Role of Carboxylate Residues Adjacent to the Conserved Core Walker B Motifs in the Catalytic Cycle of Multidrug Resistance Protein 1 (ABCC1)
2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês
10.1074/jbc.m305786200
ISSN1083-351X
AutoresLéa Payen, Mian Gao, Christopher J. Westlake, Susan P.C. Cole, Roger G. Deeley,
Tópico(s)Trace Elements in Health
ResumoMRP1 belongs to subfamily "C" of the ABC transporter superfamily. The nucleotide-binding domains (NBDs) of the C family members are relatively divergent compared with many ABC proteins. They also differ in their ability to bind and hydrolyze ATP. In MRP1, NBD1 binds ATP with high affinity, whereas NBD2 is hydrolytically more active. Furthermore, ATP binding and/or hydrolysis by NBD2 of MRP1, but not NBD1, is required for MRP1 to shift from a high to low affinity substrate binding state. Little is known of the structural basis for these functional differences. One minor structural difference between NBDs is the presence of Asp COOH-terminal to the conserved core Walker B motif in NBD1, rather than the more commonly found Glu present in NBD2. We show that the presence of Asp or Glu following the Walker B motif profoundly affects the ability of the NBDs to bind, hydrolyze, and release nucleotide. An Asp to Glu mutation in NBD1 enhances its hydrolytic capacity and affinity for ADP but markedly decreases transport activity. In contrast, mutations that eliminate the negative charge of the Asp side chain have little effect. The decrease in transport caused by the Asp to Glu mutation in NBD1 is associated with an inability of MRP1 to shift from high to low affinity substrate binding states. In contrast, mutation of Glu to Asp markedly increases the affinity of NBD2 for ATP while decreasing its ability to hydrolyze ATP and to release ADP. This mutation eliminates transport activity but potentiates the conversion from a high to low affinity binding state in the presence of nucleotide. These observations are discussed in the context of catalytic models proposed for MRP1 and other ABC drug transport proteins. MRP1 belongs to subfamily "C" of the ABC transporter superfamily. The nucleotide-binding domains (NBDs) of the C family members are relatively divergent compared with many ABC proteins. They also differ in their ability to bind and hydrolyze ATP. In MRP1, NBD1 binds ATP with high affinity, whereas NBD2 is hydrolytically more active. Furthermore, ATP binding and/or hydrolysis by NBD2 of MRP1, but not NBD1, is required for MRP1 to shift from a high to low affinity substrate binding state. Little is known of the structural basis for these functional differences. One minor structural difference between NBDs is the presence of Asp COOH-terminal to the conserved core Walker B motif in NBD1, rather than the more commonly found Glu present in NBD2. We show that the presence of Asp or Glu following the Walker B motif profoundly affects the ability of the NBDs to bind, hydrolyze, and release nucleotide. An Asp to Glu mutation in NBD1 enhances its hydrolytic capacity and affinity for ADP but markedly decreases transport activity. In contrast, mutations that eliminate the negative charge of the Asp side chain have little effect. The decrease in transport caused by the Asp to Glu mutation in NBD1 is associated with an inability of MRP1 to shift from high to low affinity substrate binding states. In contrast, mutation of Glu to Asp markedly increases the affinity of NBD2 for ATP while decreasing its ability to hydrolyze ATP and to release ADP. This mutation eliminates transport activity but potentiates the conversion from a high to low affinity binding state in the presence of nucleotide. These observations are discussed in the context of catalytic models proposed for MRP1 and other ABC drug transport proteins. ATP-binding cassette (ABC) 1The abbreviations used are: ABC, ATP-binding cassette; MRP, multidrug resistance protein; LTC4, leukotriene C4; E217βG, 17β-estradiol-17-β-(d-glucuronide); NBD, nucleotide-binding domain; MSD, membrane-spanning domain; Wt, ATPγS, adenosine 5′-O-(thiotriphosphate); AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; P-GP, P-glycoprotein; mAb, monoclonal antibody. transporters are ubiquitous transmembrane proteins that couple ATP hydrolysis to the energy-dependent transport of a wide variety of endogenous and exogenous molecules across biological membranes. Multidrug resistance protein (MRP) 1 (ABCC1) belongs to the "C" subfamily of the ABC superfamily and was discovered by virtue of its ability to cause multidrug resistance when overexpressed in a human small cell lung cancer cell line (1Cole S.P.C. Deeley R.G. Cancer Treat. Res. 1996; 87: 39-62Crossref PubMed Scopus (36) Google Scholar, 2Hipfner D.R. Deeley R.G. Cole S.P. Biochim. Biophys. Acta. 1999; 1461: 359-376Crossref PubMed Scopus (379) Google Scholar). The MRP1 multidrug resistance phenotype is similar to that resulting from overexpression of P-glycoprotein (P-GP), and involves resistance to many relatively hydrophobic, natural product type, cytotoxic agents. However, unlike P-GP, MRP1 can also transport various structurally unrelated organic anionic conjugates, including: glutathione, glucuronide, and sulfate conjugates, such as the potent mediator of inflammation, cysteinyl leukotriene LTC4, the cholestatic glucuronide-conjugated estrogen E217βG, the sulfate conjugate estrone 3-sulfate, and the glutathione epoxide conjugate of the highly mutagenic aflatoxin B1 (3Cole S.P. Sparks K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910PubMed Google Scholar, 4Cole S.P. Deeley R.G. Bioessays. 1998; 20: 931-940Crossref PubMed Scopus (333) Google Scholar, 5Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P. J. Biol. 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Typically, the functional form of ABC proteins consists of two hydrophilic nucleotide-binding domains (NBDs) located at the cytoplasmic surface of the membrane and two hydrophobic transmembrane spanning domains (MSD) that are thought to form the translocation pathway. The predicted topology of some of the ABCC transporters such as MRP1, MRP2, MRP3, MRP6, and MRP7, as well SUR1 and SUR2, is unusual in that it includes an additional NH2-terminal MSD that probably contains five transmembrane segments and has an extracellular NH2 terminus (11Hipfner D.R. Almquist K.C. Leslie E.M. Gerlach J.H. Grant C.E. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1997; 272: 23623-23630Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 12Kast C. Gros P. J. Biol. Chem. 1997; 272: 26479-26487Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 13Raab-Graham K.F. Cirilo L.J. Boettcher A.A. Radeke C.M. Vandenberg C.A. J. Biol. Chem. 1999; 274: 29122-29129Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The NBDs of ABC proteins contain three conserved motifs required for nucleotide binding and hydrolysis: the Walker A and B motifs (14Walker J.E. Eberle A. Gay N.J. Runswick M.J. Saraste M. Biochem. Soc. Trans. 1982; 10: 203-206Crossref PubMed Scopus (26) Google Scholar) and the ABC signature sequence (LSGGQ) or C-motif (15Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3375) Google Scholar). ATP hydrolysis and substrate transport is strongly dependent on cooperativity between the NBDs. In P-GP, inactivation of either NBD by mutation of essential residues in the Walker A motif completely abolishes ATP-dependent transport activity (16Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar, 17Azzaria M. Schurr E. Gros P. Mol. Cell. Biol. 1989; 9: 5289-5297Crossref PubMed Scopus (270) Google Scholar). The NBDs of P-GP can also be exchanged without loss of function and in the original model proposed for the catalytic cycle of P-GP (18Beaudet L. Gros P. J. Biol. Chem. 1995; 270: 17159-17170Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), the alternating hydrolysis of one ATP molecule by either NBD results in transport of one molecule of substrate (19Senior A.E. al Shawi M.K. Urbatsch I.L. FEBS Lett. 1995; 377: 285-289Crossref PubMed Scopus (428) Google Scholar, 20Senior A.E. Bhagat S. Biochemistry. 1998; 37: 831-836Crossref PubMed Scopus (105) Google Scholar). More recently, a variation of this model has been proposed in which one ATP hydrolysis event results in substrate transport and hydrolysis of a second ATP is required to restore the protein to a high affinity substrate binding state (21Sauna Z.E. Ambudkar S.V. J. Biol. Chem. 2001; 276: 11653-11661Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Whether or not each NBD is limited to a distinct role in the transport process has not been established. However, studies of P-GP in which positions of NBDs were exchanged suggest that the location of the NBD in the protein may influence its ability to bind and hydrolyze nucleotide. Thus although interchangeable without loss of function, the NBDs may not be functionally identical in the intact transporter (18Beaudet L. Gros P. J. Biol. Chem. 1995; 270: 17159-17170Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 22Hrycyna C.A. Ramachandra M. Germann U.A. Cheng P.W. Pastan I. Gottesman M.M. Biochemistry. 1999; 38: 13887-13899Crossref PubMed Scopus (128) Google Scholar). Recent biochemical evidence in the E. coli histidine permease suggests that its two identical HisP subunits may be functionally asymmetric (23Kreimer D.I. Chai K.P. Ferro-Luzzi A.G. Biochemistry. 2000; 39: 14183-14195Crossref PubMed Scopus (25) Google Scholar). Compared with many transporters, the NBDs of ABCC proteins are structurally relatively divergent such that there is greater similarity between comparable NBDs in different ABCC members than between NBDs in the same protein (24Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). A considerable body of evidence also indicates that NBDs within a single protein differ functionally. We and others have shown that in MRP1, the NBDs differ considerably with respect to ATP binding and vanadate-dependent trapping of ADP (24Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 25Hou Y. Cui L. Riordan J.R. Chang X. J. Biol. Chem. 2000; 275: 20280-20287Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 26Hou Y.X. Cui L. Riordan J.R. Chang X.B. J. Biol. Chem. 2002; 277: 5110-5119Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27Hou Y.X. Riordan J.R. Chang X.B. J. Biol. Chem. 2003; 278: 3599-3605Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 28Nagata K. Nishitani M. Matsuo M. Kioka N. Amachi T. Ueda K. J. Biol. Chem. 2000; 275: 17626-17630Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Furthermore, although trapping of ADP by NBD2 of MRP1 requires that NBD1 be able to bind and possibly to hydrolyze ATP, the binding of ATP by NBD1 is preserved when NBD2 is inactivated by essential mutations in its Walker A motif (24Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 25Hou Y. Cui L. Riordan J.R. Chang X. J. Biol. Chem. 2000; 275: 20280-20287Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Inactivation of each NBD also has different effects on transport activity. A number of NBD1 mutations that eliminate ATP hydrolysis and diminish ATP binding decrease transport activity by only 60–70%, whereas comparable NBD2 mutations inactivate the protein (24Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The different effects of these mutations strongly suggest that the catalytic cycle of MRP1 differs from that first proposed for P-GP and prokaryotic transporters with two essentially identical NBDs (16Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar, 29Urbatsch I.L. Sankaran B. Bhagat S. Senior A.E. J. Biol. Chem. 1995; 270: 26956-26961Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). To begin defining the structural basis of the functional differences between the two NBDs of MRP1, we have examined the role of the acidic residue immediately COOH-proximal to the highly conserved core of the Walker B motif. In NBD1, this residue is Asp, whereas in NBD2, it is Glu, as is found in the NBDs of most ABC proteins (Fig. 1). The function of this Glu residue had been studied in several ABC transporters, such as HisP and P-GP, but its role in the catalytic cycle remains controversial. In HisP, mutation of this residue to Gln results in a protein able to bind ATP but with no detectable ATPase activity (30Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (618) Google Scholar, 31Moody J.E. Millen L. Binns D. Hunt J.F. Thomas P.J. J. Biol. Chem. 2002; 277: 21111-21114Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Comparable mutations in NBD1 and NBD2 of the P-GP murine homologue, mdr3, result in loss of ATPase activity but the protein can trap ADP at the mutated NBD in the presence of vanadate, suggesting that ADP release is impaired (32Urbatsch I.L. Julien M. Carrier I. Rousseau M.E. Cayrol R. Gros P. Biochemistry. 2000; 39: 14138-14149Crossref PubMed Scopus (79) Google Scholar). In contrast, similar studies of human P-GP found that although ADP release was impaired when the corresponding Glu residues in both NBDs were mutated to Gln, single mutants in either NBD were able to release ADP but unable to proceed through a second catalytic cycle (33Sauna Z.E. Muller M. Peng X.H. Ambudkar S.V. Biochemistry. 2002; 41: 13989-14000Crossref PubMed Scopus (94) Google Scholar). To investigate the roles played by Asp793 and Glu1455 adjacent to the core Walker B NBD1 and NBD2 motifs of MRP1, respectively, we have both exchanged these residues and mutated them to a number of amino acids lacking carboxylate side chains. The mutations have been expressed singly and in combination using a baculovirus dual expression system we have described previously, which enables highly efficient reconstitution of a functional transporter from two "half-molecules" that can be readily separated by SDS-PAGE (34Gao M. Loe D.W. Grant C.E. Cole S.P. Deeley R.G. J. Biol. Chem. 1996; 271: 27782-27787Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Thus quantitative effects of the mutations on nucleotide binding, trapping, and release by individual NBDs can be easily assessed (24Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Similarly, the influence of mutations on the ability of the high affinity MRP1 substrate, LTC4, to photolabel sites in the second and third MSDs can also be determined (35Qian Y.M. Qiu W. Gao M. Westlake C.J. Cole S.P. Deeley R.G. J. Biol. Chem. 2001; 276: 38636-38644Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). These studies revealed that differences between the ATP binding and hydrolysis characteristics of the two NBDs of MRP1 are attributable in large part to the presence of Asp rather than Glu adjacent to the NBD1 Walker B motif at position 793. In addition, we demonstrate that contrary to expectations based on the alternating site model of catalysis, increasing the hydrolytic activity of NBD1 by introduction of Glu in place of Asp decreases rather than increases transport of LTC4. This decrease in transport activity is associated with an increase in ADP trapping at the mutant NBD1 and a marked reduction in trapping by the co-expressed wild-type NBD2. Finally, we present evidence that conversion of the protein from a high affinity substrate binding conformation to a low affinity transition state occurs when NBD2 is occupied by either ATP or ADP and is prevented when NBD1 is occupied by ADP. Materials—8-Azido-[α-32P]ATP, 8-azido-[γ-32P]N3ATP, and 8-azido-[α-32P]ADP were purchased from Affinity Labeling Technologies, Inc. (Lexington, KY). Orthovanadate, ATP, AMP-PNP, ADP, and ATPγS compounds were from Sigma. MRP1 cDNA Mutation—The pFBDual-MRP1 1–932/932-1531 construct (pFBDual-halves, pFBDual-D123/D45) was cloned into pFASTBAC Dual (Invitrogen). This construct, encoding the NH2- and COOH-proximal half-molecules of MRP1, has been described by Gao et al. (24Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 34Gao M. Loe D.W. Grant C.E. Cole S.P. Deeley R.G. J. Biol. Chem. 1996; 271: 27782-27787Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The Glu793 and Asp1455 mutants were generated by site-directed mutagenesis using a Clontech transformer kit. The templates used for site-directed mutagenesis, pGEM-NBD1 and pGEM-NBD2, were described previously (24Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The forward primers for D793E and E1455D were 5′-CCTCTTCGATGAGCCCCTCTCAGC-3′ and 5′-CTTGTGTTGGATGATGCCACGGCAGC-3′, respectively. The presence of the mutation and the fidelity of the sequence of the MRP1 coding region were confirmed by dideoxy sequencing (ACGT Corp., Ontario, Canada). The Bsu36I-SphI fragments bearing mutations at NBD1 were isolated from pGEM-NBD1 and used to replace the same region in pFBDual-halves to create pFBDual-halves/D793E. The EcoRI-KpnI fragments with mutations at NBD2 were isolated from pGEM-NBD2 and used to replace the equivalent region in pBS-Asp45 to generate pBS-D45/E1455D. This was then digested with NcoI and KpnI and the NcoI-KpnI fragment was used to replace the equivalent region of pFBDual-Asp45 to give pFBDual-D45/E1455D. Finally, the SalI-XbaI fragment of pFBDual-halves was isolated and cloned into pFBDual-D45/E1455D, which had been digested with the same enzymes, to generate pFBDual-halves/E1455D. In the D793E/E1455D double mutant, the SalI-XbaI fragment with the mutation in NBD1 was isolated and ligated to pFBDual-D45/E1455D, which had been digested with the same enzymes, to generate pFBDual-halves/D793E/E1455D. Other mutations (Gln793, Ser793, Asn793, Gln1455, Ser1455, Asn1455, and Leu1455) were carried out in the pFBDual-halves vector using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Oligonucleotides used for site-directed mutagenesis were synthesized by ACGT Corp. The forward primers for D793Q, D793N, D793S, E1455Q, E1455N, E1455S, and E1455L were 5′-GCTGACATTTACCTCTTCGATCAACCGCTCTCAGCAGTGGATGCC-3′, 5′-GCTGACATTTACCTCTTCGATAATCCGCTCTCAGCAGTGGATGCC-3′, 5′-GCTGACATTTACCTCTTCGATTCTCCCCTCTCAGCAGTGGATGCC-3′, 5′-CGAAGATCCTTGTGTTGGATCAGGCCACGGCGGCCGTGGACCTGG-3′, 5′-CGAAGATCCTTGTGTTGGATA ACGCCACGGCCGCCGTGGACCTGG-3′,5′-CGAAGATCCTTGTGTTGGATTCGGCCACGGCAGCCGTGGACCTGG-3′, 5′-CGAAGATCCTTGTGTTGGATTTGGCCACGGCCGCCGTGGACCTGG-3′, respectively. The templates used for site-directed mutagenesis, pFBDual-halves, were described previously (24Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The Bsu36I-SphI fragments bearing mutations at NBD1 were isolated from pFBDual-halvesmutated NBD1 and used to replace the same region in pFBDual-halves MRP1 to create pFBDual-halves/MutNBD1. The KpnI-ClaI fragment of pFBDual-halves-mutated NBD2 was isolated and used to create pFBDual-halves/MutNBD2. The presence of the mutation and the fidelity of the sequence of the MRP1 coding region were confirmed by dideoxy sequencing (ACGT Corp.). Viral Infection and Membrane Vesicle Preparation—After generation of recombinant bacmids and baculoviruses, viral infection of Sf21 cells was carried out (24Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 34Gao M. Loe D.W. Grant C.E. Cole S.P. Deeley R.G. J. Biol. Chem. 1996; 271: 27782-27787Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). To generate membrane vesicles, Sf21 cells were disrupted by nitrogen cavitation. Cell membranes were subsequently purified by 35% sucrose centrifugation (7Loe D.W. Almquist K.C. Deeley R.G. Cole S.P. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 36Leier I. Jedlitschky G. Buchholz U. Cole S.P. Deeley R.G. Keppler D. J. Biol. Chem. 1994; 269: 27807-27810Abstract Full Text PDF PubMed Google Scholar). Immunoblotting and Quantification of MRP1 Polypeptide—SDS-PAGE of membrane vesicle proteins was carried out using 7–15% gradient gels (37Hipfner D.R. Mao Q. Qiu W. Leslie E.M. Gao M. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1999; 274: 15420-15426Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA) using 25 mm Tris base, 192 mm glycine, and 20% methanol buffer. MRP1 polypeptides were detected using an enhanced chemiluminescence kit (ECL) (Amersham) and the murine mAb MRPm6 and the rat mAb MRPr1 (Pierce) (38Flens M.J. Izquierdo M.A. Scheffer G.L. Fritz J.M. Meijer C.J. Scheper R.J. Zaman G.J. Cancer Res. 1994; 54: 4557-4563PubMed Google Scholar, 39Hipfner D.R. Gao M. Scheffer G. Scheper R.J. Deeley R.G. Cole S.P. Br. J. Cancer. 1998; 78: 1134-1140Crossref PubMed Scopus (71) Google Scholar). Various MRP1 polypeptide amounts were estimated by comparison with Sf21 cell-expressed wild-type MRP1 loaded using the same conditions. Densitometry of the film images was performed using a ChemiImager™ 4000 (Alpha Innotech Corp., San Leandro, CA). The relative protein expression levels were calculated by dividing the integrated densitometry values obtained for 0.5 and 1.0 μg of total membrane protein from infected cells expressing the mutant proteins by the integrated densitometry values obtained for the comparable amounts of total membrane proteins from infected cells expressing the wild-type protein. Each comparison was performed three times in independent experiments. The results were then pooled, and the mean values were determined. Photolabeling of NBD1 and NBD2 of MRP1 Using 8-Azido-32P-nucleotides—8-Azido-[γ-32P]ATP and 8-azido-[α-32P]ATP photoaffinity labeling was performed as previously described by Gao et al. (24Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Membrane vesicles (20 μg of protein) were resuspended in transport buffer (50 mm Tris-HCl, pH 7.4, 250 mm sucrose, and 0.02% of Na3N) containing 5 mm MgCl2, and 5 μm 8-azido-[γ-32P]ATP or 8-azido-[α-32P]ATP (ALT Corp.; specific activity between 5 and 20 Ci mmol–1). After 5 min in a 96-well plate, the membranes were irradiated for 7 min on ice in a Stratalinker UV cross-linker (λ = 302 nm). After addition of 150 μl of ice-cold buffer (50 mm Tris-HCl, pH 7.4, 0.1 mm EGTA, and 5 mm MgCl2), the membranes were centrifuged at 14,000 rpm for 15 min at 4 °C. A second wash was performed and the pellets were resuspended in 14 μl of ice-cold buffer. After addition of Laemmli buffer (4×) containing dithiothreitol (100 mm final), membrane vesicles were electrophoresed on gradient SDS-PAGE (7–15%). After drying for 2 h at 80 °C, gels were autoradiographed in a PhosphorImager (Amersham Biosciences) and autoradiographed using Kodak Bio-Max MS films. Vanadate-induced Trapping of 8-Azido-[α-32P]ADP by MRP1—The 100 mm vanadate solution was prepared by dissolving 184 mg of sodium orthovanadate in 9.35 ml of water. The pH of vanadate solution was adjusted to 10 using 2 m HCl and boiled for 10 min. The solution must become colorless. Just before using, this solution must be boiled 10 min and diluted to 10 mm with transport buffer. Membrane vesicles (20 μg of protein) were resuspended in transport buffer containing 5 mm MgCl2, and 1–15 μm 8-azido-32P-nucleotide (ALT Corp.; specific activity between 5 and 20 Ci mmol–1). Unless otherwise indicated in the figure legend, the 15-min incubation at 37 °C was performed in the presence or absence of 1 mm vanadate. The reaction was started by addition of 8-azido-[γ-32P]ATP, 8-azido-[α-32P]ATP, or 8-azido-[α-32P]ADP and stopped by transfer on ice and addition of ice-cold buffer as previously described. Unreacted nucleotides were then removed (2×) by addition of 150 μl of ice-cold buffer followed by centrifugation. Pellets were resuspended in 14 μl of ice-cold buffer and vesicle membranes were irradiated for 7 min on ice in a Stratalinker UV cross-linker (λ = 302 nm) as previously described by Gao et al. (24Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). After the addition of Laemmli buffer (4×), containing dithiothreitol (100 mm final), membrane vesicles were electrophoresed on gradient SDS-PAGE (7–15%). After drying for 2 h at 80 °C, gels were autoradiographed in a PhosphorImager (Amersham Biosciences) and autoradiographed using Kodak Bio-Max MS films. Transport of [3H]LTC4 into Insect Membrane Vesicles—Uptake of [3H]LTC4 (50 nm, 182 Ci mmol–1, PerkinElmer Life Sciences) into membrane vesicles was measured at 23 °C in the presence of ATP (4 mm) or AMP (4 mm) using a rapid filtration technique as previously described (7Loe D.W. Almquist K.C. Deeley R.G. Cole S.P. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). Photoaffinity Labeling by MRP1 with [3H]LTC4—Unless otherwise indicated in the figure legend, insect membrane vesicles (75 μg of protein in 20 μl) were incubated with [3H]LTC4 (0.13 μCi, 200 nm) at room temperature for 20 min. Cells were then frozen in liquid nitrogen (1 min), followed by UV irradiation (1 min). This was repeated 10 times. Radiolabeled vesicles were analyzed on SDS-PAGE (7–15%). Proteins were fixed by 25% isopropyl alcohol, 65% water, and 10% acid acetic, for 30 min, and gels were dried at 80 °C for 2 h and autoradiographed on Kodak Bio-Max MS films (35Qian Y.M. Qiu W. Gao M. Westlake C.J. Cole S.P. Deeley R.G. J. Biol. Chem. 2001; 276: 38636-38644Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Wild-type and various mutant MRP1 half-molecules were co-expressed in Sf21 cells infected with baculovirus dual-expression vectors. The levels of co-expressed MRP1 fragments in membrane vesicles from infected cells were then determined by immunoblotting, using the rat mAb MRPr1 (epitope amino-acids 238–247) and murine mAb MRPm6 (epitope amino-acids 1511–1520) to detect NH2- and COOH-proximal fragments, respectively. Membrane vesicles from insect cells infected with a β-Gus baculovirus construct were used as a negative control. No MRP1 specific binding was detected by mAb MRPr1 and mAb MRPm6 in cells infected with the β-Gus vector. Effect of D793E and E1455D Single and Double Mutations on LTC4 Transport Activity—LTC4 uptake by membrane vesicles from Sf21 cells expressing wild-type and mutant MRP1 half-molecules was determined at 23 °C, as described by Loe et al. (7Loe D.W. Almquist K.C. Deeley R.G. Cole S.P. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). The expression levels of mutant and wild-type proteins were determined to be comparable by immunoblotting (Fig. 2A) and transport activity values have not been normalized. Uptake at 3 min, by vesicles containing the D793E mutant NH2-terminal fragment and the wild-type COOH-proximal half was ∼20% of that obtained with vesicles containing two wild-type fragments. The transport activity of vesicles containing either the E1455D mutant fragment and the wild-type NH2-terminal half, or co-expressed D793E and E1455D mutant fragments was similar to that of the β-Gus control (Fig. 2B). Thus the effects of the D793E and E1455D mutations on LTC4 transport were similar to previously described mutations of the Walker A motif that eliminate ATP hydrolysis and decrease ATP binding by NBD1 and NBD2, respectively (24Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 25Hou Y. Cui L. Riordan J.R. Chang X. J. Biol. Chem. 2000; 275: 20280-20287Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Photolabeling with 8-Azido-[γ-32P]ATP in the Presence of AMP-PNP at 4 °C—To determine whether or not the D793E and E1455D mutations altered ATP binding, studies were carried out at 4 °C using the radioactive photoactivable analog of ATP, 8-azido-[γ-32P]ATP, hydrolysis of which results in loss of the γ-32P label (24Gao M. Cui H.R. Loe D
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