The Wilson Disease Protein ATP7B Resides in the Late Endosomes with Rab7 and the Niemann-Pick C1 Protein
2005; Elsevier BV; Volume: 166; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)62272-9
ISSN1525-2191
AutoresMasaru Harada, Takumi Kawaguchi, Hiroto Kumemura, Kunihiko Terada, Haruaki Ninomiya, Eitaro Taniguchi, Shinichiro Hanada, Shinji Baba, Michiko Maeyama, Hironori Koga, Takato Ueno, Koh Furuta, Tatsuo Suganuma, Toshihiro Sugiyama, Michio Sata,
Tópico(s)Prion Diseases and Protein Misfolding
ResumoWilson disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. Although the Wilson disease gene has been cloned, the cellular localization of the gene product (ATP7B) has not been fully clarified. Therefore, the precise physiological action of ATP7B is still unknown. We examined the distribution of ATP7B using an anti-ATP7B antibody, green fluorescent protein (GFP)-ATP7B (GFP-ATP7B) and ATP7B-DsRed in various cultured cells. Intracellular organelles were visualized by fluorescence microscopy. The distribution of ATP7B was compared with that of Rab7 and Niemann-Pick C1 (NPC1), proteins that localize in the late endosomes. U18666A, which induces the NPC phenotype, was used to modulate the intracellular vesicle traffic. GFP-ATP7B colocalized with various late endosome markers including Rab7 and NPC1 but not with Golgi or lysosome markers. U18666A induced the formation of late endosome-lysosome hybrid organelles, with GFP-ATP7B localized with NPC1 in these structures. We have confirmed that ATP7B is a late endosome-associated membrane protein. ATP7B appears to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes. Thus, defective copper ATPase activity of ATP7B in the late endosomes appears to be the main defect of Wilson disease. Wilson disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. Although the Wilson disease gene has been cloned, the cellular localization of the gene product (ATP7B) has not been fully clarified. Therefore, the precise physiological action of ATP7B is still unknown. We examined the distribution of ATP7B using an anti-ATP7B antibody, green fluorescent protein (GFP)-ATP7B (GFP-ATP7B) and ATP7B-DsRed in various cultured cells. Intracellular organelles were visualized by fluorescence microscopy. The distribution of ATP7B was compared with that of Rab7 and Niemann-Pick C1 (NPC1), proteins that localize in the late endosomes. U18666A, which induces the NPC phenotype, was used to modulate the intracellular vesicle traffic. GFP-ATP7B colocalized with various late endosome markers including Rab7 and NPC1 but not with Golgi or lysosome markers. U18666A induced the formation of late endosome-lysosome hybrid organelles, with GFP-ATP7B localized with NPC1 in these structures. We have confirmed that ATP7B is a late endosome-associated membrane protein. ATP7B appears to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes. Thus, defective copper ATPase activity of ATP7B in the late endosomes appears to be the main defect of Wilson disease. Wilson disease is an autosomal recessive disorder characterized by the progressive accumulation of copper in the body. 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The localization of ATP7B has important implications in how it functions in biliary copper excretion and copper incorporation into ceruloplasmin. 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In the present study, we examined the relationship between ATP7B and these late endosome-associated proteins in various cultured cells. Furthermore, we examined the effect of U18666A, a sterol derivative affecting vesicle movements and causing the NPC phenotype,29Neufeld EB Wastney M Patel S Suresh S Cooney AM Dwyer NK Roff CF Ohno K Morris JA Carstea ED Incardona JP Strauss III, JF Vanier MT Patterson MC Brady RO Pentchev PG Blanchette-Mackie EJ The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo.J Biol Chem. 1999; 274: 9627-9635Crossref PubMed Scopus (337) Google Scholar on the distribution of ATP7B. We now show that ATP7B is really a late endosome-associated membrane protein. Huh7 (a hepatoma cell line),31Nakabayashi H Taketa K Miyano K Yamane T Sato J Growth of human hepatoma cells lines with differentiated functions in chemically defined medium.Cancer Res. 1982; 42: 3858-3863PubMed Google Scholar MDCK, and OUMS29 (a human hepatocyte cell line)32Kobayashi N Miyazaki M Fukaya K Inoue Y Sakaguchi M Uemura T Noguchi H Kondo A Tanaka N Namba M Transplantation of highly differentiated immortalized human hepatocytes to treat acute liver failure.Transplantation. 2000; 69: 202-207Crossref PubMed Scopus (74) Google Scholar cells were cultured in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum (Wako Pure Chemical Industries, Ltd., Osaka, Japan), penicillin (100 U/ml, crystalline penicillin G Meiji, Meiji Seika Kaisya, Tokyo, Japan) and streptomycin (0.1 mg/ml, Meiji Seika Kaisya) at 37°C in 5% CO.2Camakaris J Voskoboinik I Mercer JF Molecular mechanisms of copper homeostasis.Biochem Biophys Res Commun. 1999; 261: 225-232Crossref PubMed Scopus (212) Google Scholar Copper concentration in the culture medium measured by direct colorimetric assay was below the detectable level (< 5 μg/dL). cDNAs were transiently transfected into cultured cells using Effecten Transfection Reagent (Qiagen GmbH, Hilden, Germany) according to the manufacturer's recommendations at 24 hours after plating. To produce the green fluorescent protein (GFP)-ATP7B fusion protein, the BamH I-XbaI fragment, including the entire coding region of human ATP7B cDNA, was ligated into a pEGFP-C2 vector (CLONTECH Laboratories Japan Ltd., Tokyo, Japan) digested with BglII and XbaI, the site located at the 3′ end of EGFP cDNA.11Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Taniguchi E Sasatomi K Miura N Suganuma T Fujita H Furuta K Tanikawa K Sugiyama T Sata M Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes.Gastroenterology. 2000; 118: 921-928Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 12Harada M Sakisaka S Kawaguchi T Kimura R Taniguchi E Koga H Hanada S Baba S Furuta K Kumashiro R Sugiyama T Sata M Copper does not alter the intracellular distribution of ATP7B, a copper-transporting ATPase.Biochem Biophys Res Commun. 2000; 275: 871-876Crossref PubMed Scopus (28) Google Scholar, 13Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Kim M Taniguchi E Hanada S Suganuma T Furuta K Sugiyama T Sata M A mutation of the Wilson disease protein, ATP7B, is degraded in the proteasomes and forms protein aggregates.Gastroenterology. 2001; 120: 967-974Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 14Harada M Kumemura H Sakisaka S Shishido S Taniguchi E Kawaguchi T Hanada S Koga H Kumashiro R Ueno T Suganuma T Furuta K Namba M Sugiyama T Sata M Wilson disease protein ATP7B is localized in the late endosomes in a polarized human hepatocyte cell line.Int J Mol Med. 2003; 11: 293-298PubMed Google Scholar ATP7B-DsRed was produced by ligating the SalI-SmaI fragment of ATP7B cDNA into pDsRed-N1 (CLONTECH) digested with SalI and SmaI. Myc-Rab7 was produced by ligating the SalI -KpnI fragment of Rab7 cDNA (a kind gift from Dr. A. Wandinger-Ness)33Feng Y Press B Wandinger-Ness A Rab 7: an important regulator of late endocytic membrane traffic.J Cell Biol. 1995; 131: 1435-1452Crossref PubMed Scopus (541) Google Scholar, 34Press B Feng Y Hoflack B Wandinger-Ness A Mutant Rab7 causes the accumulation of cathepsin D and cation-independent mannose 6-phosphate receptor in an early endocytic compartment.J Cell Biol. 1998; 140: 1075-1089Crossref PubMed Scopus (227) Google Scholar into pCMV-Myc (CLONTECH) digested with SalI and KpnI. PAsC9/flag-NPC1 was produced by insertion of a flag epitope into the ClaI site of human NPC cDNA.35Sugimoto Y Ninomiya H Ohsaki Y Higaki K Davies JP Ioannou YA Accumulation of cholera toxin and GM1 ganglioside in the early endosome of Niemann-Pick C1-deficient cells.Proc Natl Acad Sci USA. 2001; 98: 12391-12396Crossref PubMed Scopus (91) Google Scholar The following antibodies were used: mouse anti-γ-adaptin antibody (Sigma); mouse monoclonal anti-ATP7B antibody;16Yang XL Miura N Kawarada Y Terada K Petrukhin K Gilliam TC Sugiyama T Two forms of Wilson disease protein produced by alternative splicing are localized in distinct cellular compartments.Biochem J. 1997; 326: 897-902Crossref PubMed Scopus (88) Google Scholar rabbit polyclonal anti-human cathepsin D antibody (Upstate Biotechnology Incorporated, New York, NY); mouse monoclonal anti-flag antibody (Sigma); mouse monoclonal anti-β1, 4-galactosyltransferase (GalT) antibody;36Kawano J Ide S Oinuma T Suganuma T A protein-specific monoclonal antibody to rat liver β1,4 galactolsyltransferase and its application to immunohistochemistry.J Histochem Cytochem. 1994; 42: 363-369Crossref PubMed Scopus (39) Google Scholar mouse monoclonal anti-58-kd Golgi protein antibody (Sigma); mouse monoclonal anti-lysosome-associated membrane protein (Lamp) 1 and 2 antibodies37Chen JW Murphy TL Willingham MC Pastan I August JT Identification of two lysosomal membrane glycoproteins.J Cell Biol. 1985; 101: 85-95Crossref PubMed Scopus (417) Google Scholar (kind gifts from Prof. J.T. August, Johns Hopkins University, Baltimore, MD); mouse monoclonal anti-myc antibody (CLONTECH); fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin (Dako Japan, Kyoto, Japan); tetramethylrhodamine isothiocyanate (TRITC)-conjugated rabbit anti-mouse immunoglobulin (Dako Japan); TRITC-conjugated swine anti-rabbit immunoglobulin (Dako Japan); Alexa Flour 488-conjugated rabbit anti-mouse immunoglobulin (Molecular Probes, Eugene, OR). Rhodamine-dextran (R-dextran) was used to stain the endocytic compartments (1 mg/ml, Sigma). U18666A (2 μg/ml, Biomol, Plymouth Meeting, PA) was used to manipulate the vesicle traffic in the late endocytic structures. At 48 hours after the transfection, cells on glass coverslips were fixed in freshly prepared 3% paraformaldehyde in phosphate-buffered saline (PBS) for 30 minutes and permeabilized in 0.2% Triton X-100 in PBS for 10 minutes or 0.1% saponin in PBS for 30 minutes. For the detection of NPC1 in flag-NPC 1-transfected cells, cells were fixed with cold ethanol (−20°C) for 20 minutes. Nonspecific binding was blocked by incubation with Protein Block Serum Free (Dako Japan) for 30 minutes followed by incubation with the primary antibodies for 1 hour and the secondary antibodies for 1 hour, respectively. A confocal laser scanning microscope (Fluoview FV 300, Olympus, Tokyo, Japan) equipped with an Argon/Krypton laser was used for observation. For double-labeling analysis, images were acquired sequentially using separate excitation wavelengths of 488 nm for GFP and FITC and 568 nm for TRITC and DsRed, and then merged. At 48 hours after seeding, the cells were treated with bathocuproine disulfonate (Sigma), a copper chelator, or copper sulfate (Wako Pure Chemical Industries, Ltd.) at the concentration of 40 μmol/L or 200 μmol/L for 2 hours, respectively. Cells were fixed with 1% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated in a graded ethanol series, and embedded in Quetol 812 (Nisshin EM, Tokyo, Japan). Ultra-thin sections were stained with uranyl acetate and lead citrate, and examined with a transmission electron microscope (JEM 2000-EX, JEOL, Tokyo, Japan). Immunofluorescent signals of ATP7B of Huh7 cells were shown in Figure 1A, they were observed as a punctate vesicular pattern around the nucleus. ATP7B was localized in the similar manner in cells treated with bathocuproine disulfonate (Figure 1B) or copper sulfate (Figure 1C). GalT has a polarized distribution toward the trans-Golgi and TGN,36Kawano J Ide S Oinuma T Suganuma T A protein-specific monoclonal antibody to rat liver β1,4 galactolsyltransferase and its application to immunohistochemistry.J Histochem Cytochem. 1994; 42: 363-369Crossref PubMed Scopus (39) Google Scholar and it was visualized with the anti-GalT antibody (Figure 1D). GalT was distributed as a compact Golgi ribbon, and the morphologies of the TGN and ATP7B-containing structures were apparently different. Lamp1 is a membrane protein localized in the late endosome and lysosomes, and it was visualized with the anti-Lamp1 antibody.37Chen JW Murphy TL Willingham MC Pastan I August JT Identification of two lysosomal membrane glycoproteins.J Cell Biol. 1985; 101: 85-95Crossref PubMed Scopus (417) Google Scholar Lamp1 was distributed as a vesicular pattern similar to ATP7B, however, ATP7B was more restricted to the perinuclear region (Figure 1E). Immunofluorescent signals of ATP7B in ATP7B cDNA-transfected cells showed a juxtanuclear punctate pattern similar to endogeneous ATP7B (data not shown).11Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Taniguchi E Sasatomi K Miura N Suganuma T Fujita H Furuta K Tanikawa K Sugiyama T Sata M Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes.Gastroenterology. 2000; 118: 921-928Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 12Harada M Sakisaka S Kawaguchi T Kimura R Taniguchi E Koga H Hanada S Baba S Furuta K Kumashiro R Sugiyama T Sata M Copper does not alter the intracellular distribution of ATP7B, a copper-transporting ATPase.Biochem Biophys Res Commun. 2000; 275: 871-876Crossref PubMed Scopus (28) Google Scholar In GFP-ATP7B-transfected cells, GFP signals were observed as a punctate pattern similar to endogeneous ATP7B (Figure 2A).11Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Taniguchi E Sasatomi K Miura N Suganuma T Fujita H Furuta K Tanikawa K Sugiyama T Sata M Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes.Gastroenterology. 2000; 118: 921-928Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 12Harada M Sakisaka S Kawaguchi T Kimura R Taniguchi E Koga H Hanada S Baba S Furuta K Kumashiro R Sugiyama T Sata M Copper does not alter the intracellular distribution of ATP7B, a copper-transporting ATPase.Biochem Biophys Res Commun. 2000; 275: 871-876Crossref PubMed Scopus (28) Google Scholar, 13Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Kim M Taniguchi E Hanada S Suganuma T Furuta K Sugiyama T Sata M A mutation of the Wilson disease protein, ATP7B, is degraded in the proteasomes and forms protein aggregates.Gastroenterology. 2001; 120: 967-974Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 14Harada M Kumemura H Sakisaka S Shishido S Taniguchi E Kawaguchi T Hanada S Koga H Kumashiro R Ueno T Suganuma T Furuta K Namba M Sugiyama T Sata M Wilson disease protein ATP7B is localized in the late endosomes in a polarized human hepatocyte cell line.Int J Mol Med. 2003; 11: 293-298PubMed Google Scholar When GFP-ATP7B transfected cells were labeled with the anti-ATP7B antibody, the GFP signals were identical to the signals produced by the anti-ATP7B antibody (data not shown).11Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Taniguchi E Sasatomi K Miura N Suganuma T Fujita H Furuta K Tanikawa K Sugiyama T Sata M Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes.Gastroenterology. 2000; 118: 921-928Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar GFP alone was observed throughout the cells when pEGFP-C2 was transfected (data not shown).11Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Taniguchi E Sasatomi K Miura N Suganuma T Fujita H Furuta K Tanikawa K Sugiyama T Sata M Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes.Gastroenterology. 2000; 118: 921-928Abstract Full Text Full Text PDF PubMed Scopus (57) Google ScholarFigure 2Confocal laser scanning microscopic images of ATP7B-DsRed-transfected Huh7 cells (A–L). Some cells were cotransfected with GFP-ATP7B (A–C). Green: GFP-ATP7B (A), galactosyltransferase (GalT) (D), lysosome-associated membrane protein (lamp) 1 (G), cathepsin D (J). Red: ATP7B-DsRed (B, E, H, and K). Bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previously, we examined the relation between GFP-ATP7B and various organelles using antibodies to various marker proteins of intracellular organelles and R-dextran. GFP-ATP7B was colocalized with R-dextran, and Lamp 1 and 2, but not with calnexin, γ-adaptin, GalT, or cathepsin D in primary cultured rat hepatocytes, Huh7, Hep3B, HEK293, and OUMS29 cells (Table 1).11Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Taniguchi E Sasatomi K Miura N Suganuma T Fujita H Furuta K Tanikawa K Sugiyama T Sata M Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes.Gastroenterology. 2000; 118: 921-928Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 12Harada M Sakisaka S Kawaguchi T Kimura R Taniguchi E Koga H Hanada S Baba S Furuta K Kumashiro R Sugiyama T Sata M Copper does not alter the intracellular distribution of ATP7B, a copper-transporting ATPase.Biochem Biophys Res Commun. 2000; 275: 871-876Crossref PubMed Scopus (28) Google Scholar, 13Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Kim M Taniguchi E Hanada S Suganuma T Furuta K Sugiyama T Sata M A mutation of the Wilson disease protein, ATP7B, is degraded in the proteasomes and forms protein aggregates.Gastroenterology. 2001; 120: 967-974Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 14Harada M Kumemura H Sakisaka S Shishido S Taniguchi E Kawaguchi T Hanada S Koga H Kumashiro R Ueno T Suganuma T Furuta K Namba M Sugiyama T Sata M Wilson disease protein ATP7B is localized in the late endosomes in a polarized human hepatocyte cell line.Int J Mol Med. 2003; 11: 293-298PubMed Google ScholarTable 1Relation between ATP7B and Markers for Intracellular OrganellesCellMarkerTreatmentLocalization of the markerColocalizationReferenceIsolated rat hepatocytesrhodamine dextran—endocytic structures+11Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Taniguchi E Sasatomi K Miura N Suganuma T Fujita H Furuta K Tanikawa K Sugiyama T Sata M Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes.Gastroenterology. 2000; 118: 921-928Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholargalactosyltransferase—TGN−11Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Taniguchi E Sasatomi K Miura N Suganuma T Fujita H Furuta K Tanikawa K Sugiyama T Sata M Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes.Gastroenterology. 2000; 118: 921-928Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholarlysosomal glycoprotein 8—lysosome−11Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Taniguchi E Sasatomi K Miura N Suganuma T Fujita H Furuta K Tanikawa K Sugiyama T Sata M Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes.Gastroenterology. 2000; 118: 921-928Abstract Full Text Full Text PDF PubMed Scopus (57) Google ScholarZO-1—tight junction−11Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Taniguchi E Sasatomi K Miura N Suganuma T Fujita H Furuta K Tanikawa K Sugiyama T Sata M Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes.Gastroenterology. 2000; 118: 921-928Abstract Full Text Full Text PDF PubMed Scopus (57) Google ScholarHuh7rhodamine dextran—endocytic structures+11Harada M Sakisaka S Terada K Kimura R Kawaguchi T Koga H Taniguchi E Sasatomi K Miura N Suganuma T Fujita H Furuta K Tanikawa K Sugiyama T Sata M Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal ra
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