were used to avoid frequent isolation of the Z65-type, PEX2-defective mutants. P9OH/UV-resistant cell colonies were examined for the intracellular location of catalase, a peroxisomal matrix enzyme, by immunofluorescence microscopy and using anti-catalase antibody. As six mutant cell clones showed cytosolic catalase, there was likely to be a deficiency in peroxisome assembly. These mutants also showed the typical peroxisome assembly-defective phenotype, including significant decrease of dihydroxyacetonephosphate acyltransferase, the first step key enzyme in plasmalogen synthesis, and loss of resistance to 12-(1'-pyrene)dodecanoic acid/UV treatment. By transfection of Pex2p and Pex6p (formerly PAF-2) cDNAs and cell fusion analysis between the CHO cell mutants, two mutants, ZP104 and ZP109, were found to belong to a novel complementation group. Further complementation analysis using fibroblasts from patients with peroxisome biogenesis disorders revealed that the mutants belonged to human complementation group III. Taken together, ZP104 and ZP109 are in a newly identified fifth complementation group in CHO mutants reported to date and represent the human complementation group III.
Asunto(s)
Adenosina Trifosfatasas/genética , Microcuerpos/genética , Mutación , Trastorno Peroxisomal/genética , Síndrome de Zellweger/genética , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Células CHO , Cricetinae , Resistencia a Medicamentos , Prueba de Complementación Genética , Humanos , Ácidos Láuricos/farmacología , Trastorno Peroxisomal/clasificación , Síndrome de Zellweger/clasificaciónRESUMEN
For the study of mechanism of peroxisome biogenesis, we attempted to isolate CHO cell mutants deficient in peroxisome biogenesis. We used as the parent strain a stable CHO transformant of rat PEX2 (formerly named peroxisome assembly factor-1) cDNA, to avoid unusually frequent isolation of Pex2 mutants. Among the three peroxisome-deficient mutants obtained, ZP102 was a new CHO mutant of complementation group 2, and was restored for peroxisome assembly by the transfection of human PEX5 (formerly called PXR1 or PTS1R) cDNA. This approach would facilitate the isolation of new complementation gorups of peroxisome-deficient CHO mutants and the identification of essential genes for peroxisome biogenesis.
Asunto(s)
Proteínas de la Membrana/biosíntesis , Microcuerpos/fisiología , Receptores Citoplasmáticos y Nucleares/biosíntesis , Animales , Células CHO , Catalasa/metabolismo , Fusión Celular , Cricetinae , Cartilla de ADN , Digitonina/farmacología , Prueba de Complementación Genética , Humanos , Microcuerpos/ultraestructura , Mutación , Factor 2 de la Biogénesis del Peroxisoma , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Reacción en Cadena de la Polimerasa , Ratas , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes/biosíntesis , TransfecciónRESUMEN
Rat peroxisome assembly factor-2 (PAF-2) cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP92, using transient transfection assay. This cDNA encodes a 978-amino acid protein with two putative ATP-binding sites. PAF-2 is a member of a putative ATPase family, including two yeast gene products essential for peroxisome assembly. A stable transformant of ZP92 with the cDNA was morphologically and biochemically restored for peroxisome biogenesis. Fibroblasts derived from patients deficient in peroxisome biogenesis (complementation group C) were also complemented with PAF-2 cDNA, indicating that PAF-2 is a strong candidate for the pathogenic gene of group C peroxisome deficiency.