Abstract
We isolated peroxisome biogenesis-defective mutants from Chinese hamster ovary cells by the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) method. Seven cell mutants, ZP116, ZP119, ZP160, ZP161, ZP162, ZP164, and ZP165, of 11 P9OH/UV-resistant cell clones showed cytosolic localization of catalase, a peroxisomal matrix enzyme, apparently indicating a defect of peroxisome biogenesis. By transfection of PEX cDNAs and cell fusion analysis, mutants ZP119 and ZP165 were found to belong to a novel complementation group (CG), distinct from earlier mutants. CG analysis by cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome indicated that ZP119 and ZP165 were in the same CG as the most recently identified human CG-J. The peroxisomal matrix proteins examined, including PTS1 proteins as well as a PTS2 protein, 3-ketoacyl-CoA thiolase, were also found in the cytosol in ZP119 and ZP165. Furthermore, these mutants showed typical peroxisome assembly-defective phenotype such as severe loss of resistance to 12-(1'-pyrene)dodecanoic acid/UV treatment. Most strikingly, peroxisomal reminiscent vesicular structures, so-called peroxisomal ghosts noted in all CGs of earlier Chinese hamster ovary cell mutants as well as in eight CGs of patients' fibroblasts, were not discernible in ZP119 and ZP165, despite normal synthesis of peroxisomal membrane proteins. Accordingly, ZP119 and ZP165 are the first cell mutants defective in import of both soluble and membrane proteins, representing the 14th peroxisome-deficient CG in mammals, including humans.
Highlights
Ferent subcellular compartments is one of the major characteristics of eukaryotic cells
These results demonstrate that ZP119, ZP161, and ZP165 belong to complementation groups (CGs)(s) different from those of Z65, ZP92, and ZP105
ZP119 and ZP165 are considered to be the first mammalian cell mutants absent from peroxisomal reminiscent membrane ghosts that have been detected in all seven CGs of CHO mutants [12, 17,18,19, 46] and eight CGs of the fibroblasts from patients with peroxisome deficiency disease such as Zellweger syndrome [22,23,24,25]
Summary
Rat PEX2-transformed CHO-K1, TKa cells [17,18,19, 42], were cultured in a Ham’s F12 medium supplemented with 10% fetal calf serum. TKa cells were mutagenized with N-methyl-NЈ-nitro-N-nitrosoguanidine (Nacalai Tesque, Kyoto, Japan) as described [42]. Cells resistant to 9-(1Ј-pyrene)nonanol/ultraviolet (P9OH/UV) treatment were selected in F12-containing 10% fetal calf serum, 6 M P9OH (Molecular Probes, Eugene, OR) followed by exposure to long UV wavelength as described [12]
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