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• W2023864554 abstract "Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the half-type ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral cells. Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the half-type ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral cells. Cholesterol is important to the body as a component of cellular membranes and a precursor of steroid hormones. However, excess cholesterol is a risk factor for atherosclerosis. Thus, cholesterol levels are strictly regulated by synthesis and circulation in the body. Many ABC proteins are reported to function in lipid homeostasis (1Takahashi K. Kimura Y. Nagata K. Yamamoto A. Matsuo M. Ueda K. ABC proteins, key molecules for lipid homeostasis.Med. Mol. Morphol. 2005; 38: 2-12Crossref PubMed Scopus (72) Google Scholar). For example, ABCG5 and ABCG8 mediate the efflux of cholesterol and sitosterol from intestine and hepatocytes into intestinal lumen and bile duct (2Yu L. Li-Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol.J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (607) Google Scholar, 3Yu L. Hammer R.E. Li-Hawkins J. von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion.Proc. Natl. Acad. Sci. USA. 2002; 99: 16237-16242Crossref PubMed Scopus (608) Google Scholar). ABCB4 (multidrug resistance 3) is a phosphatidylcholine (PC) flippase and functions in the secretion of PC into bile duct from hepatocytes (4Borst P. Zelcer N. van Helvoort A. ABC transporters in lipid transport.Biochim. Biophys. Acta. 2000; 1486: 128-144Crossref PubMed Scopus (261) Google Scholar). ABCA1 mediates the efflux of cholesterol and phospholipids from macrophages to form HDL (5Lee J.Y. Parks J.S. ATP-binding cassette transporter AI and its role in HDL formation.Curr. Opin. Lipidol. 2005; 16: 19-25Crossref PubMed Scopus (163) Google Scholar). ABCG1 is a half-type ABC protein, with a nucleotide binding fold (NBF) in its N-terminal half and a transmembrane region in its C-terminal half. Human ABCG1 cDNA was cloned as a gene homologous to white in Drosophila (6Chen H. Rossier C. Lalioti M.D. Lynn A. Chakravarti A. Perrin G. Antonarakis S.E. Cloning of the cDNA for a human homologue of the Drosophila white gene and mapping to chromosome 21q22.3.Am. J. Hum. Genet. 1996; 59: 66-75PubMed Google Scholar), a transporter of eye pigments, and many N-terminal variant forms of ABCG1 have been reported (7Kennedy M.A. Venkateswaran A. Tarr P.T. Xenarios I. Kudoh J. Shimizu N. Edwards P.A. Characterization of the human ABCG1 gene. Liver X receptor activates an internal promoter that produces a novel transcript encoding an alternative form of the protein.J. Biol. Chem. 2001; 276: 39438-39447Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 8Lorkowski S. Kratz M. Wenner C. Schmidt R. Weitkamp B. Fobker M. Reinhardt J. Rauterberg J. Galinski E.A. Cullen P. Expression of the ATP-binding cassette transporter gene ABCG1 (ABC8) in Tangier disease.Biochem. Biophys. Res. Commun. 2001; 283: 821-830Crossref PubMed Scopus (64) Google Scholar). Other members of the ABCG subfamily form a dimer to function. For example, ABCG2 forms a homodimer to participate in multidrug resistance in breast cancer cells (9Kage K. Tsukahara S. Sugiyama T. Asada S. Ishikawa E. Tsuruo T. Sugimoto Y. Dominant-negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S-S dependent homodimerization.Int. J. Cancer. 2002; 97: 626-630Crossref PubMed Scopus (268) Google Scholar), whereas ABCG5 and ABCG8 form a heterodimer to function in the efflux of sterol from cells in the small intestine and in hepatocytes (10Graf G.A. Li W-P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface.J. Clin. Invest. 2002; 110: 659-669Crossref PubMed Scopus (297) Google Scholar, 11Graf G.A. Yu L. Li W-P. Gerard R. Tuma P.L. Cohen J.C. Hobbs H.H. ABCG5 and ABCG8 are obligate heterodimers for protein trafficking and biliary cholesterol excretion.J. Biol. Chem. 2003; 278: 48275-48282Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Cserepes et al. (12Cserepes J. Szentpetery Z. Seres L. Ozvegy-Laczka C. Langmann T. Schmitz G. Glavinas H. Klein I. Homolya L. Varadi A. et al.Functional expression and characterization of the human ABCG1 and ABCG4 proteins: indications for heterodimerization.Biochem. Biophys. Res. Commun. 2004; 320: 860-867Crossref PubMed Scopus (86) Google Scholar) showed that a WalkerA lysine mutant of ABCG4 inhibited the ATPase activity of ABCG1 in a dominant-negative manner and suggested that ABCG1 can heterodimerize with ABCG4, which is most homologous to ABCG1 among the ABCG subfamily. Cross-linking experiments suggested that ABCG1 forms a homodimer (13Vaughan A.M. Oram J.F. ABCG1 redistributes cell cholesterol to domains removable by high density lipoprotein but not by lipid-depleted apolipoproteins.J. Biol. Chem. 2005; 280: 30150-30157Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). However, it has not been clearly shown that ABCG1 functions as a homodimer. It is thought that ABCG1 is involved in transporting cholesterol, because it is induced upon the loading of macrophages with cholesterol via a pathway of nuclear hormone receptors, liver X receptor (LXR) and retinoid X receptor (RXR) (7Kennedy M.A. Venkateswaran A. Tarr P.T. Xenarios I. Kudoh J. Shimizu N. Edwards P.A. Characterization of the human ABCG1 gene. Liver X receptor activates an internal promoter that produces a novel transcript encoding an alternative form of the protein.J. Biol. Chem. 2001; 276: 39438-39447Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 14Klucken J. Buchler C. Orso E. Kaminski W.E. Porsch-Ozcurumez M. Liebisch G. Kapinsky M. Diederich W. Drobnik W. Dean M. et al.ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport.Proc. Natl. Acad. Sci. USA. 2000; 97: 817-822Crossref PubMed Scopus (474) Google Scholar, 15Venkateswaran A. Repa J.J. Lobaccaro J-M. A. Bronson A. Mangelsdorf D.J. Edwards P.A. Human white/murine ABC8 mRNA levels are highly induced in lipid-loaded macrophages. A transcriptional role for specific oxysterols.J. Biol. Chem. 2000; 275: 14700-14707Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). The ABCG1 gene is expressed in lung, brain, spleen, and macrophages. In liver, ABCG1 is expressed mainly in Kupffer cells (16Hoekstra M. Kruijt J.K. Van Eck M. van Berkel T. J.C. Specific gene expression of ATP-binding cassette transporters and nuclear hormone receptors in rat liver parenchymal, endothelial, and Kupffer cells.J. Biol. Chem. 2003; 278: 25448-25453Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Expression of ABCG1 was increased in macrophages from patients with Tangier disease compared with control macrophages (17Lorkowski S. Rust S. Engel T. Jung E. Tegelkamp K. Galinski E.A. Assmann G. Cullen P. Genomic sequence and structure of the human ABCG1 (ABC8) gene.Biochem. Biophys. Res. Commun. 2001; 280: 121-131Crossref PubMed Scopus (63) Google Scholar). Kennedy et al. (18Kennedy M.A. Barrera G.C. Nakamura K. Baldan A. Tarr P. Fishbein M.C. Frank J. Francone O.L. Edwards P.A. ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation.Cell Metabolism. 2005; 1: 121-131Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar) reported that disruption of ABCG1 in mice on a high-fat, high-cholesterol diet showed accumulation of both neutral lipids and phospholipids in hepatocytes and macrophages, whereas overexpression of ABCG1 protected murine tissues from lipid accumulation. Endogenous ABCG1 is reported to localize to the perinuclear region and, in some cases, is distributed in the plasma membrane of macrophage-derived foam cells (14Klucken J. Buchler C. Orso E. Kaminski W.E. Porsch-Ozcurumez M. Liebisch G. Kapinsky M. Diederich W. Drobnik W. Dean M. et al.ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport.Proc. Natl. Acad. Sci. USA. 2000; 97: 817-822Crossref PubMed Scopus (474) Google Scholar, 17Lorkowski S. Rust S. Engel T. Jung E. Tegelkamp K. Galinski E.A. Assmann G. Cullen P. Genomic sequence and structure of the human ABCG1 (ABC8) gene.Biochem. Biophys. Res. Commun. 2001; 280: 121-131Crossref PubMed Scopus (63) Google Scholar). It was reported that ABCG1 mediates the efflux of cholesterol from cells to HDL-2 or HDL-3 but not to lipid-poor apolipoprotein A-I (apoA-I) (19Wang N. Lan D. Chen W. Matsuura F. Tall A.R. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins.Proc. Natl. Acad. Sci. USA. 2004; 101: 9774-9779Crossref PubMed Scopus (886) Google Scholar) and that ABCG1 redistributes cholesterol to cell surface domains accessible for removal by HDL (13Vaughan A.M. Oram J.F. ABCG1 redistributes cell cholesterol to domains removable by high density lipoprotein but not by lipid-depleted apolipoproteins.J. Biol. Chem. 2005; 280: 30150-30157Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Consistent with these reports, inhibition of ABCG1 protein expression resulted in reduced HDL-3-dependent efflux of cholesterol and phospholipids in macrophages (14Klucken J. Buchler C. Orso E. Kaminski W.E. Porsch-Ozcurumez M. Liebisch G. Kapinsky M. Diederich W. Drobnik W. Dean M. et al.ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport.Proc. Natl. Acad. Sci. USA. 2000; 97: 817-822Crossref PubMed Scopus (474) Google Scholar). These findings suggest that ABCG1 is involved in lipid efflux in peripheral cells, like ABCA1. However, the mechanism of efflux by ABCG1 is not clear. In this study, we investigated the subcellular localization and function of ABCG1. We demonstrated that ABCG1 localized to the plasma membrane in a HEK293 stable cell line and that ABCG1 mediates the efflux of cholesterol and phospholipids [preferentially sphingomyelin (SM)] from cells. Rabbit polyclonal anti-ABCG1 antibody, goat polyclonal anti-ABCG1 antibody, mouse monoclonal anti-myc antibody, goat polyclonal anti-myc antibody, and mouse monoclonal anti-FLAG antibody were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-ABCG8 antibody and mouse monoclonal anti-PentaHis antibody were obtained from Novus Biologicals and Qiagen, respectively. Rabbit polyclonal anti-FLAG and anti-BSA antibodies were purchased from Sigma. Mouse monoclonal anti-ABCA1 antibody was prepared as described previously (20Tanaka A.R. Abe-Dohmae S. Ohnishi T. Aoki R. Morinaga G. Okuhira K.I. Ikeda Y. Kano F. Matsuo M. Kioka N. et al.Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis.J. Biol. Chem. 2003; 278: 8815-8819Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). ABCG5 and ABCG8 cDNA were cloned from a human liver cDNA library. Dithiobis (succinimidylpropionate) (DSP), dimethyl 3,3′-dithiobispropionimidate-HCl (DTBP), and sulfo-N-hydroxysuccinimidobiotin (sulfo-NHS-biotin) were purchased from Pierce. Marathon cDNA libraries from fetal liver or placenta were obtained from Clontech. 8-Azido-[α-32P]ATP was purchased from Affinity Labeling Technologies. Other chemicals were purchased from Sigma, Amersham Biosciences, Wako Pure Chemical Industries, and Nacalai Tesque. ABCG1 cDNA was cloned from a human fetal liver and placental cDNA library. The cDNA sequence cloned in this study was the same as that reported by Kennedy et al. (7Kennedy M.A. Venkateswaran A. Tarr P.T. Xenarios I. Kudoh J. Shimizu N. Edwards P.A. Characterization of the human ABCG1 gene. Liver X receptor activates an internal promoter that produces a novel transcript encoding an alternative form of the protein.J. Biol. Chem. 2001; 276: 39438-39447Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar) (GenBank accession number NM_207630) except for its N terminus, which corresponded to a variant reported by Chen et al. (6Chen H. Rossier C. Lalioti M.D. Lynn A. Chakravarti A. Perrin G. Antonarakis S.E. Cloning of the cDNA for a human homologue of the Drosophila white gene and mapping to chromosome 21q22.3.Am. J. Hum. Genet. 1996; 59: 66-75PubMed Google Scholar) (GenBank accession number X91249). A WalkerA lysine mutant ABCG1 (ABCG1-K120M) was prepared with the QuikChange II Site-Directed Mutagenesis Kit (Stratagene) as described by the manufacturer. The cloned cDNA was inserted into the Not I site of pcDNA3.1(+) (Invitrogen) to make an expression vector for pcDNA3.1(+)/ABCG1 and pcDNA3.1(+)/ABCG1-K120M, or into the NotI-XbaI site of pcDNA3.1(+)A-mycHis (Invitrogen) or pcDNA3.1(+)A-FLAG [prepared by inserting FLAG tag sequences instead of myc and His tag sequences of pcDNA3.1(+)A-mycHis]to make an expression vector for pcDNA3.1(+)A-mycHis/ABCG1 or pcDNA3.1(+)A-FLAG/ABCG1. HEK293 cells were grown in DMEM supplemented with 10% (v/v) FBS in 5% CO2 at 37°C. THP-1 cells were grown in RPMI 1640 medium supplemented with 10% (v/v) FBS in 5% CO2 at 37°C. The differentiation of THP-1 monocytes into macrophages was induced with 0.2 μg/ml phorbol 12-myristrate 13-acetate (Wako Pure Chemical) for 4 days. The differentiated cells were cultured in RPMI 1640 medium and 0.2% BSA for 24 h, and ABCG1 expression was induced for 24 h by adding TO901317 (Cayman). HEK293 cells were transfected with pcDNA3.1(+)/ABCG1 or pcDNA3.1(+)/ABCG1-K120M using LipofectAMINE (Invitrogen) according to the manufacturer's instructions. Cells were selected with 1 mg/ml geneticin (G418) for 2 weeks. Single colonies were isolated, and the expression of ABCG1 was examined by Western blotting and immunofluorescent staining with rabbit polyclonal anti-ABCG1 antibody. Digestion with endoglycosidase H and peptide N-glycosidase F (New England Biolabs, Beverly, MA) was done as described by the manufacturer. In brief, 20 μg of membrane protein from cells was treated with 500 units of endoglycosidase H or 0.3 units of peptide N-glycosidase F for 1 h at 37°C. The deglycosylated proteins were electrophoresed on a 10% SDS-polyacrylamide gel and immunodetected using rabbit polyclonal anti-ABCG1 antibody. Cells were cultured on glass cover slips, fixed with 4% paraformaldehyde in PBS+ (phosphate-buffered saline containing 0.1 g/l CaCl2 and MgCl26H2O), and permeabilized with 0.4% Triton X-100 in PBS+ for 5 min. To diminish the nonspecific binding of antibodies, the cells were incubated in 10% goat serum in PBS+. Cells were incubated for 1 h with rabbit polyclonal anti-ABCG1 antibody diluted 1:500 in PBS+ containing 10% goat serum and then incubated with fluorescent Alexa 488-conjugated anti-rabbit IgG (Molecular Probes) for 1 h. Cells were directly viewed with a 63× Plan-Neofluar oil-immersion objective using a Zeiss confocal microscope (LSM5 Pascal). Cells were washed with ice-cold PBS+ and incubated with 0.5 mg/ml sulfo-NHS-biotin solubilized in PBS+ for 30 min on ice in the dark. Cells were washed with PBS+ to remove unbound sulfo-NHS-biotin and lysed in RIPA buffer [20 mM Tris-Cl (pH 7.5), 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate] containing protease inhibitors [100 μg/ml (p-amidinophenyl)methanesulfonyl fluoride, 2 μg/ml leupeptin, and 2 μg/ml aprotinin). ImmunoPure Immobilized Monomeric Avidin Gel (Pierce) was added to the cell lysate to precipitate the biotinylated proteins. The biotinylated proteins were electrophoresed on a 10% SDS-polyacrylamide gel and immunodetected. After 48 h of transfection with pcDNA3.1(+)A-mycHis/ABCG1 or pcDNA3.1(+)A-FLAG/ABCG1, cells were washed with PBS and lysed in Nonidet P-40 lysis buffer [50 mM Tris-Cl (pH 7.5), 150 mM NaCl, and 1% Nonidet P-40] containing protease inhibitors. The lysates were incubated with antibodies and immunoprecipitated with protein G-Sepharose 4B Fast Flow (Sigma). The immunoprecipitated proteins were washed with Nonidet P-40 lysis buffer and electrophoresed on a 10% SDS-polyacrylamide gel. Cells were washed with cold PBS and incubated with 250 μM DSP or DTBP at room temperature for 30 min. The cross-linking reaction was terminated by the addition of Tris-Cl buffer (pH 7.5) to 20 mM, and cells were incubated at room temperature for 15 min. Cells were washed with PBS and lysed in Nonidet P-40 lysis buffer. Samples were denatured in SDS sample buffer with or without DTT, electrophoresed on a 7% SDS-polyacrylamide gel, and immunodetected. Membranes (20 μg of proteins) from HEK293 cells, prepared as described previously (21Matsuo M. Kioka N. Amachi T. Ueda K. ATP binding properties of the nucleotide binding folds of SUR1.J. Biol. Chem. 1999; 274: 37479-37482Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), were incubated with 50 μM 8-azido-[α-32P]ATP in 3 μl of TEM buffer [40 mM Tris-Cl (pH 7.5), 0.1 mM EGTA, and 1 mM MgCl2] containing 2 mM ouabain for 10 min on ice. Proteins were ultraviolet light-irradiated for 3 min (at 254 nm, 5.5 mW/cm2) on ice. To remove free 8-azido-[α-32P]ATP, ice-cold TEM buffer was added to the sample and the supernatant was removed after centrifugation (15,000 g, 5 min, 2°C). Pellets were resuspended in 100 μl of RIPA buffer containing 100 μg/ml (p-amidinophenyl)methanesulfonyl fluoride, and membrane proteins were solubilized for 30 min at 4°C. After centrifugation for 15 min at 15,000 g, the lysates were incubated with antibodies and immunoprecipitated with protein G-Sepharose 4B Fast Flow (Sigma). The immunoprecipitated proteins were washed with RIPA buffer, and samples were electrophoresed on a 10% SDS-polyacrylamide gel and autoradiographed. Cells were subcultured on 12-well plates at a density of 5.0 × 105 cells. After incubation for 24 h, the cells were labeled with 2 μCi/ml [3H]cholesterol or [3H]choline for 24 h in DMEM containing 10% FBS. The cells were washed with fresh medium and incubated with DMEM containing 0.02% BSA in the absence or presence of 10 μg/ml apoA-I (Calbiochem) or 20 μg/ml HDL (Calbiochem) for 4 h. For cholesterol efflux, the medium was collected and the cells were lysed with 0.1 N NaOH and 0.1% SDS. For phospholipid efflux, phospholipids were extracted from the medium with chloroform-methanol (2:1) or from the cells with hexane-isopropanol (3:2). PC and SM were separated by TLC on silica gel 60 plates (Merck) developed in chloroform-methanol-acetic acid-water (60:30:10:5). The radioactivity was counted by liquid scintillation counting. Cells were subcultured on six-well plates at a density of 1.0 × 106 cells. After incubation for 24 h, the cells were washed with fresh medium and incubated with DMEM containing 0.02% BSA in the absence or presence of 10 μg/ml apoA-I. The lipid content in the medium was determined after 24 or 48 h of incubation as described previously (22Abe-Dohmae S. Suzuki S. Wada Y. Aburatani H. Vance D.E. Yokoyama S. Characterization of apolipoprotein-mediated HDL generation induced by cAMP in a murine macrophage cell line.Biochemistry. 2000; 39: 11092-11099Crossref PubMed Scopus (97) Google Scholar). Cells were subcultured on six-well plates at a density of 1.0 × 106 cells. After incubation for 24 h, the cells were washed with fresh medium and incubated with DMEM containing 0.02% BSA in the absence or presence of 10 μg/ml apoA-I for 48 h. The medium was centrifuged for 15 min at 7,000 g to remove cell debris twice. The lipids were extracted from 12 ml of medium by the method of Bligh and Dyer (23Bligh E.C. Dyer W.F. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1956; 37: 911-917Crossref Scopus (42863) Google Scholar) after the addition of di-14:1 PC as an internal standard. The content of choline phospholipids in the medium was analyzed by MS. Mass spectrometric analyses were performed with a triple quadrupole instrument model Quattro micro (Micromass, Manchester, UK) equipped with an electrospray source as described previously (24Hiramatsu T. Sonoda H. Takanezawa Y. Morikawa R. Ishida M. Kasahara K. Sanai Y. Taguchi R. Aoki J. Arai H. Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A1s, mPA-PLA1α and mPA-PLA1β.J. Biol. Chem. 2003; 278: 49438-49447Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The samples were provided by the UltiMate high-performance liquid chromatography system (LC Packings, San Francisco, CA) into the electrospray interface at a flow rate of 4 μl/min in a solvent system of acetonitrile-methanol-water (2:3:1) containing 0.1% ammonium formate (pH 6.4). The mass spectrometer was operated in the positive and negative scan modes. The flow rate of the nitrogen drying gas was 12 l/min at 80°C. The capillary and cone voltages were set at 3.7 kV and 30 V, respectively, argon at 3–4 × 104 Torr was used as the collision gas, and a collision energy of 30–40 V was used to obtain fragment ions for precursor ions. The relationship between peak height and amount of SM was examined using bovine brain SM (Avanti). Values are presented as means ± SD. Statistical significance was determined by Student’s t-test. A value of P < 0.05 was considered statistically significant. To analyze the subcellular localization and function of human ABCG1, the ABCG1 expression vector was introduced into HEK293 cells, and a cell line stably expressing ABCG1 (HEK/ABCG1) was established. HEK293 cells stably expressing ABCG1-K120M (HEK/ABCG1-K120M), a WalkerA lysine mutant, were also established. Both cell lines expressed similar amounts of ABCG1 migrating as ∼60 kDa proteins on SDS-PAGE (Fig. 1A, lanes 4, 5). THP-1 cells, differentiated by phorbol 12-myristrate, faintly expressed ABCG1 migrating at ∼60 kDa, and ABCG1 was induced by TO901317, a LXR ligand (Fig. 1A, lanes 1, 2). The amount of ABCG1 expressed in HEK/ABCG1 cells was comparable to that in THP-1 cells induced by TO901317. Other members of the ABCG subfamily (ABCG2, ABCG5, and ABCG8) have been reported to be glycosylated (10Graf G.A. Li W-P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface.J. Clin. Invest. 2002; 110: 659-669Crossref PubMed Scopus (297) Google Scholar, 25Maliepaard M. Scheffer G.L. Faneyte I.F. van Gastelen M.A. Pijnenborg A.C.L.M. Schinkel A.H. van de Vijver M.J. Scheper R.J. Schellens J.H.M. Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues.Cancer Res. 2001; 61: 3458-3464PubMed Google Scholar). Indeed, ABCG5, expressed without ABCG8 and modified with high-mannose-type N-linked oligosaccharide, migrated faster after treatment with N-glycosidase F and endoglycosidase H (Fig. 1B, lanes 8–10), as reported (10Graf G.A. Li W-P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface.J. Clin. Invest. 2002; 110: 659-669Crossref PubMed Scopus (297) Google Scholar). However, the migration of ABCG1, expressed in both THP-1 and HEK/ABCG1 cells, was not changed by treatment with either glycosidase (Fig. 1B, lanes 1–3, 5–7). ABCG1 was reported to be distributed mainly in the perinuclear region and only partly in the plasma membrane in macrophages (14Klucken J. Buchler C. Orso E. Kaminski W.E. Porsch-Ozcurumez M. Liebisch G. Kapinsky M. Diederich W. Drobnik W. Dean M. et al.ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport.Proc. Natl. Acad. Sci. USA. 2000; 97: 817-822Crossref PubMed Scopus (474) Google Scholar, 17Lorkowski S. Rust S. Engel T. Jung E. Tegelkamp K. Galinski E.A. Assmann G. Cullen P. Genomic sequence and structure of the human ABCG1 (ABC8) gene.Biochem. Biophys. Res. Commun. 2001; 280: 121-131Crossref PubMed Scopus (63) Google Scholar). However, immunostaining with anti-ABCG1 antibody suggested that the exogenously expressed ABCG1 was distributed mainly in the plasma membrane of HEK293 cells (Fig. 2B,E). No signal was detected in host HEK293 cells (Fig. 2A). ABCG1-K120M was also detected mainly in the plasma membrane (Fig. 2C, F), suggesting that the WalkerA lysine mutation did not affect the subcellular localization of ABCG1. To confirm the cell surface expression of ABCG1, membrane proteins were biotinylated by sulfo-NHS-biotin and precipitated by avidin agarose (Fig. 3, upper panel). We also examined ABCA1 and ABCG8 as a positive and a negative control, respectively. ABCA1, which is localized mainly in the plasma membrane (20Tanaka A.R. Abe-Dohmae S. Ohnishi T. Aoki R. Morinaga G. Okuhira K.I. Ikeda Y. Kano F. Matsuo M. Kioka N. et al.Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis.J. Biol. Chem. 2003; 278: 8815-8819Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), was precipitated by avidin agarose after biotinylation (lane 2) but not without biotinylation (lane 1). On the other hand, ABCG8, which is distributed to the endoplasmic reticulum when expressed alone without ABCG5 (10Graf G.A. Li W-P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface.J. Clin. Invest. 2002; 110: 659-669Crossref PubMed Scopus (297) Google Scholar), was not precipitated by avidin agarose (lane 4). ABCG1 and ABCG1-K120M were precipitated by avidin agarose after biotinylation (lanes 8, 10) but not without biotinylation (lanes 7, 9). These results indicate that ABCG1 localizes to the plasma membrane. Half-type ABC proteins function as homodimers or heterodimers. ABCG2 localizes to the plasma membrane (25Maliepaard M. Scheffer G.L. Faneyte I.F. van Gastelen M.A. Pijnenborg A.C.L.M. Schinkel A.H. van de Vijver M.J. Scheper R.J. Schellens J.H.M. Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues.Cancer Res. 2001; 61: 3458-3464PubMed Google Scholar, 26Rocchi E. Khodjakov A. Volk E.L. Yang C-H. Litman T. Bates S.E. Schneider E. The product of the ABC half-transporter gene ABCG2 (BCRP/MXR/ABCP) is expressed in the plasma membrane.Biochem. Biophys. Res. Commun. 2000; 271: 42-46Crossref PubMed Scopus (167) Google Scholar) and functions as a homodimer (9Kage K. Tsukahara S. Sugiyama T. Asada S. Ishikawa E. Tsuruo T. Sugimoto Y. Dominant-negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S-S dependent homodimerization.Int. J. Cancer. 2002; 97: 626-630Crossref PubMed Scopus (268) Google Scholar), whereas ABCG5 and ABCG8 localize to the plasma membrane and function as heterodimers (10Graf G.A. Li W-P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface.J. Clin. Invest. 2002; 110: 659-669Crossref PubMed Scopus (297) Google Scholar, 11Graf G.A. Yu L. Li W-P. Gerard R. Tuma P.L. Cohen J.C. Hobbs H.H. ABCG5 and ABCG8 are obligate heterodimers for protein trafficking and biliary cholesterol excretio" @default.
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• W2023864554 title "Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1" @default.
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