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• W2034398538 abstract "The ATP-binding cassette half-transporters ABCG5 (G5) and ABCG8 (G8) promote secretion of neutral sterols into bile, a major pathway for elimination of sterols. Mutations in either ABCG5 or ABCG8 cause sitosterolemia, a recessive disorder characterized by impaired biliary and intestinal sterol secretion, sterol accumulation, and premature atherosclerosis. The mechanism by which the G5G8 heterodimer couples ATP hydrolysis to sterol transport is not known. Here we examined the roles of the Walker A, Walker B, and signature motifs in the nucleotide-binding domains (NBD) of G5 and G8 using recombinant adenoviruses to reconstitute biliary sterol transport in G5G8-deficient mice. Mutant forms of each half-transporter were co-expressed with their wild-type partners. Mutations at crucial residues in the Walker A and Walker B domains of G5 prevented biliary sterol secretion, whereas mutations of the corresponding residues in G8 did not. The opposite result was obtained when mutations were introduced into the signature motif; mutations in the signature domain of G8 prevented sterol transport, but substitution of the corresponding residues in G5 did not. Taken together, these findings indicate that the NBDs of G5 and G8 are not functionally equivalent. The integrity of the canonical NBD formed by the Walker A and Walker B motifs of G5 and the signature motif of G8 is essential for G5G8-mediated sterol transport. In contrast, mutations in key residues of the NBD formed by the Walker A and B motifs of G8 and the signature sequence of G5 did not affect sterol secretion. The ATP-binding cassette half-transporters ABCG5 (G5) and ABCG8 (G8) promote secretion of neutral sterols into bile, a major pathway for elimination of sterols. Mutations in either ABCG5 or ABCG8 cause sitosterolemia, a recessive disorder characterized by impaired biliary and intestinal sterol secretion, sterol accumulation, and premature atherosclerosis. The mechanism by which the G5G8 heterodimer couples ATP hydrolysis to sterol transport is not known. Here we examined the roles of the Walker A, Walker B, and signature motifs in the nucleotide-binding domains (NBD) of G5 and G8 using recombinant adenoviruses to reconstitute biliary sterol transport in G5G8-deficient mice. Mutant forms of each half-transporter were co-expressed with their wild-type partners. Mutations at crucial residues in the Walker A and Walker B domains of G5 prevented biliary sterol secretion, whereas mutations of the corresponding residues in G8 did not. The opposite result was obtained when mutations were introduced into the signature motif; mutations in the signature domain of G8 prevented sterol transport, but substitution of the corresponding residues in G5 did not. Taken together, these findings indicate that the NBDs of G5 and G8 are not functionally equivalent. The integrity of the canonical NBD formed by the Walker A and Walker B motifs of G5 and the signature motif of G8 is essential for G5G8-mediated sterol transport. In contrast, mutations in key residues of the NBD formed by the Walker A and B motifs of G8 and the signature sequence of G5 did not affect sterol secretion. ABC 2The abbreviations used are: ABC, ATP-binding cassette; G5, ABCG5; G8, ABCG8; TAP, antigen processing; NBD, nucleotide-binding domain; BeFx, beryllium fluoride; AlFx, aluminum fluoride; Vi, orthovanadate.2The abbreviations used are: ABC, ATP-binding cassette; G5, ABCG5; G8, ABCG8; TAP, antigen processing; NBD, nucleotide-binding domain; BeFx, beryllium fluoride; AlFx, aluminum fluoride; Vi, orthovanadate. transporters comprise a large family of polytopic membrane proteins that use the energy of ATP hydrolysis to translocate a wide variety of substrates across biological membranes (1.Davidson A.L. Chen J. Annu. Rev. Biochem. 2004; 73: 241-268Crossref PubMed Scopus (484) Google Scholar). Most ABC transporters share a common architecture that includes two NBDs arranged in series with two membrane-spanning domains. The G family of ABC transporters includes five half-transporters that each contain a single hydrophilic NBD at the NH2 terminus and a membrane-spanning domain consisting of six transmembrane α-helices at the COOH terminus. Some of the G family members, such as ABCG2, function as homodimers, whereas other family members, such as ABCG5 (G5) and ABCG8 (G8), function as heterodimers (2.Graf G.A. Li W.-P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. J. Clin. Investig. 2002; 110: 659-669Crossref PubMed Scopus (297) Google Scholar).G5 and G8 are expressed almost exclusively in hepatocytes and enterocytes, where they promote excretion of cholesterol and plant sterols into bile and into the gut lumen, respectively (3.Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1338) Google Scholar). Expression of the two proteins is coordinately regulated at the transcriptional level (3.Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1338) Google Scholar); ABCG5 and ABCG8 are juxtaposed on chromosome 2 and up-regulated by the nuclear hormone receptor liver X receptor (4.Repa J.J. Berge K.E. Pomajzl C. Richardson J.A. Hobbs H. Mangelsdorf D.J. J. Biol. Chem. 2002; 277: 18793-18800Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar), which promotes the expression of numerous other proteins involved in the centripetal movement of sterols from peripheral tissues to the liver (5.Repa J.J. Mangelsdorf D.J. Nat. Med. 2002; 8: 1243-1248Crossref PubMed Scopus (338) Google Scholar). Expression of both G5 and G8 is required for either protein to be transported out of the endoplasmic reticulum (2.Graf G.A. Li W.-P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. J. Clin. Investig. 2002; 110: 659-669Crossref PubMed Scopus (297) Google Scholar, 6.Graf G.A. Yu L. Li W.P. Gerard R. Tuma P.L. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2003; 278: 48275-48282Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar), and mutations in either ABCG5 or ABCG8 cause sitosterolemia, a rare autosomal recessive disorder characterized by sterol accumulation and premature coronary atherosclerosis (3.Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1338) Google Scholar, 7.Lee M.H. Lu K. Hazard S. Yu H. Shulenin S. Hidaka H. Kojima H. Allikmets R. Sakuma N. Pegoraro R. Srivastava A.K. Salen G. Dean M. Patel S.B. Nat. Genet. 2001; 27: 79-83Crossref PubMed Scopus (0) Google Scholar). Mice lacking G5 and G8 (G5G8-/-) have a markedly reduced capacity to secrete sterols into bile (8.Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16237-16242Crossref PubMed Scopus (601) Google Scholar), whereas overexpression of G5 and G8 in the liver dramatically increases biliary cholesterol levels (9.Yu L. Li-Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. J. Clin. Investig. 2002; 110: 671-680Crossref PubMed Scopus (600) Google Scholar).The mechanism used by ABC transporters to couple ATP hydrolysis to sterol transport has not been fully elucidated. Three highly conserved sequence elements in the NBDs of ABC transporters: the Walker A motif, the Walker B motif, and a signature motif, play critical roles in nucleotide binding and hydrolysis (10.Higgins C. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar). The x-ray crystallographic structures of several bacterial ABC transporters have revealed that the two NBDs of each transporter are formed by association between the Walker A and B motifs of one subunit and the signature motif of the other (11.Chen J. Lu G. Lin J. Davidson A.L. Quiocho F.A. Mol. Cell. 2003; 12: 651-661Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 12.Hopfner K.P. Karcher A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar, 13.Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (929) Google Scholar, 14.Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). ATP binds at the interface between the two subunits. A highly conserved basic residue (usually lysine) within the Walker A motif stabilizes the interaction between the NBD and the nucleotide (15.Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar), and the aspartate of the Walker B motif orients the magnesium ion in the nucleotide-binding site. The glutamate immediately downstream of the Walker B motif is highly conserved and plays a critical role in transport function (16.Payen L.F. Gao M. Westlake C.J. Cole S.P. Deeley R.G. J. Biol. Chem. 2003; 278: 38537-38547Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 17.Sauna Z.E. Muller M. Peng X.H. Ambudkar S.V. Biochemistry. 2002; 41: 13989-14000Crossref PubMed Scopus (93) Google Scholar, 18.Tombline G. Bartholomew L.A. Tyndall G.A. Gimi K. Urbatsch I.L. Senior A.E. J. Biol. Chem. 2004; 279: 46518-46526Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), although the exact mechanistic role of this residue remains controversial. It has been suggested that the carboxylic group of the glutamate coordinates a water molecule that initiates ATP hydrolysis by promoting nucleophilic attack on the γ-phosphate of the bound ATP (15.Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar, 19.Verdon G. Albers S.V. Dijkstra B.W. Driessen A.J. Thunnissen A.M. J. Mol. Biol. 2003; 330: 343-358Crossref PubMed Scopus (133) Google Scholar). However, in ABCB1(P-glycoprotein), the conserved glutamate is not critical for the initial cleavage of the bond between the β and γ phosphates (17.Sauna Z.E. Muller M. Peng X.H. Ambudkar S.V. Biochemistry. 2002; 41: 13989-14000Crossref PubMed Scopus (93) Google Scholar, 18.Tombline G. Bartholomew L.A. Tyndall G.A. Gimi K. Urbatsch I.L. Senior A.E. J. Biol. Chem. 2004; 279: 46518-46526Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). A conserved glycine at the fourth position of the signature motif interacts with the oxygen of the γ-phosphate of the ATP (11.Chen J. Lu G. Lin J. Davidson A.L. Quiocho F.A. Mol. Cell. 2003; 12: 651-661Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 12.Hopfner K.P. Karcher A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar, 14.Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). Based on the available structural and functional data from other ABC transporters (11.Chen J. Lu G. Lin J. Davidson A.L. Quiocho F.A. Mol. Cell. 2003; 12: 651-661Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 12.Hopfner K.P. Karcher A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar, 13.Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (929) Google Scholar, 14.Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar, 15.Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar), we have developed a working model of the NBDs in the G5G8 heterodimer (Fig. 1).In some transporters, such as ABCB1 (MDR1 or P-glycoprotein), the binding of ATP to one NBD precedes the binding of a second nucleotide to the other NBD, and ATP hydrolysis at both sites is required for efficient substrate transport (20.Senior A.E. Acta Physiol. Scand. Suppl. 1998; 643: 213-218PubMed Google Scholar, 21.Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar). In other transporters, such as ABCC7 (cystic fibrosis transmembrane conductance regulator), ATP hydrolysis at only one NBD is required to energize transport (22.Berger A.L. Ikuma M. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 455-460Crossref PubMed Scopus (76) Google Scholar). The precise roles of the two NBDs in G5 and G8 are not known. Development of an in vitro system to characterize the ATPase activity and sterol transport activity of G5 and G8 has proved to be challenging, and to date no such assay has been developed. Here we have expressed recombinant G5 and G8 in cultured cells and in genetically modified mice expressing no G5 and G8 to characterize the relative functional roles of the Walker A, Walker B, and signature motifs in each half-transporter. The results presented demonstrate that motifs in both NBDs are required for biliary sterol transport, but the actions of the two ATP-binding sites of G5 and G8 are not functionally equivalent.EXPERIMENTAL PROCEDURESMaterials—Culture medium and fetal bovine serum were obtained from Invitrogen. 8-Azido-ATP, 8-azido[α-32P]ATP (14.3 Ci/mmol), and 8-azido[α-32P]ADP (15.4 Ci/mmol) were purchased from Affinity Labeling Technologies, Inc. (Lexington, KY). BeCl2, AlCl3, NaF, and Vi were obtained from Sigma. Complete EDTA-free protease inhibitors were purchased from Roche Applied Science; Nonidet P-40 was purchased from Calbiochem (La Jolla, CA). All other chemicals and reagents were obtained from Sigma-Aldrich unless otherwise indicated.Site-directed Mutagenesis—The mutations were generated using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The template used was a shuttle vector, pACCMVpLpA(-)loxP-SSP, containing G5 or G8 cDNAs. Oligonucleotides bearing mismatched bases at the residues to be mutated (underlined) were synthesized by IDT, Inc. (Coralville, IA). They are as follows: for G5, K93M-forward (5′-TCA GGC TCA GGG ATG ACC ACG CTG-3′), K93M-reverse (5′-CAG CGT GGT CAT CCC TGA GCC TGA-3′), K93R-forward (5′-TCA GGC TCA GGG AGG ACC ACG CTG-3′), K93R-reverse (5′-CAG CGT GGT CCT CCC TGA GCC TGA-3′), G197D-forward (5′-GGA ATT TCC AGT GAC GAG CGG CGC CGA-3′), G197D-reverse (5′-TCG GCG CCG CTC GTC ACT GGA AAT TCC-3′), I194VS196G-forward (5′-AAT TTT GGG GGA GTT TCC GGT GGC GAG CGG CGC-3′), I194VS196G-reverse (5′-GCG CCG CTC GCC ACC GGA AAC TCC CCC AAA ATT-3′), E219D-forward (5′-ATG ATG CTA GAT GAC CCA ACC ACA GGA-3′), E219D-reverse (5′TCC TGT GGT TGG GTC ATC TAG CAT CAT-3′), E219Q-forward (5′-ATG ATG CTA GAT CAG CCA ACC ACA GGA-3′), E219Q-reverse (5′-TCC TGT GGT TGG CTG ATC TAG CAT CAT-3′). For G8, R111M-forward (5′-TCA GGC TGC GGG ATG GCC TCA CTA CTC-3′), R111M-reverse (5′GAG TAG TGA GGC CAT CCC GCA GCC TGA-3′), R111K-forward (5′-TCA GGC TGC GGG AAG GCC TCA CTA CTC-3′), R111K-reverse (5′GAG TAG TGA GGC CTT CCC GCA GCC TGA-3′), G216D-forward (5′-GGG GTG TCC GGG GAT GAG CGC CGA CGA-3′), G216D-reverse (5′-TCG TCG GCG CTC ATC CCC GGA CAC CCC-3′), V214IG215S-forward (5′-TAT GTA CGT GGG ATC TCC TCG GGT GAG CGC CGA-3′), V214IG215S-reverse (5′-TCG GCG CTC ACC CGA GGA GAT CCC ACG TAC ATA-3′), E238D-forward (5′-CTC ATT CTG GAT GAC CCC ACT TCT GGC-3′), E238D-reverse (5′-GCC AGA AGT GGG GTC ATC CAG AAT GAG-3′), E238Q-forward (5′-CTC ATT CTG GAT CAG CCC ACT TCT GGC-3′), and E238D-reverse (5′-GCC AGA AGT GGG CTG ATC CAG AAT GAG-3′). The presence of the desired mutation and the integrity of each construct were verified by DNA sequencing.Infection of CRL-1601 Cells with Recombinant Adenoviral Vectors—Recombinant adenoviral vectors containing cDNAs for wild-type and mutant G5 and G8 were generated by in vitro cre-lox recombination as described previously (2.Graf G.A. Li W.-P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. J. Clin. Investig. 2002; 110: 659-669Crossref PubMed Scopus (297) Google Scholar). Cultured rat hepatoma cells (CRL-1601) were maintained in Dulbecco's modified Eagle's medium (glucose, 1 g/liter) containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. On day one, the cells were seeded at 5 × 106 cells/150-mm plate. After 24 h, the cells were infected with recombinant adenoviral vectors containing cDNAs for wild-type or mutant G5 and G8 (8 × 1010 particles/plate). After 60 h, the cells were harvested, and a plasma membrane-enriched fraction was isolated by centrifugation through sucrose, as described (23.Zhang D.W. Cole S.P. Deeley R.G. J. Biol. Chem. 2001; 276: 13231-13239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Protein concentrations were determined using the BCA assay (Pierce) according to the manufacturer's protocol, and the membrane fractions were aliquotted and stored at -80 °C.Expression of Recombinant G5 and G8 in Mice Using Adenovirus—Mice homozygous for disrupted alleles at Abcg5 and Abcg8 (G5G8-/-) were generated as described (8.Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16237-16242Crossref PubMed Scopus (601) Google Scholar) and maintained on a regular chow diet (Harlan Teklad, Madison, WI). Adenoviral particles (5 × 1012 particles/kg) were injected into the tail veins of the mice. After 72 h, the mice were fasted for 4 h, anesthetized with halothane, and killed by exsanguination. Bile was collected, and neutral sterol levels were measured using gas liquid chromatography and mass spectrometry as described (6.Graf G.A. Yu L. Li W.P. Gerard R. Tuma P.L. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2003; 278: 48275-48282Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Liver tissue was snap frozen in liquid nitrogen and stored at -80 °C.SDS-PAGE and Immunoblot Analysis of G5 and G8—The membrane proteins were subjected to SDS-PAGE and then transferred to nitrocellulose membranes (Amersham Biosciences) by electroblotting. Immunoblotting was performed using an anti-G8 monoclonal antibody (1B10A5) or a rabbit polyclonal antibody (5161) directed against a recombinant peptide corresponding to amino acids 1–350 of G8 (6.Graf G.A. Yu L. Li W.P. Gerard R. Tuma P.L. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2003; 278: 48275-48282Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar); G5 was detected using rabbit anti-mouse polyclonal antibody (antibody 5155 or 4591) directed against the corresponding NH2-terminal peptide from G5 (6.Graf G.A. Yu L. Li W.P. Gerard R. Tuma P.L. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2003; 278: 48275-48282Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Antibody binding was detected using horseradish peroxidase-conjugated goat anti-mouse or with donkey anti-rabbit IgG (Amersham Biosciences), followed by enhanced chemiluminescence detection (Pierce). The membranes were then exposed to F-BX810™ Blue X-Ray films (Phoenix Research Products, Hayward, CA).Immunoprecipitation of G5 and G8—The membrane fractions of cells were solubilized in 400 μl of lysis buffer (50 mm HEPES, 100 mm NaCl, 1.5 mm MgCl2, 5 mm dithiothreitol, 1% (v/v) Nonidet P-40, 0.1% SDS, 5 mm EDTA) containing 1× protease inhibitors (Complete EDTA-free protease inhibitors; Roche Applied Science) for 2 h at 4°C. The cell lysates were precleared by adding 10 μl of a 50% (v/v) suspension of protein A-agarose (RepliGen Corp., Waltham, MA) and 10 μlof a 50% (v/v) suspension of protein G-agarose (Upstate, Lake Placid, NY), and the samples were centrifuged at 16,000 × g for 5 min. The precleared lysates were incubated with an anti-G8 monoclonal antibody (20 μg) and 20 μl of a 50% (v/v) suspension of protein G-agarose overnight at 4 °C. After centrifugation at 3,000 × g for 5 min, the pellets were washed in lysis buffer three times for 10 min at 4 °C, and then the proteins were eluted using 1× SDS loading buffer (31 mm Tris-HCl, pH 6.8, 1%SDS, 12.5% glycerol, 0.0025% bromphenol) containing 5% β-mercaptoethanol. The supernatants were transferred into new microcentrifuge tubes and incubated with 2 μl of anti-G5 polyclonal antibody (1:200), and 20 μl of a 50% (v/v) suspension of protein A-agarose for 6 h at 4°C. After extensive washing, the proteins were eluted from the beads with 1× SDS loading buffer containing 5% β-mercaptoethanol. The proteins were then analyzed by SDS-PAGE and immunoblotting. The antibodies were stripped from the membrane by incubation for 40 min at 55 °C in stripping buffer (Pierce). Stripped membranes were confirmed to be free of signal by repeat autoradiography before incubating the filter with another antibody.Photolabeling of G5 and G8 with 8-Azido[α-32P]ATP and BeFx-induced Trapping of 8-Azido[α-32P]ADP by G5 and G8—ATP binding was examined by photolabeling with 8-azido[α-32P]ATP using membrane proteins isolated from CRL-1601 cells expressing recombinant G5, G8, or G5 plus G8 as described previously (24.Zhang D.W. Gu H.M. Situ D. Haimeur A. Cole S.P. Deeley R.G. J. Biol. Chem. 2003; 278: 46052-46063Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Briefly, membrane fractions (10–40 μg) were incubated in 30 μl of labeling buffer (250 mm sucrose, 50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 0.02% NaN3) containing 10 μm 8-azido[α-32P]ATP on ice for 5 min and then subjected to UV irradiation for 3 min at 4 °C. The reactions were terminated by the addition of 400 μl of ice-cold stop buffer (50 mm Tris-HCl, pH 7.4, 0.1 mm EGTA, 5 mm MgCl2), and the membranes were centrifuged at 16,000 × g for 30 min at 4 °C. The pellets were solubilized in 400 μl of lysis buffer, and G5 and G8 were immunoprecipitated as described above. The samples were size-fractionated on 8% gels by SDS-PAGE and then transferred to nitrocellulose membranes by electroblotting. The membranes were dried and exposed to F-BX810™ Blue X-ray films overnight at -80 °C.An identical protocol was used for the ATP competition experiment except that membrane fractions were incubated in labeling buffer containing 10 μm 8-azido[α-32P]ATP and various amounts of unlabeled ATP (0, 1, 10, 50 mm). To determine the binding affinity of G5 and G8 for 8-azido[α-32P]ATP, the experiment was performed in the presence of increasing concentrations of 8-azido[α-32P]ATP (ranging from 0.5 to 160 μm). The radioactivity incorporated into the G5 and G8 bands was quantified using a STORM 820 PhosphorImager system (Amersham Biosciences) and the software ImageQuant 5.0. Apparent Kd values were obtained from the best fit of the data to a hyperbolic curve using Graphpad software and the equation Y = BmaxX/(Kd + X), where Bmax is the maximal binding, and Kd is the concentration of ligand required to reach half-maximal binding.BeFx, AlFx, and Vi-induced trapping of 8-azido[α-32P]ADP was performed as described (25.Sankaran B. Bhagat S. Senior A.E. Arch. Biochem. Biophys. 1997; 341: 160-169Crossref PubMed Scopus (33) Google Scholar, 26.Sankaran B. Bhagat S. Senior A.E. Biochemistry. 1997; 36: 6847-6853Crossref PubMed Scopus (62) Google Scholar). Briefly, membrane proteins (10–40 μg) were incubated in labeling buffer (30 μl) containing 10 μm 8-azido[α-32P]ATP, and 1 mm (or 5 mm) Vi or 5 mm NaF plus either 100 μm BeCl2 or 100 μm AlCl3 at 37 °C for 15 min. The samples were incubated on ice for 5 min in the presence of 30 mm cold ATP, followed by UV irradiation. The membranes were centrifuged at 16,000 × g for 30 min at 4 °C, and the pellets were washed twice in 400 μl of ice-cold stop buffer and resuspended in 400 μl of lysis buffer, and G5 and G8 were separately immunoprecipitated as described. The proteins were size-fractionated on 8% gels by SDS-PAGE and subsequently transferred to nitrocellulose membranes by electroblotting. The membranes were then dried and exposed to F-BX810™ Blue X-Ray films overnight at -80 °C. The same membranes were also used for immunoblot analysis of G5 and G8.RESULTS8-Azido[α-32P]ATP Binding to G5 and G8 Does Not Require G5G8 Dimerization—To examine the binding of ATP to G5 and G8, we used recombinant adenoviruses to express the two proteins in cultured rat hepatoma cells (CRL-1601), either individually or together, and then immunoprecipitated the proteins from a solubilized membrane fraction. Only the lower molecular weight, precursor (P) forms of the proteins were seen when either G5 or G8 was expressed alone in cells (Fig. 2A). The mature (M), fully glycosylated forms of G5 and G8 were present only when G5 and G8 were co-expressed. These results are consistent with our prior findings that G5 and G8 must heterodimerize to exit the endoplasmic reticulum and transit through the Golgi complex to the cell surface (6.Graf G.A. Yu L. Li W.P. Gerard R. Tuma P.L. Cohen J.C. Hobbs H.H. J. Biol. Chem. 2003; 278: 48275-48282Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar).FIGURE 2Immunoblot analysis and 8-azido[α-32P]ATP binding to recombinant G5 and G8 in cultured rat hepatocytes (CRL-1601 cells). A, membrane proteins (30 μg) were isolated from CRL-1601 cells expressing wild-type G5, wild-type G8, G5 plus G8, or vector alone and incubated on ice with 8-azido[α-32P]ATP (10 μm) in labeling buffer for 5 min. The membranes were subjected to UV irradiation, and then G5 and G8 were immunoprecipitated using anti-G5 and anti-G8 specific antibodies (G5, left panel; G8, right panel). The immunoprecipitates were size-fractionated on SDS-PAGE gels, and the proteins were transferred to nitrocellulose membranes. The membranes were exposed to autoradiography (bottom panels). Immunoblotting was performed on the same membrane using anti-G5 (top panels) and anti-G8 (middle panels) polyclonal antibodies. B, competition of 8-azido[α-32P]ATP binding by cold ATP. The binding of 8-azido[α-32P]ATP to G5 (top) and G8 (bottom) was measured as described for A except that the reaction was performed in the presence of unlabeled ATP (0, 1, 10, and 50 mm). C, binding of 8-azido[α-32P]ATP to G5 (top panel) and G8 (bottom panel) from membrane proteins (30 μg) was measured in the presence or absence of MgCl2 (5 mm). Molecular mass standards (in kDa) are provided. The experiment was repeated twice, and the results were similar.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next we examined whether heterodimerization and transport of G5 and G8 out of the endoplasmic reticulum is required for the proteins to bind an analog of ATP, 8-azido[α-32P]ATP. In cells expressing both G5 and G8, the precursor and mature forms of G5 and G8 photolabeled with 8-azido[α-32P]ATP. The degree of labeling was proportional to the amount of protein present (Fig. 2A). Azido-ATP also bound to G5 and G8 when they were expressed individually in cells. Thus, heterodimization of G5 and G8 was not required for ATP binding. Binding to both the precursor and mature forms of G5 and G8 was competed by the addition of excess cold nucleotide (Fig. 2B).Binding of 8-azido[α-32P]ATP required Mg2+ (Fig. 2C) and was inhibited by the addition of EDTA (data not shown) in cells expressing both half-transporters. Magnesium ions were also required to photolabel G5 with 8-azido[α32-P]ATP when the half-transporter was expressed individually in cells (Fig. 2C). In contrast to these findings, magnesium was not required for 8-azido[α-32P]ATP to bind to G8 when it was expressed in the absence of G5 (Fig. 2C).Next we examined the relative affinity of G5 and G8 for 8-azido[α-32P]ATP by measuring the amount of nucleotide bound to each half-transporter over a range of concentrations (Fig. 3). When G5 and G8 were expressed individually in cells, G5 bound ATP in a saturable manner with an apparent Kd of 66 μm, whereas ATP binding to G8 was not fully saturable up to a concentration of 160 μm (Fig. 3A). Co-expression of G5 and G8 did not affect the affinity of ATP binding to G5 (apparent Kd = 57 μm; Fig. 3B), whereas it increased the affinity of G8 for ATP. The binding of ATP to G8 became saturable with an apparent Kd of 174 μm. These data demonstrated that expressing G8 together with G5 resulted in a higher affinity, magnesium-dependent ATP binding to G8.FIGURE 3Binding affinity of G5 and G8 for 8-azido[α-32P]ATP. Cellular membranes containing either G5 or G8 (A) or both G5 and G8 (B) were incubated with increasing concentrations of 8-azido-[α32P]ATP and processed as described in the legend to Fig. 2A. The amount of radioactivity in the G5 and G8 bands was quantified using ImageQuaNT (29.Sauna Z.E. Nandigama K. Ambudkar S.V. J. Biol. Chem. 2004; 279: 48855-48864Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The apparent Kd values for G5 and G8 were obtained from the best fit of the binding data to a hyperbolic curve using GraphPad Prizm 4 software. The experiment was repeated, and similar results were obtained.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nucleotide Trapping by BeFx and AlFx—Currently, no assays are available to detect the ATPase activity of G5 and G8. We therefore examined the ability of the G5G8 heterodimer to bind and hydrolyze ATP by determining whether the protein complex can trap ADP after hydrolysis in the presence of various p" @default.
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• W2034398538 title "Functional Asymmetry of Nucleotide-binding Domains in ABCG5 and ABCG8" @default.
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