Newly Identified Chinese Hamster Ovary Cell Mutants Are Defective in Biogenesis of Peroxisomal Membrane Vesicles (Peroxisomal Ghosts), Representing a Novel Complementation Group in Mammals
1998; Elsevier BV; Volume: 273; Issue: 37 Linguagem: Inglês
10.1074/jbc.273.37.24122
ISSN1083-351X
AutoresNaohiko Kinoshita, Kamran Ghaedi, Nobuyuki Shimozawa, Ronald J. A. Wanders, Yuji Matsuzono, Tsuneo Imanaka, Kanji Okumoto, Yasuyuki Suzuki, Naomi Kondo, Yukio Fujiki,
Tópico(s)Virus-based gene therapy research
ResumoWe 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. 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. peroxisome targeting signal types 1 and 2 acyl-CoA oxidase complementation group Chinese hamster ovary dihydroxyacetonephosphate acyltransferase enhanced green fluorescent protein 9-(1′-pyrene)nonanol/ultraviolet 12-(1′-pyrene)dodecanoic acid peroxisomal 70-kDa integral membrane protein resistant to 6-thioguanine Zellweger syndrome. The hierarchy of the highly organized biogenesis of the different subcellular compartments is one of the major characteristics of eukaryotic cells. Peroxisome, a single membrane-bounded organelle, functions in various metabolic pathways such as β-oxidation of very long chain fatty acids and synthesis of ether phospholipids, plasmalogens (1van den Bosch H. Schutgens R.B.H. Wanders R.J.A. Tager J.M. Annu. Rev. Biochem. 1992; 61: 157-197Crossref PubMed Scopus (733) Google Scholar). Significant progress has recently been made in our understanding of the biogenesis of peroxisomes by findings such as those of targeting signals and protein factors, peroxins (2Distel B. Erdmann R. Gould S.J. Blobel G. Crane D.I. Cregg J.M. Dodt G. Fujiki Y. Goodman J.M. Just W.W. Kiel J.A.K.W. Kunau W.-H. Lazarow P.B. Mannaerts G.P. Moser H. Osumi T. Rachubinski R.A. Roscher A. Subramani S. Tabak H.F. Tsukamoto T. Valle D. van der Klei I. van Veldhoven P.P. Veenhuis M. J. Cell Biol. 1996; 135: 1-3Crossref PubMed Scopus (313) Google Scholar) required for peroxisome assembly, and through disparate studies using yeast and mammalian cells (3Lazarow P.B. Fujiki Y. Annu. Rev. Cell Biol. 1985; 1: 489-530Crossref PubMed Scopus (877) Google Scholar, 4Subramani S. Annu. Rev. Cell Biol. 1993; 9: 445-478Crossref PubMed Scopus (359) Google Scholar, 5Fujiki Y. Biochim. Biophys. Acta. 1997; 1361: 235-250Crossref PubMed Scopus (61) Google Scholar). Both peroxisomal membrane and matrix proteins are imported post-translationally into peroxisomes (3Lazarow P.B. Fujiki Y. Annu. Rev. Cell Biol. 1985; 1: 489-530Crossref PubMed Scopus (877) Google Scholar). New peroxisomes are then formed by growth and division of preexisting peroxisomes. Peroxisomal targeting signals, type 1 (PTS1)1 and type 2 (PTS2) independently function in evolutionarily diverse organisms from yeasts to humans (5Fujiki Y. Biochim. Biophys. Acta. 1997; 1361: 235-250Crossref PubMed Scopus (61) Google Scholar). Over 15 peroxisome biogenesis factors, peroxins, have been cloned (5Fujiki Y. Biochim. Biophys. Acta. 1997; 1361: 235-250Crossref PubMed Scopus (61) Google Scholar, 6Waterham H.R. Cregg J. BioEssays. 1997; 19: 57-66Crossref PubMed Scopus (53) Google Scholar, 7Subramani S. Nat. Genet. 1997; 15: 331-333Crossref PubMed Scopus (82) Google Scholar, 8Erdmann R. Veenhuis M. Kunau W.-H. Trends Cell Biol. 1997; 7: 400-407Abstract Full Text PDF PubMed Scopus (117) Google Scholar).Human peroxisome-deficient disorders are autosomal recessive (9Lazarow P.B. Moser H.W. Scriver C.R. Beaudet A.I. Sly W.S. Valle D. Disorders of Peroxisome Biogenesis. 7th Ed. McGraw-Hill Inc., New York1995: 2287-2324Google Scholar) and include at least 11 different genotypes (10Brul S. Westerweld A. Strijland A. Wanders R.J.A. Schram A.W. Heymans H.S.A. Schutgens R.B.H. Bosch H.V. d. Tager J.M. J. Clin. Invest. 1988; 81: 1710-1715Crossref PubMed Scopus (134) Google Scholar, 11Yajima S. Suzuki Y. Shimozawa N. Yamaguchi S. Orii T. Fujiki Y. Osumi T. Hashimoto T. Moser H.W. Hum. Genet. 1992; 88: 491-499Crossref PubMed Scopus (71) Google Scholar, 12Shimozawa N. Tsukamoto T. Suzuki Y. Orii T. Fujiki Y. J. Clin. Invest. 1992; 90: 1864-1870Crossref PubMed Scopus (71) Google Scholar, 13Moser A.B. Rasmussen M. Naidu S. Watkins P.A. McGuiness M. Hajra A.K. Chen G. Raymond G. Liu A. Gordon D. Garnaas K. Walton D.S. Skjeldal O.H. Guggenheim M.A. Jackson L.G. Elias E.R. Moser H.W. J. Pediatr. 1995; 127: 13-22Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 14Poulos A. Christodoulou J. Chow C.W. Goldblatt J. Paton B.C. Orii T. Suzuki Y. Shimozawa N. J. Pediatr. 1995; 127: 596-599Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 15Shimozawa N. Suzuki Y. Zhang Z. Imamura A. Tsukamoto T. Osumi T. Tateishi K. Okumoto K. Fujiki Y. Orii T. Barth P.G. Wanders R.J.A. Kondo N. Biochem. Biophys. Res. Commun. 1998; 243: 368-371Crossref PubMed Scopus (21) Google Scholar), with three clinically distinct phenotypes of diseases, cerebro-hepato-renal Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease (1van den Bosch H. Schutgens R.B.H. Wanders R.J.A. Tager J.M. Annu. Rev. Biochem. 1992; 61: 157-197Crossref PubMed Scopus (733) Google Scholar, 9Lazarow P.B. Moser H.W. Scriver C.R. Beaudet A.I. Sly W.S. Valle D. Disorders of Peroxisome Biogenesis. 7th Ed. McGraw-Hill Inc., New York1995: 2287-2324Google Scholar). So far we have isolated seven complementation groups (CGs) of peroxisome-deficient CHO cell mutants, including Z24/ZP107 (16Tsukamoto T. Yokota S. Fujiki Y. J. Cell Biol. 1990; 110: 651-660Crossref PubMed Scopus (186) Google Scholar, 17Okumoto K. Bogaki A. Tateishi K. Tsukamoto T. Osumi T. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Exp. Cell Res. 1997; 233: 11-20Crossref PubMed Scopus (38) Google Scholar), Z65 (16Tsukamoto T. Yokota S. Fujiki Y. J. Cell Biol. 1990; 110: 651-660Crossref PubMed Scopus (186) Google Scholar), ZP92 (12Shimozawa N. Tsukamoto T. Suzuki Y. Orii T. Fujiki Y. J. Clin. Invest. 1992; 90: 1864-1870Crossref PubMed Scopus (71) Google Scholar), ZP105/ZP139 (17Okumoto K. Bogaki A. Tateishi K. Tsukamoto T. Osumi T. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Exp. Cell Res. 1997; 233: 11-20Crossref PubMed Scopus (38) Google Scholar, 18Otera H. Tateishi K. Okumoto K. Ikoma Y. Matsuda E. Nishimura M. Tsukamoto T. Osumi T. Ohashi K. Higuchi O. Fujiki Y. Mol. Cell. Biol. 1998; 18: 388-399Crossref PubMed Google Scholar), ZP109 (17Okumoto K. Bogaki A. Tateishi K. Tsukamoto T. Osumi T. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Exp. Cell Res. 1997; 233: 11-20Crossref PubMed Scopus (38) Google Scholar), ZP110 (19Tateishi K. Okumoto K. Shimozawa N. Tsukamoto T. Osumi T. Suzuki Y. Kondo N. Okano I. Fujiki Y. Eur. J. Cell Biol. 1997; 73: 352-359PubMed Google Scholar), and ZP114 (19Tateishi K. Okumoto K. Shimozawa N. Tsukamoto T. Osumi T. Suzuki Y. Kondo N. Okano I. Fujiki Y. Eur. J. Cell Biol. 1997; 73: 352-359PubMed Google Scholar), by colony-autoradiographic screening (20Zoeller R.A. Raetz C.R.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5170-5174Crossref PubMed Scopus (111) Google Scholar) and the 9-(1′-pyrene)nonanol (P9OH)/UV selection method (21Morand O.H. Allen L.-A.H. Zoeller R.A. Raetz C.R.H. Biochim. Biophys. Acta. 1990; 1034: 132-141Crossref PubMed Scopus (54) Google Scholar). All mutants resemble fibroblasts from patients with peroxisome-deficiency disease, in defects of biogenesis and function of peroxisomes (5Fujiki Y. Biochim. Biophys. Acta. 1997; 1361: 235-250Crossref PubMed Scopus (61) Google Scholar). All CGs of CHO mutants are impaired in import of peroxisomal matrix proteins but contain peroxisome-reminiscent ghosts (12Shimozawa N. Tsukamoto T. Suzuki Y. Orii T. Fujiki Y. J. Clin. Invest. 1992; 90: 1864-1870Crossref PubMed Scopus (71) Google Scholar, 17Okumoto K. Bogaki A. Tateishi K. Tsukamoto T. Osumi T. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Exp. Cell Res. 1997; 233: 11-20Crossref PubMed Scopus (38) Google Scholar, 19Tateishi K. Okumoto K. Shimozawa N. Tsukamoto T. Osumi T. Suzuki Y. Kondo N. Okano I. Fujiki Y. Eur. J. Cell Biol. 1997; 73: 352-359PubMed Google Scholar), as noted in fibroblasts from the patients (22Santos M.J. Imanaka T. Shio H. Lazarow P.B. J. Biol. Chem. 1988; 263: 10502-10509Abstract Full Text PDF PubMed Google Scholar, 23Wiemer E.A.C. Brul S. Just W.W. van Driel R. Brouwer-Kelder E. van den Berg M. Weijers P.J. Schutgens R.B.H. van den Bosch H. Schram A. Wanders R.J.A. Tager J.M. Eur. J. Cell Biol. 1989; 50: 407-417PubMed Google Scholar, 24Santos M.J. Hoefler S. Moser A.B. Moser H.W. Lazarow P.B. J. Cell. Physiol. 1992; 151: 103-112Crossref PubMed Scopus (42) Google Scholar, 25Wendland M. Subramani S. J. Clin. Invest. 1993; 92: 2462-2468Crossref PubMed Scopus (32) Google Scholar). This finding was interpreted to mean that transport of peroxisomal membrane polypeptides is normal in CHO cell mutants and fibroblasts from the patients (4Subramani S. Annu. Rev. Cell Biol. 1993; 9: 445-478Crossref PubMed Scopus (359) Google Scholar, 5Fujiki Y. Biochim. Biophys. Acta. 1997; 1361: 235-250Crossref PubMed Scopus (61) Google Scholar, 12Shimozawa N. Tsukamoto T. Suzuki Y. Orii T. Fujiki Y. J. Clin. Invest. 1992; 90: 1864-1870Crossref PubMed Scopus (71) Google Scholar, 17Okumoto K. Bogaki A. Tateishi K. Tsukamoto T. Osumi T. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Exp. Cell Res. 1997; 233: 11-20Crossref PubMed Scopus (38) Google Scholar, 19Tateishi K. Okumoto K. Shimozawa N. Tsukamoto T. Osumi T. Suzuki Y. Kondo N. Okano I. Fujiki Y. Eur. J. Cell Biol. 1997; 73: 352-359PubMed Google Scholar,22Santos M.J. Imanaka T. Shio H. Lazarow P.B. J. Biol. Chem. 1988; 263: 10502-10509Abstract Full Text PDF PubMed Google Scholar, 23Wiemer E.A.C. Brul S. Just W.W. van Driel R. Brouwer-Kelder E. van den Berg M. Weijers P.J. Schutgens R.B.H. van den Bosch H. Schram A. Wanders R.J.A. Tager J.M. Eur. J. Cell Biol. 1989; 50: 407-417PubMed Google Scholar, 24Santos M.J. Hoefler S. Moser A.B. Moser H.W. Lazarow P.B. J. Cell. Physiol. 1992; 151: 103-112Crossref PubMed Scopus (42) Google Scholar, 25Wendland M. Subramani S. J. Clin. Invest. 1993; 92: 2462-2468Crossref PubMed Scopus (32) Google Scholar). CHO cell mutants, Z24/ZP107, Z65, ZP92, ZP105/ZP139, and ZP104/ZP109, were classified to five CGs of 11 human CGs (12Shimozawa N. Tsukamoto T. Suzuki Y. Orii T. Fujiki Y. J. Clin. Invest. 1992; 90: 1864-1870Crossref PubMed Scopus (71) Google Scholar, 17Okumoto K. Bogaki A. Tateishi K. Tsukamoto T. Osumi T. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Exp. Cell Res. 1997; 233: 11-20Crossref PubMed Scopus (38) Google Scholar, 18Otera H. Tateishi K. Okumoto K. Ikoma Y. Matsuda E. Nishimura M. Tsukamoto T. Osumi T. Ohashi K. Higuchi O. Fujiki Y. Mol. Cell. Biol. 1998; 18: 388-399Crossref PubMed Google Scholar); others such as ZP110 and ZP114 represent two distinct CGs besides 11 human CGs (19Tateishi K. Okumoto K. Shimozawa N. Tsukamoto T. Osumi T. Suzuki Y. Kondo N. Okano I. Fujiki Y. Eur. J. Cell Biol. 1997; 73: 352-359PubMed Google Scholar). Therefore, mammalian peroxisome assembly apparently requires at least 13 genes or their products of which only five,PEX1, PEX2 (formerly PAF-1), PEX5, PEX6 (formerly PAF-2), and PEX12, are known (26Tamura S. Okumoto K. Toyama R. Shimozawa N. Tsukamoto T. Suzuki Y. Osumi T. Kondo N. Fujiki Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4350-4355Crossref PubMed Scopus (86) Google Scholar, 27Reuber B.E. Germain-Lee E. Collins C.S. Morrell J.C. Ameritunga R. Moser H.W. Valle D. Gould S.J. Nat. Genet. 1997; 17: 445-448Crossref PubMed Scopus (191) Google Scholar, 28Portsteffen H. Beyer A. Becker E. Epplen C. Pawlak A. Kunau W.-H. Dodt G. Nat. Genet. 1997; 17: 449-452Crossref PubMed Scopus (121) Google Scholar, 29Tsukamoto T. Miura S. Fujiki Y. Nature. 1991; 350: 77-81Crossref PubMed Scopus (220) Google Scholar, 30Dodt G. Braverman N. Wong C.S. Moser A. Moser H.W. Watkins P. Valle D. Gould S.J. Nat. Genet. 1995; 9: 115-125Crossref PubMed Scopus (383) Google Scholar, 31Wiemer E.A. Nuttley W.M. Bertolaet B.L. Li X. Francke U. Wheelock M.J. Anne U.K. Johnson K.R. Subramani S. J. Cell Biol. 1995; 130: 51-65Crossref PubMed Scopus (164) Google Scholar, 32Tsukamoto T. Miura S. Nakai T. Yokota S. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Sakai F. Bogaki A. Yasumo H. Osumi T. Nat. Genet. 1995; 11: 395-401Crossref PubMed Scopus (101) Google Scholar, 33Yahraus T. Braverman N. Dodt G. Kalish J.E. Morrell J.C. Moser H.W. Valle D. Gould S.J. EMBO J. 1996; 15: 2914-2923Crossref PubMed Scopus (161) Google Scholar, 34Chang C.-C. Lee W.-H. Moser H. Valle D. Gould S.J. Nat. Genet. 1997; 15: 385-388Crossref PubMed Scopus (128) Google Scholar, 35Okumoto K. Fujiki Y. Nat. Genet. 1997; 17: 265-266Crossref PubMed Scopus (61) Google Scholar, 36Okumoto K. Shimozawa N. Kawai A. Tamura S. Tsukamoto T. Osumi T. Moser H. Wanders R.J.A. Suzuki Y. Kondo N. Fujiki Y. Mol. Cell. Biol. 1998; 18: 4324-4336Crossref PubMed Scopus (92) Google Scholar). PEX1, PEX2, PEX6, and PEX12 were isolated by functional phenotype-complementation assay, using ZP107, Z65, ZP92, and ZP109, respectively (26Tamura S. Okumoto K. Toyama R. Shimozawa N. Tsukamoto T. Suzuki Y. Osumi T. Kondo N. Fujiki Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4350-4355Crossref PubMed Scopus (86) Google Scholar, 29Tsukamoto T. Miura S. Fujiki Y. Nature. 1991; 350: 77-81Crossref PubMed Scopus (220) Google Scholar, 32Tsukamoto T. Miura S. Nakai T. Yokota S. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Sakai F. Bogaki A. Yasumo H. Osumi T. Nat. Genet. 1995; 11: 395-401Crossref PubMed Scopus (101) Google Scholar, 35Okumoto K. Fujiki Y. Nat. Genet. 1997; 17: 265-266Crossref PubMed Scopus (61) Google Scholar,36Okumoto K. Shimozawa N. Kawai A. Tamura S. Tsukamoto T. Osumi T. Moser H. Wanders R.J.A. Suzuki Y. Kondo N. Fujiki Y. Mol. Cell. Biol. 1998; 18: 4324-4336Crossref PubMed Scopus (92) Google Scholar). These PEXs, including recently identifiedPEX7 (37Braverman N. Steel G. Obie C. Moser A. Moser H. Gould S.J. Valle D. Nat. Genet. 1997; 15: 369-376Crossref PubMed Scopus (356) Google Scholar, 38Motley A.M. Hettema E.H. Hogenhout E.M. Brites P. ten Asbroek A.L.M.A. Wijburg F.A. Baas F. Heijmans H.S. Tabak H.F. Wanders R.J.A. Distel B. Nat. Genet. 1997; 15: 377-380Crossref PubMed Scopus (222) Google Scholar, 39Purdue P.E. Zhang J.W. Skoneczny M. Lazarow P.B. Nat. Genet. 1997; 15: 381-384Crossref PubMed Scopus (223) Google Scholar), have been shown to be responsible for the genetic cause in patients with peroxisome biogenesis disorders (26Tamura S. Okumoto K. Toyama R. Shimozawa N. Tsukamoto T. Suzuki Y. Osumi T. Kondo N. Fujiki Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4350-4355Crossref PubMed Scopus (86) Google Scholar, 27Reuber B.E. Germain-Lee E. Collins C.S. Morrell J.C. Ameritunga R. Moser H.W. Valle D. Gould S.J. Nat. Genet. 1997; 17: 445-448Crossref PubMed Scopus (191) Google Scholar, 28Portsteffen H. Beyer A. Becker E. Epplen C. Pawlak A. Kunau W.-H. Dodt G. Nat. Genet. 1997; 17: 449-452Crossref PubMed Scopus (121) Google Scholar,30Dodt G. Braverman N. Wong C.S. Moser A. Moser H.W. Watkins P. Valle D. Gould S.J. Nat. Genet. 1995; 9: 115-125Crossref PubMed Scopus (383) Google Scholar, 31Wiemer E.A. Nuttley W.M. Bertolaet B.L. Li X. Francke U. Wheelock M.J. Anne U.K. Johnson K.R. Subramani S. J. Cell Biol. 1995; 130: 51-65Crossref PubMed Scopus (164) Google Scholar, 33Yahraus T. Braverman N. Dodt G. Kalish J.E. Morrell J.C. Moser H.W. Valle D. Gould S.J. EMBO J. 1996; 15: 2914-2923Crossref PubMed Scopus (161) Google Scholar, 34Chang C.-C. Lee W.-H. Moser H. Valle D. Gould S.J. Nat. Genet. 1997; 15: 385-388Crossref PubMed Scopus (128) Google Scholar, 35Okumoto K. Fujiki Y. Nat. Genet. 1997; 17: 265-266Crossref PubMed Scopus (61) Google Scholar, 36Okumoto K. Shimozawa N. Kawai A. Tamura S. Tsukamoto T. Osumi T. Moser H. Wanders R.J.A. Suzuki Y. Kondo N. Fujiki Y. Mol. Cell. Biol. 1998; 18: 4324-4336Crossref PubMed Scopus (92) Google Scholar, 40Shimozawa N. Tsukamoto T. Suzuki Y. Orii T. Shirayoshi Y. Mori T. Fujiki Y. Science. 1992; 255: 1132-1134Crossref PubMed Scopus (311) Google Scholar, 41Fukuda S. Shimozawa N. Suzuki Y. Tomatsu S. Tsukamoto T. Hashiguchi N. Osumi T. Masuno M. Imaizumi K. Kuroki Y. Fujiki Y. Orii T. Kondo N. Am. J. Hum. Genet. 1996; 59: 1210-1220PubMed Google Scholar). Thus, peroxisome biogenesis-defective CHO cell mutants are proven as a useful mammalian somatic cell system for investigation of peroxisome assembly at molecular and cellular levels, as well as for elucidation of genetic cause of peroxisome biogenesis disorders (5Fujiki Y. Biochim. Biophys. Acta. 1997; 1361: 235-250Crossref PubMed Scopus (61) Google Scholar).However, the process of peroxisomal membrane vesicle formation is little understood, despite the development of genetic approaches described above. We report here isolation and characterization of a newly identified CG of CHO cell mutants defective in peroxisome membrane biogenesis, apparently at the initial stage of peroxisome assembly. These CHO mutants apparently represent the most recently classified human CG-J.EXPERIMENTAL PROCEDURESSelection of Peroxisome-deficient CHO Cell MutantsRat PEX2-transformed CHO-K1, TKa cells (17Okumoto K. Bogaki A. Tateishi K. Tsukamoto T. Osumi T. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Exp. Cell Res. 1997; 233: 11-20Crossref PubMed Scopus (38) Google Scholar, 18Otera H. Tateishi K. Okumoto K. Ikoma Y. Matsuda E. Nishimura M. Tsukamoto T. Osumi T. Ohashi K. Higuchi O. Fujiki Y. Mol. Cell. Biol. 1998; 18: 388-399Crossref PubMed Google Scholar, 19Tateishi K. Okumoto K. Shimozawa N. Tsukamoto T. Osumi T. Suzuki Y. Kondo N. Okano I. Fujiki Y. Eur. J. Cell Biol. 1997; 73: 352-359PubMed Google Scholar, 42Tsukamoto T. Bogaki A. Okumoto K. Tateishi K. Fujiki Y. Shimozawa N. Suzuki Y. Kondo N. Osumi T. Biochem. Biophys. Res. Commun. 1997; 230: 402-406Crossref PubMed Scopus (36) Google Scholar), were cultured in a Ham's F12 medium supplemented with 10% fetal calf serum. TKa cells were mutagenized withN-methyl-N′-nitro-N-nitrosoguanidine (Nacalai Tesque, Kyoto, Japan) as described (42Tsukamoto T. Bogaki A. Okumoto K. Tateishi K. Fujiki Y. Shimozawa N. Suzuki Y. Kondo N. Osumi T. Biochem. Biophys. Res. Commun. 1997; 230: 402-406Crossref PubMed Scopus (36) Google Scholar). Cells resistant to 9-(1′-pyrene)nonanol/ultraviolet (P9OH/UV) treatment were selected in F12-containing 10% fetal calf serum, 6 μm P9OH (Molecular Probes, Eugene, OR) followed by exposure to long UV wavelength as described (12Shimozawa N. Tsukamoto T. Suzuki Y. Orii T. Fujiki Y. J. Clin. Invest. 1992; 90: 1864-1870Crossref PubMed Scopus (71) Google Scholar).Morphological AnalysisPeroxisomes in CHO cells were visualized by indirect immunofluorescence light microscopy using rabbit anti-rat catalase antibody, and those in fused cells of CHO mutants with human fibroblasts were detected with either rabbit anti-human catalase antibody or anti-rat catalase antibody, as described (12Shimozawa N. Tsukamoto T. Suzuki Y. Orii T. Fujiki Y. J. Clin. Invest. 1992; 90: 1864-1870Crossref PubMed Scopus (71) Google Scholar). Several CHO mutant cell clones were stained with rabbit antibodies specific for PTS1 peptide comprising the C-terminal, 10 amino acid residues of rat acyl-CoA oxidase (AOx) (18Otera H. Tateishi K. Okumoto K. Ikoma Y. Matsuda E. Nishimura M. Tsukamoto T. Osumi T. Ohashi K. Higuchi O. Fujiki Y. Mol. Cell. Biol. 1998; 18: 388-399Crossref PubMed Google Scholar), rat 3-ketoacyl-CoA thiolase (16Tsukamoto T. Yokota S. Fujiki Y. J. Cell Biol. 1990; 110: 651-660Crossref PubMed Scopus (186) Google Scholar), C-terminal 15-amino acid peptide of rat peroxisomal 70-kDa integral membrane protein (PMP70) (43Imanaka T. Shiina Y. Takano T. Hashimoto T. Osumi T. J. Biol. Chem. 1996; 271: 3706-3713Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), and C-terminal 20-amino acid peptide of Pex12p (35Okumoto K. Fujiki Y. Nat. Genet. 1997; 17: 265-266Crossref PubMed Scopus (61) Google Scholar). Antigen-antibody complex was detected by fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin G antibody (Cappel, Durham, NC) under a Carl Zeiss Axioskop FL microscope (Oberkochen, Germany) using a number 17 filter.AssaysThe latency of catalase (12Shimozawa N. Tsukamoto T. Suzuki Y. Orii T. Fujiki Y. J. Clin. Invest. 1992; 90: 1864-1870Crossref PubMed Scopus (71) Google Scholar), dihydroxyacetonephosphate acyltransferase (DHAP-ATase) (17Okumoto K. Bogaki A. Tateishi K. Tsukamoto T. Osumi T. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Exp. Cell Res. 1997; 233: 11-20Crossref PubMed Scopus (38) Google Scholar), and resistance to P9OH/UV and 12-(1′-pyrene)dodecanoic acid (P12)/UV treatments (12Shimozawa N. Tsukamoto T. Suzuki Y. Orii T. Fujiki Y. J. Clin. Invest. 1992; 90: 1864-1870Crossref PubMed Scopus (71) Google Scholar) were determined as described.DNA TransfectioncDNA transfection to CHO mutant cells was done by means of liposome-mediated transfection with plasmids of rat PEX2, human PEX5, rat PEX6 expressing vector (17Okumoto K. Bogaki A. Tateishi K. Tsukamoto T. Osumi T. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Exp. Cell Res. 1997; 233: 11-20Crossref PubMed Scopus (38) Google Scholar), and rat PEX12 (35Okumoto K. Fujiki Y. Nat. Genet. 1997; 17: 265-266Crossref PubMed Scopus (61) Google Scholar). Transfection was carried out for 3 h with 1 μg of cDNA, using 12 μg of LipofectAMINE (Life Technologies, Inc.), according to the procedure recommended by the manufacturer. The cells were cultured for 2 days and then incubated overnight in 2 ml of serum-free F12 medium before immunostaining.Expression of GFP Fusion ProteinPlasmid expressing “enhanced” green fluorescent protein (EGFP) (44Cormack B.P. Valdivia R.H. Falkow S. Gene (Amst.). 1996; 173: 33-38Crossref PubMed Scopus (2489) Google Scholar) fused at the C terminus with human PMP70 (EGFP-PMP70) was constructed as follows. Human PMP70 cDNA (45Kamijo K. Kamijo T. Ueno I. Osumi T. Hashimoto T. Biophys. Biochim. Acta. 1992; 1129: 323-327Crossref PubMed Scopus (74) Google Scholar) was inserted into theNotI site in a mammalian expression vector, pUcD2SRαMCSHyg (36Okumoto K. Shimozawa N. Kawai A. Tamura S. Tsukamoto T. Osumi T. Moser H. Wanders R.J.A. Suzuki Y. Kondo N. Fujiki Y. Mol. Cell. Biol. 1998; 18: 4324-4336Crossref PubMed Scopus (92) Google Scholar), containing hyg gene in pUcD2SRαMCS (32Tsukamoto T. Miura S. Nakai T. Yokota S. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Sakai F. Bogaki A. Yasumo H. Osumi T. Nat. Genet. 1995; 11: 395-401Crossref PubMed Scopus (101) Google Scholar). ASacII-BamHI fragment obtained from this plasmid was inserted into SacII-BamHI site in pEGFP-C1 vector (CLONTECH, Palo Alto, CA). Plasmid expressing a fusion protein of EGFP with rat Pex12p (EGFP-Pex12p) was likewise constructed. The BamHI site was created immediately upstream from the initiator methionine in rat Pex12p cDNA (RnPEX12) (35Okumoto K. Fujiki Y. Nat. Genet. 1997; 17: 265-266Crossref PubMed Scopus (61) Google Scholar) by polymerase chain reaction using a forward primer, 5′-CGCGGATCCCTACTATGGCTGAGCATGG-3′ (initiation codon is underlined), and a reverse primer, 5′-GTATTCAGAAAATGAGGC-3′ (residues 239–256, starting from the first nucleotide of the initiator methionine codon of RnPEX12 open reading frame). pBS·RnPEX12(B-A) was constructed by replacing aBamHI-SpeI fragment of pBS·RnPEX12(35Okumoto K. Fujiki Y. Nat. Genet. 1997; 17: 265-266Crossref PubMed Scopus (61) Google Scholar) with a BamHI-SpeI fragment of the polymerase chain reaction fragment. A BamHI (blunted)-ApaI fragment of pBS·RnPEX12(B-A) was replaced intoBglII (blunted)/ApaI-digested part of pEGFP-C1, thereby constructing pEGFP·RnPEX12. Plasmids for EGFP-PMP70 and EGFP-Pex12p contained deduced sequence at a linker part, SGLRSRALAAA and FGLRSIP, respectively. EGFP-PMP70 and EGFP-Pex12p were expressed in CHO cells by transient transfection. Transfectants grown on cover glass were fixed and observed under a Carl Zeiss Axioplan 2 microscope using a number 17 filter.Cell FusionParent CHO cells and cells to be fused were co-cultured for 1 day and then fused with polyethylene glycol as described (16Tsukamoto T. Yokota S. Fujiki Y. J. Cell Biol. 1990; 110: 651-660Crossref PubMed Scopus (186) Google Scholar). Selection of fused cells was carried out with 1 mm ouabain or 100 μg/ml hygromycin B (Sigma) (16Tsukamoto T. Yokota S. Fujiki Y. J. Cell Biol. 1990; 110: 651-660Crossref PubMed Scopus (186) Google Scholar, 17Okumoto K. Bogaki A. Tateishi K. Tsukamoto T. Osumi T. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Exp. Cell Res. 1997; 233: 11-20Crossref PubMed Scopus (38) Google Scholar). Cell fusion of CHO mutant variants resistant to 6-thioguanine (TGr) with human fibroblasts was done as described (12Shimozawa N. Tsukamoto T. Suzuki Y. Orii T. Fujiki Y. J. Clin. Invest. 1992; 90: 1864-1870Crossref PubMed Scopus (71) Google Scholar).Subcellular FractionationCHO-K1, Z65, and ZP119 cells were homogenized, as described (16Tsukamoto T. Yokota S. Fujiki Y. J. Cell Biol. 1990; 110: 651-660Crossref PubMed Scopus (186) Google Scholar), except that protease/inhibitor mixture containing 20 μg/ml each of antipain and pepstatin, 20 μg/ml of leupeptin, and 50 units/ml aprotinin was added to the homogenization buffer. A postnuclear supernatant fraction, prepared by centrifugation of homogenates at 750 × g for 10 min, was then centrifuged at 100,000 × g for 1 h to separate organelles (heavy and light mitochondrial and microsomal fractions) and cytosol. Each fraction was analyzed by SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to polyvinylidene difluoride membrane (Bio-Rad), and probed with specific antibody. Antigen-antibody complex was visualized with ECL Western blotting detection reagent (Amersham Pharmacia Biotech, Tokyo, Japan).Radiolabeling of CellsContinuous LabelingMetabolic labeling of cells with 10 μCi/ml [35S]methionine plus [35S]cysteine (NEN Life Science Products) for 1 or 24 h in F12 medium and immunoprecipitation of peroxisomal proteins from cell lysates were done as described (16Tsukamoto T. Yokota S. Fujiki Y. J. Cell Biol. 1990; 110: 651-660Crossref PubMed Scopus (186) Google Scholar), in the presence of protease/inhibitor mixture.Pulse-Chase ExperimentsCells growing in a 35-mm dish were pulse-labeled for 1 h with 100 μCi/ml [35S]methionine plus [35S]cysteine (Amersham). The medium was removed, and the cells were washed twice with phosphate-buffered saline and fed 2 ml of F12 plus 10% fetal calf serum medium. At selected intervals, cells were washed three times with phosphate-buffered saline and lysed as described (16Tsukamoto T. Yokota S. Fujiki Y. J. Cell Biol. 1990; 110: 651-660Crossref PubMed Scopus (186) Google Scholar), in the presence of protease/inhibitor mixture plus 2 mmphenylmethylsulfonyl fluoride.Northern Blot AnalysisRNA blot of poly(A)+ RNA from wild-type CHO-K1, Z65, and ZP119 cells was hybridized with a 32P-labeled probe of 0.6-kb NcoI-PvuII fragment of human PMP70 cDNA, under conditions of high stringency. Probe labeling was done with [32P]dCTP using a Megaprime DNA labeling system (Amersham). The membrane was also hybridized with the32P-labeled, 1.3-kb fragment of cDNA for human glyceraldehyde-3-phosphate dehydrogenase, as a control for load and integrity of the RNA. Washing of the membrane was done tw
Referência(s)