Isolation and Characterization of Novel Peroxisome Biogenesis-Defective Chinese Hamster Ovary Cell Mutants Using Green Fluorescent Protein

Ghaedi, Kamran; Kawai, Atsushi; Okumoto, Kanji; Tamura, Shigehiko; Shimozawa, Nobuyuki; Suzuki, Yasuyuki; Kondo, Naomi; Fujiki, Yukio

doi:10.1006/excr.1999.4413

Cited by 35 publications

(65 citation statements)

References 28 publications

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“…GFP-transformed CHO Cells and Isolation of Peroxisome Biogenesisdefective Mutants-TKaG2 cells (22), rat PEX2-transformed CHO-K1 cells stably expressing N-terminally PTS2-tagged GFP, and selection of peroxisome biogenesis mutants (22,24) were as described.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, in this study, we have searched for a CHO cell mutant showing such a pex5 phenotype with PTS2 import deficiency using the improved mutant isolation method making use of green fluorescent protein (GFP) tagged with PTS2 (22). We isolated 10 CHO cell mutant clones by the P9OH/UV method using PTS2-GFP (22) and classified them in five previously identified CGs.…”

mentioning

confidence: 99%

“…We isolated 10 CHO cell mutant clones by the P9OH/UV method using PTS2-GFP (22) and classified them in five previously identified CGs. To our surprise, a mutant cell line (ZPG231) of CG2 showed a novel phenotype, PEX5-defective, but impaired solely in PTS2 import.…”

mentioning

confidence: 99%

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Disruption of the Interaction of the Longer Isoform of Pex5p, Pex5pL, with Pex7p Abolishes Peroxisome Targeting Signal Type 2 Protein Import in Mammals

Matsumura

Otera

Fujiki

2000

Journal of Biological Chemistry

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104

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We isolated peroxisome biogenesis-defective Chinese hamster ovary cell mutants from TKaG2 cells, wild-type CHO-K1 cells transformed with two cDNAs encoding rat Pex2p and peroxisome targeting signal (PTS) type 2-tagged green fluorescent protein, by the 9-(1-pyrene)nonanol/UV selection method. Ten mutant clones showed cytosolic PTS2-green fluorescent protein, indicative of a defect in PTS2 import, and were classified in five complementation groups, i.e. pex1, pex2, pex5, pex14, and group A. One PEX5-deficient mutant, ZPG231, showed a novel phenotype: PTS2 proteins in the cytosol, but PTS1 proteins and catalase in peroxisomes. In ZPG231, two isoforms of the PTS1 receptor Pex5p, a shorter Pex5pS and a longer Pex5pL, were expressed as in wild-type cells, but possessed the missense point mutation S214F in both Pex5p isoforms, termed Pex5pS-S214F and Pex5pL-S214F, respectively. The S214F mutation was located only one amino acid upstream of the Pex5pL-specific 37-amino acid insertion site. Pex5pS-S214F and Pex5pL-S214F interacted with peroxisomal proteins, including PTS1 protein, catalase, and Pex14p, as efficiently as normal Pex5p. In contrast, the S214F mutation severely affected the binding of Pex5pL to the PTS2 receptor Pex7p. Expression of Pex5pL-S214F in pex5 cell mutants defective in PTS1 and PTS2 transport restored peroxisomal import of PTS1, but not PTS2. Together, the results indicate that ZPG231 is the first cell mutant providing evidence that disruption of the Pex5pL-Pex7p interaction completely abolishes PTS2 import in mammals.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Disruption of the Interaction of the Longer Isoform of Pex5p, Pex5pL, with Pex7p Abolishes Peroxisome Targeting Signal Type 2 Protein Import in Mammals

Matsumura

Otera

Fujiki

2000

Journal of Biological Chemistry

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104

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“…Bound fractions were eluted with 50 l of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. An in vitro binding assay was also performed using cell lysates of 207P7, a stable human PEX7 transfectant of pex7 CHO mutant ZPG207 (19) expressing a higher level of Pex7p (29). 207P7 cells (10 6 ) were lysed on ice with the binding assay buffer for 1 h and centrifuged at 100,000 ϫ g for 40 min at 4°C.…”

Section: Biochemicalsmentioning

confidence: 99%

Peroxisomal Targeting Signal Receptor Pex5p Interacts with Cargoes and Import Machinery Components in a Spatiotemporally Differentiated Manner: Conserved Pex5p WXXXF/Y Motifs Are Critical for Matrix Protein Import

Otera

Setoguchi

Hamasaki

et al. 2002

Molecular and Cellular Biology

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197

287

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“…Genetic heterogeneity, comprising 16 different complementation groups (CGs), has been identified in mammals, including 13CGs of PBDs and 3 distinct CGs of mutant Chinese hamster ovary (CHO) cell lines, as determined by cellfusion and/or peroxin gene (PEX) transfection, using fibroblasts from patients with PBDs and peroxisome-deficient CHO cell mutants (Ghaedi et al 1999;Fujiki 2000) (see Table 1). The primary cause responsible for the peroxisome deficiency in the 13 CGs of PBDs is a failure in peroxisome biogenesis (Fujiki 2000;Gould and Valle 2000).…”

Section: Introductionmentioning

confidence: 99%

The peroxin Pex6p gene is impaired in peroxisomal biogenesis disorders of complementation group 6

et al. 2001

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Human genetic peroxisomal biogenesis disorders (PBDs), such as Zellweger syndrome, comprise 13 different complementation groups (CGs). Eleven peroxin genes, termed PEXs, responsible for PBDs have been identified, whereas pathogenic genes for PBDs of 2 CGs, CG-A (the same CG as CG8 in the United States and Europe) and CG6, remained unidentified. We herein provide several lines of novel evidence indicating that PEX6, the pathogenic gene for CG4, is impaired in PBD of CG6. Expression of PEX6 restored peroxisome assembly in fibroblasts from a CG6 PBD patient. This patient was a compound heterozygote for PEX6 gene alleles. Accordingly, by merging CG6 with CG4, human PBDs are now classified into 12 CGs.

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Isolation and Characterization of Novel Peroxisome Biogenesis-Defective Chinese Hamster Ovary Cell Mutants Using Green Fluorescent Protein

Cited by 35 publications

References 28 publications

Disruption of the Interaction of the Longer Isoform of Pex5p, Pex5pL, with Pex7p Abolishes Peroxisome Targeting Signal Type 2 Protein Import in Mammals

Disruption of the Interaction of the Longer Isoform of Pex5p, Pex5pL, with Pex7p Abolishes Peroxisome Targeting Signal Type 2 Protein Import in Mammals

Peroxisomal Targeting Signal Receptor Pex5p Interacts with Cargoes and Import Machinery Components in a Spatiotemporally Differentiated Manner: Conserved Pex5p WXXXF/Y Motifs Are Critical for Matrix Protein Import

The peroxin Pex6p gene is impaired in peroxisomal biogenesis disorders of complementation group 6

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