ABCMdb

PMID: 12882957

Payen LF, Gao M, Westlake CJ, Cole SP, Deeley RG
Role of carboxylate residues adjacent to the conserved core Walker B motifs in the catalytic cycle of multidrug resistance protein 1 (ABCC1).
J Biol Chem. 2003 Oct 3;278(40):38537-47. Epub 2003 Jul 27., 2003-10-03 [PubMed]

Sentences

No. Mutations Sentence Comment

69 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp The forward primers for D793E and E1455D were 5Ј-CCTCTTCGATGAGCCCCTCTCAGC-3Ј and 5Ј-CTTGTGTTG- GATGATGCCACGGCAGC-3Ј, respectively. Login to comment 71 ABCC1 p.Asp793Glu 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. Login to comment 72 ABCC1 p.Glu1455Asp 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. Login to comment 73 ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp 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. Login to comment 74 ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp 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. Login to comment 75 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp 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. Login to comment 78 ABCC1 p.Glu1455Leu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Ser ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Asn ABCC1 p.Asp793Gln ABCC1 p.Asp793Ser The forward primers for D793Q, D793N, D793S, E1455Q, E1455N, E1455S, and E1455L were 5Ј-GCTGACATTTACCTCTTCGATCAACCGCTCTC- AGCAGTGGATGCC-3Ј, 5Ј-GCTGACATTTACCTCTTCGATAATCCGC- TCTCAGCAGTGGATGCC-3Ј, 5Ј-GCTGACATTTACCTCTTCGATTCT- CCCCTCTCAGCAGTGGATGCC-3Ј, 5Ј-CGAAGATCCTTGTGTTGGA- TCAGGCCACGGCGGCCGTGGACCTGG-3Ј, 5Ј-CGAAGATCCTTGTG- TTGGATA ACGCCACGGCCGCCGTGGACCTGG-3Ј, 5Ј-CGAAGATCC- TTGTGTTGGATTCGGCCACGGCAGCCGTGGACCTGG-3Ј, 5Ј-CGAA- GATCCTTGTGTTGGATTTGGCCACGGCCGCCGTGGACCTGG-3Ј, respectively. Login to comment 122 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp 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. (7). Login to comment 124 ABCC1 p.Asp793Glu Uptake at 3 min, by vesicles containing the D793E mutant NH2-terminal fragment and the wild-type COOH-proximal half was ϳ20% of that obtained with vesicles containing two wild-type fragments. Login to comment 125 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp 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 beta-Gus control (Fig. 2B). Login to comment 126 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp 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 (24, 25). Login to comment 127 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Photolabeling with 8-Azido-[␥-32 P]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-[␥-32 P]ATP, hydrolysis of which results in loss of the ␥-32 P label (24, 33). Login to comment 130 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ATP labeling of wild-type MRP1, D793E, E1455D single mutant proteins was similar and occurred preferentially at NBD1, whereas it was slightly decreased at NBD1 of the double mutant protein. Login to comment 132 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp Labeling at NBD2 in the E1455D and D793E/E1455D mutant proteins was strongly and moderately enhanced, respectively, relative to the wild-type NBDs. Login to comment 134 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Nucleotide Trapping by Wild-type MRP1 and D793E, E1455D Single and Double Mutant Proteins Using 8-Azido-[␣- 32 P]ATP-To determine whether ATP hydrolysis by the mutant NBDs was altered, ADP trapping experiments were performed at 37 °C using various concentrations of 8-azido-[␣- 32 P]ATP in the presence or absence of vanadate. Login to comment 139 ABCC1 p.Asp793Glu In comparison to wild-type MRP1, ADP trapping was increased at the D793E mutant NBD1 and decreased at the co-expressed wild-type NBD2. Login to comment 140 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Increased labeling of the mutant NBD1 was observed at all 8-azido-[␣-32 P]ATP concentrations tested and unlike the wild-type NBD1, was readily detectable at 2.5 ␮M 8-azido-[␣-32 P]ATP with both the D793E and D793E/E1455D mutants (Fig. 3, A and C). Login to comment 143 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Effect of D793E and E1455D single and double mutations on ATP-dependent LTC4 transport activity (B) and on ATP binding at 4 °C (C). Login to comment 144 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp A, membrane proteins (1 ␮g from Sf21 cells expressing both halves of either MRP1 (MRP1 dh), mutant proteins (D793E, E1455D, and D793E/E1455D)) were separated by SDS-PAGE on gradient gels and transferred to Immobilon-P membranes. Login to comment 148 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp B, membrane vesicles (2 ␮g) containing wild-type MRP1 (f), MRP1 mutants D793E (Œ), E1455D (), D793E/E1455D (q), and control beta-Gus vector (ࡗ) were assayed for ATP-dependent LTC4 transport activity at 23 °C for up to 3 min in transport buffer containing [3 H]LTC4 (50 nM, 0.13 ␮Ci), as described under "Experimental Procedures." Login to comment 151 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp C, at 4 °C, 8-azido-[␥-32 P]ATP photolabeling by wild-type MRP1 and mutant D793E, E1455D, and D793E/E1455D was carried out. Login to comment 156 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Catalytic Cycle of MRP138540 The E1455D mutation dramatically increased photolabeling of the mutant NBD2 and unlike the D793E mutation, increased rather than decreased labeling of the co-expressed wild-type NBD1. Login to comment 159 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp Despite the striking increase in photolabeling of the E1455D mutant NBD2, in the double D793E/E1455D mutant, labeling occurred predominantly at NBD1 and was markedly diminished at NBD2 relative to the E1455D single mutant. Login to comment 161 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp Photolabeling with 8-Azido-[␥-32 P]ATP or 8-Azido-[␣-32 P]ADP at 37 °C-The relatively weak vanadate dependence of photolabeling observed when using 8-azido-[␣-32 P]ATP, particularly with the E1455D and D793E/E1455D mutants, raised the possibility that the mutant NBD2 may be capable of tight binding of both ATP and ADP. Login to comment 163 ABCC1 p.Asp793Glu In the presence of 1 mM vanadate, photolabeling of wild-type and the D793E mutant NBD1 by 8-azido-[␥-32 P]ATP was barely detectable, presumably as a result of the hydrolytic loss of the [␥-32 P]PO4 (Fig. 4A). Login to comment 164 ABCC1 p.Asp793Glu These data combined with trapping using 8-azido-[␣-32 P]ATP indicate that the nucleotide "trapped" by the NBDs of the wild-type and D793E mutant proteins was indeed ADP. Login to comment 165 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp In contrast, both NBDs of the E1455D mutant were strongly labeled by 8-azido-[␥-32 P]ATP, while in the D793E/E1455D double mutant labeling of NBD2 was much reduced and labeling of NBD1 was essentially eliminated (Fig. 4A). Login to comment 166 ABCC1 p.Glu1455Asp In contrast to the ATP binding observed at 4 °C, photolabeling at 37 °C of the E1455D mutant NBD2 was stronger than the co-expressed wild-type NBD1 and was only slightly competed by 1 mM AMP-PNP. Login to comment 167 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp These experiments suggest that the tight binding of ATP by the E1455D mutant NBD2 stimulates the binding of ATP by the co-expressed wild-type NBD1 and conversely, that the increased trapping of ADP at the D793E mutant NBD1, diminishes ATP binding by both the co-expressed wild-type and E1455D mutant NBD2. Login to comment 169 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Nucleotide trapping by D793E, E1455D single and double mutant proteins using 8-azido-[␣-32 P]ATP. Login to comment 170 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp A-C, at 37 °C, under hydrolytic conditions, the effect of the 8-azido-[␣-32 P]ATP concentration on ADP trapping by wild-type MRP1 and D793E (A), E1455D (B), and D793E/E1455D (C) mutant proteins was evaluated. Membrane vesicles (20 ␮g) were incubated with 8-azido-[␣-32 P]ATP (1-15 ␮M) in the absence (-) or presence (ϩ) of 1 mM vanadate for 15 min in transport buffer containing 5 mM MgCl2. Login to comment 176 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Effect of D793E, E1455D single and double mutations on ATP binding and ADP labeling under hydrolytic conditions. Login to comment 177 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp A, at 37 °C, 8-azido-[␥-32 P]ATP photolabeling by wild-type MRP1 and D793E, E1455D single and double mutations was carried out. Login to comment 181 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp The position of the labeled MRP1 NH2-half and COOH-half polypeptides are indicated, and endogenous proteins labeled are indicated by E followed by arrows. B, at 37 °C, 8-azido-[␣- 32 P]ADP photolabeling by wild-type MRP1 and mutant D793E, E1455D, and D793E/E1455D proteins was evaluated. Login to comment 190 ABCC1 p.Asp793Glu In comparison to wild-type, ADP labeling of the D793E mutant NBD1 was increased and was almost maximal using 5 ␮M 8-azido-[␣-32 P]ADP (Fig. 4B, lanes 2, 4, and 5). Login to comment 191 ABCC1 p.Asp793Glu In contrast, photolabeling by 15 ␮M 8-azido-[␣- 32 P]ADP was clearly decreased at the wild-type NBD2 co-expressed with the D793E mutant NBD1, when compared with the co-expressed wild-type half-molecules (Fig. 4B, lanes 2-5). Login to comment 192 ABCC1 p.Glu1455Asp ADP binding by the E1455D single mutant was strongly increased at both NBDs relative to the co-expressed wild-type fragments although the majority of photolabeling occurred at the mutant NBD2. Login to comment 193 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp In the D793E/E1455D double mutant, photolabeling of NBD2 was diminished and labeling of NBD1 was increased relative to the single D793E and E1455D single mutants, so that ADP labeling was similar at both NBDs (Fig. 4B, lanes 2-9). Login to comment 194 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Thus as observed with 8-azido-[␥-32 P]ATP, the increased binding of ADP by the E1455D mutant NBD2 appears to stimulate tight binding of ADP at both the wild-type and D793E mutant NBD1. Login to comment 195 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp In contrast, the increased ADP binding of the D793E mutant NBD1 decreases ADP binding by both the wild-type and E1455D mutant NBD2, as would be predicted by an alternating site model of catalysis. Login to comment 196 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Evaluation of ADP Release by Wild-type and D793E, E1455D Single and Double Mutant Proteins-It has been demonstrated that ADP release constitutes the rate-limiting step in the normal catalytic cycle of P-GP (41). Login to comment 200 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp Consequently, we investigated the influence of D793E, E1455D, and D793E/ E1455D MRP1 mutants on ADP release by each NBD. Login to comment 205 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp As described above, weak photolabeling of NBD1 in samples cross-linked immediately after washing could be detected with the D793E and D793E/E1455D mutants (Fig. 5, lanes 3 and 5). Login to comment 207 ABCC1 p.Glu1455Asp In contrast, the E1455D mutation displayed very strong labeling of the mutant NBD2 that persisted following the reincubation period (Fig. 5, lanes 4 and 10). Login to comment 209 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp Unlike the single E1455D mutant, very little photolabeling of NBD2 was retained following reincubation of the D793E/E1455D double mutant (Fig. 5, lanes 5 and 11). Login to comment 210 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp To further investigate the impairment of ADP release by the E1455D and the D793E/E1455D mutants, cold ADP (1 mM) was added 3 min before the end of the initial nucleotide labeling with 8-azido-[␣-32 P]ATP (Fig. 5, lanes 6-8). Login to comment 211 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ADP competed completely for 8-azido-␣-32 P-nucleotide trapping at both NBDs of the D793E mutant and at NBD1 of the D793E/E1455D double mutant indicating that the bound nucleotide was readily exchangeable (Fig. 5, lanes 6 and 8). Login to comment 212 ABCC1 p.Glu1455Asp However, relatively strong photolabeling of both NBDs was retained in the E1455D mutant. Login to comment 213 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp In the D793E/E1455D double mutant all labeling at NBD1 was lost and labeling of NBD2 was markedly decreased compared with the E1455D single mutant (Fig. 5, lanes 7 and 8). Login to comment 215 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Thus the results indicate that the release of ADP by the E1455D NBD2 is severely impaired and that this also decreases the ability of the co-expressed wild-type NBD1, but not the D793E mutant, to exchange nucleotide. Login to comment 216 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp LTC4 Photolabeling by Wild-type and D793E, E1455D Single, and D793E/E1455D Double Mutant Proteins Under Hydrolytic and Non-hydrolytic Conditions-We have shown previously that LTC4 can photolabel MRP1 at sites in MSD2 and MSD3 and that photolabeling, particularly of the site in MSD2, is strongly attenuated under ADP trapping conditions. Login to comment 219 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp To examine this prediction further, we investigated the effect of the D793E and E1455D mutations on the binding of LTC4 because the former, unlike the Walker A mutations, increases ADP trapping at NBD1, whereas the latter increases nucleotide binding and markedly slows down nucleotide release at NBD2. Login to comment 221 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Evaluation of ADP release by wild-type and D793E, E1455D single and double mutations using 8-azido-[␣-32 P]ATP. Login to comment 230 ABCC1 p.Asp793Glu Photolabeling of the D793E mutant in the absence of nucleotide was indistinguishable from that of the co-expressed wild-type half-molecules. Login to comment 231 ABCC1 p.Asp793Glu However, in contrast to the previously described Walker A mutations, the D793E mutation essentially abolished the effect of ATP, and ATP plus vanadate on LTC4 binding (Fig. 6A, lanes 7-10). Login to comment 234 ABCC1 p.Glu1455Asp The LTC4 labeling profile of the E1455D mutant in the absence of nucleotide was also similar to that observed for wild-type MRP1 (Fig. 6B, lanes 2 and 11). Login to comment 238 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp The double D793E/E1455D mutant behaved in a manner very similar to that of the D793E single mutation, with the exception that ATP␥S retained some ability to diminish LTC4 labeling (Fig. 6B, lanes 6-11). Login to comment 239 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Thus these results together with those of vanadate trapping experiments are again consistent with the suggestion that increased trapping of ADP by the D793E mutant NBD1 decreases the nucleotide binding and hydrolysis by wild-type and E1455D mutant NBD2 and prevents conversion to a low affinity transition state. Login to comment 241 ABCC1 p.Asp793Asn ABCC1 p.Glu1455Ser ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Asn To determine whether there may be additional consequences to eliminating the carboxylate side chain, we mutated Asp793 and Glu1455 to Asn, Gln, and Ser as observed in cystic fibrosis transmembrane conductance regulator. Login to comment 242 ABCC1 p.Glu1455Leu In addition, Glu1455 was mutated to Leu. Login to comment 245 ABCC1 p.Glu1455Gln The exception was the NH2-proximal of the E1455Q mutant that was expressed at a level 1.75 times higher than its COOH-proximal half (Fig. 7A). Login to comment 246 ABCC1 p.Glu1455Gln Because the ATP-dependent uptake of LTC4 relies upon association of both halves of the protein and the levels of all COOH-proximal fragments were comparable, no adjustment was made to compensate for the increased expression of the NH2-proximal half of E1455Q (Fig. 7A). Login to comment 248 ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Asp793Gln ABCC1 p.Asp793Ser Unlike the D793E mutation, which decreased LTC4 transport activity by 80%, NBD1 mutations (D793S, D793N, and D793Q) had little effect on ATP-dependent LTC4 uptake (Fig. 7B). Login to comment 249 ABCC1 p.Glu1455Leu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Ser ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Asn In contrast, the NBD2 mutations (E1455S, E1455N, E1455Q, and E1455L) like E1455D completely abolished LTC4 transport (Fig. 7B). Login to comment 251 ABCC1 p.Asp793Gln ABCC1 p.Asp793Ser At 4 °C, mutation of Asp793 to Ser, Gln, or Asn decreased photolabeling of NBD1 by 8-azido-[␣-32 P]ATP to a similar extent. Login to comment 252 ABCC1 p.Asp793Glu However, NBD1 was still more strongly photolabeled than NBD2, as observed with the wild-type co-expressed halves and, unlike the D793E mutation that diminished labeling of NBD2, labeling was unaffected by substitution with the three polar uncharged residues (Fig. 8A, lanes 3, 5, and 7). Login to comment 254 ABCC1 p.Glu1455Asn Mutation to Ser, Gln, and Leu essentially eliminated labeling of NBD2, whereas labeling of the E1455N mutation was similar to that of the FIG. 6. Login to comment 255 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp Effect of ATP␥S or ADP trapping on [3 H]LTC4 photolabeling by wild-type MRP1, D793E, E1455D, and D793E/E1455D mutant proteins. Login to comment 256 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Asp Wild-type MRP1, D793E (A) and E1455D, D793E/ E1455D (B) membrane proteins (75 ␮g) were incubated in transport buffer containing 5 mM MgCl2 at 23 °C for 20 min in the absence (-) or presence (ϩ) of ATP (1 mM), vanadate (1 mM), or ATP␥S (4 mM), alone or in combination, prior to addition of [3 H]LTC4 (200 nM, 0.13 ␮Ci). Login to comment 261 ABCC1 p.Glu1455Gln Labeling of the wild-type NBD1 co-expressed with the E1455Q mutation appears to be increased, but we cannot completely exclude the possibility that this is attributable at least in part to the 1.75-fold higher level of expression of this fragment. Login to comment 263 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp The effect of the non-conservative mutations on nucleotide binding and ADP trapping was examined at 37 °C using 5 ␮M 8-azido-[␣-32 P]ATP in the presence or absence of vanadate, exactly as described for the D793E and E1455D mutations. Login to comment 264 ABCC1 p.Asp793Asn ABCC1 p.Asp793Gln ABCC1 p.Asp793Ser Similar to wild-type, in the absence of vanadate, D793S, D793Q, and D793N mutant proteins did not display any nucleotide binding at either NBD (Fig. 8B). Login to comment 265 ABCC1 p.Asp793Glu This contrasts with the D793E mutation in which photolabeling of NBD1 was readily detectable in the presence of 5 ␮M 8-azido-[␣-32 P]ATP and in the absence of vanadate. Login to comment 266 ABCC1 p.Asp793Ser In the presence of vanadate, the labeling profile of the D793S mutation was very similar to wild-type with nucleotide being bound relatively strongly by NBD2 (Fig. 8B, lanes 3 and 5). Login to comment 267 ABCC1 p.Asp793Asn ABCC1 p.Asp793Gln Although labeling at NBD2 was decreased in the D793Q and D793N mutants, this domain remained the predominant site of photolabeling (Fig. 8B, lanes 7 and 9). Login to comment 268 ABCC1 p.Glu1455Leu ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Ser ABCC1 p.Glu1455Asn Like the E1455D mutant, the E1455S, E1455N, and E1455L mutations resulted in strong vanadate-independent photolabeling of NBD2 and increased vanadate-dependent photolabeling of NBD1 (Fig. 8C, lanes 3, 4, and 7-10). Login to comment 269 ABCC1 p.Glu1455Gln The E1455Q FIG. 7. Login to comment 270 ABCC1 p.Glu1455Leu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Ser ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Asn ABCC1 p.Asp793Gln ABCC1 p.Asp793Ser Effect of D793Q, D793N, D793S, E1455S, E1455Q, E1455N, E1455L mutations on [3 H]LTC4 transport activity. Login to comment 271 ABCC1 p.Glu1455Leu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Ser ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Asn ABCC1 p.Asp793Gln ABCC1 p.Asp793Ser A, membrane proteins (1 ␮g) from Sf21 cells expressing both halves of either MRP1 (MRP1 dh) or mutant proteins (D793Q, D793N, D793S, E1455S, E1455Q, E1455N, E1455L) were separated by SDS-PAGE on gradient gels and transferred to Immobilon-P membranes. Login to comment 275 ABCC1 p.Glu1455Leu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Ser ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Asn ABCC1 p.Asp793Gln ABCC1 p.Asp793Ser B, membrane vesicles (2 ␮g) containing MRP1 dh, D793Q, D793S, D793N, E1455Q, E1455S, E1455N, E1455L, or beta-Gus were assayed for ATP-dependent LTC4 transport activity at 23 °C for up to 3 min in transport buffer containing [3 H]LTC4 (50 nM, 0.13 ␮Ci), as described under "Experimental Procedures." Login to comment 280 ABCC1 p.Glu1455Gln These observations are consistent with NBD2 of the E1455Q mutant being occupied predominantly by ADP in the presence of vanadate, whereas in the other mutations that display significant levels of vanadate independent binding, the photolabeling of NBD2 may be the result of occupancy by either ATP or ADP. Login to comment 285 ABCC1 p.Asp793Glu Based on the behavior of NBD2 of MRP1 and other NBDs containing Glu adjacent to the Walker B motif, we anticipated that the D793E mutation in NBD1 would increase ATP hydrolysis and possibly decrease the affinity for ATP. Login to comment 290 ABCC1 p.Asp793Glu In contrast to the wild-type NBD1, vanadate did not further increase nucleotide trapping by the D793E mutant when photolabeling was carried out with 8-azido-[␣-32 P]ATP at 37 °C (Fig. 3A). Login to comment 292 ABCC1 p.Asp793Glu In addition, the D793E mutation slightly decreased binding of 8-azido-[␥-32 P]ATP at 4 °C by the co-expressed wild-type NBD2 (Fig. 2C) and caused a major decrease in the trapping of ADP at 37 °C in the presence of vanadate (Fig. 3A). Login to comment 294 ABCC1 p.Asp793Glu Consistent with the decrease in ATP binding and ADP trapping at the co-expressed wild-type NBD2, the decrease in LTC4 binding observed with the wild-type protein in the presence of ATP and vanadate was completely abrogated by the D793E mutation (Fig. 6A, lanes 7-10). Login to comment 295 ABCC1 p.Asp793Glu Thus the D793E mutation decreases LTC4 transport by preventing the transition from a high to low affinity binding state. Login to comment 296 ABCC1 p.Glu1455Asp The E1455D mutation essentially eliminated transport of LTC4 as observed previously with mutations that inactivate NBD2 (Fig. 2B). Login to comment 302 ABCC1 p.Glu1455Asp Thus the E1455D mutation substantially increased affinity for both ATP and ADP. Login to comment 304 ABCC1 p.Glu1455Leu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Ser ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Asn ABCC1 p.Asp793Gln ABCC1 p.Asp793Ser Comparison of nucleotide binding and vanadate trapping by wild-type MRP1 and mutant proteins (D793Q, D793N, D793S, E1455S, E1455Q, E1455N, and E1455L). Login to comment 305 ABCC1 p.Glu1455Leu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Ser ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Asn ABCC1 p.Asp793Gln ABCC1 p.Asp793Ser A, at 4 °C, 8-azido- [␣-32 P]ATP photolabeling by wild-type MRP1 and mutant proteins (D793Q, D793N, D793S, E1455S, E1455Q, E1455N, and E1455L) was evaluated. Membrane vesicles (20 ␮g) were incubated with 5 ␮M 8-azido-[␣-32 P]ATP for 5 min on ice in transport buffer containing 5 mM MgCl2. Login to comment 308 ABCC1 p.Glu1455Leu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Ser ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Asn ABCC1 p.Asp793Gln ABCC1 p.Asp793Ser The position of the labeled MRP1 NH2-half and COOH-half polypeptides are indicated, and endogenous proteins labeled are indicated by E followed by arrows. B and C, at 37 °C under trapping conditions, 8-azido-[␣-32 P]ADP trapping by wild-type MRP1 mutant proteins (D793Q, D793N, D793S, E1455S, E1455Q, E1455N, and E1455L) was studied. Login to comment 312 ABCC1 p.Glu1455Asp The E1455D mutation also strongly enhanced nucleotide binding and vanadate-dependent trapping at the co-expressed wild-type NBD1 (Fig. 3, B and C). Login to comment 318 ABCC1 p.Asp793Glu Consistent with such a prediction, the D793E mutation increased ADP trapping at the mutant NBD1 and decreased vanadate-dependent trapping by the co-expressed wild-type NBD2. Login to comment 319 ABCC1 p.Glu1455Asp Contrary to expectation, the markedly increased 8-azido-[␣-32 P]ADP binding by the E1455D mutant NBD2 was accompanied by increased rather than decreased ADP photolabeling of the co-expressed wild-type NBD1, suggesting that both NBDs could be simultaneously occupied by ADP. Login to comment 322 ABCC1 p.Glu1455Asp We also found that the E1455D mutation drastically decreased nucleotide release at 37 °C, as evidenced by an inability to displace either prebound 8-azido-[␣- 32 P]ATP (Fig. 5) or 8-azido-[␣-32 P]ADP (data not shown) with a large excess of cold nucleotide. Login to comment 324 ABCC1 p.Glu1455Asp In the absence of nucleotides, LTC4 photolabeling was unaffected by the E1455D mutation. Login to comment 327 ABCC1 p.Glu1455Asp This is consistent with stable vanadate-independent binding of nucleotide by the E1455D mutant NBD2 (Fig. 3B). Login to comment 328 ABCC1 p.Glu1455Asp In addition, ATP␥S was much more effective at reducing binding by the E1455D mutant when compared with the wild-type protein. Login to comment 330 ABCC1 p.Glu1455Asp Thus the loss of LTC4 transport activity appears to be attributable to the impaired ability of the E1455D mutant to release nucleotide from NBD2 and to re-establish a high affinity binding state. Login to comment 331 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Like the single mutations, the D793E/E1455D mutation had no effect on LTC4 binding in the absence of nucleotides. Login to comment 332 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp However, in the presence of ATP or ATP plus vanadate, the D793E mutation in the double mutant abrogated the shift from a high to a low affinity binding state, despite the potentiating effect observed with the E1455D single mutation. Login to comment 333 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Asp Paradoxically, a decrease of LTC4 binding by the D793E/E1455D double mutant protein was still observed in the presence of ATP␥S (Fig. 6B). Login to comment 339 ABCC1 p.Asp793Asn ABCC1 p.Asp793Gln Mutation of Asp793 to Asn, Gln, and also Ser, which is the residue found at the comparable position of cystic fibrosis transmembrane conductance regulator, all decreased slightly LTC4 transport activity, whereas the comparable mutations of Glu1455 inactivated the protein (Fig. 7B). Login to comment 342 ABCC1 p.Glu1455Asp ABCC1 p.Glu1455Gln Like the E1455D mutation, the substitution of Glu1455 with Gln, Asn, Leu, and Ser completely abolished LTC4 transport activity (Fig. 7B). Login to comment 343 ABCC1 p.Glu1455Asp However, in contrast to the E1455D mutation, which at 4 °C increased ATP binding at NBD2, binding was decreased by the Gln, Ser, and Leu mutations, whereas the Asn mutation had little or no effect relative to wild-type (Fig. 8A, lanes 4, 6, 8, and 9). Login to comment 345 ABCC1 p.Glu1455Asp At 37 °C, Ser, Asn, and Leu mutations of Glu1455 strongly increased nucleotide binding and trapping at both NBDs in the presence and absence of vanadate, as observed with the E1455D mutation, whereas binding by the Gln mutant protein, although increased relative to wild-type MRP1, remained strongly vanadate-dependent (Fig. 8C). Login to comment 346 ABCC1 p.Glu1455Asp This suggests that the Gln mutant, unlike the other mutant proteins including the E1455D mutant, is able to hydrolyze ATP and probably release ADP in the absence of vanadate. Login to comment 349 ABCC1 p.Glu1455Gln Thus the behavior of the MRP1 Glu1455 to Gln mutation is similar to that of the comparable inactivating mutation in human P-GP, whereas the other mutations appear to block ADP release, as reported in studies of murine mdr3 (32, 33). Login to comment 355 ABCC1 p.Asp793Glu However, a requirement for such a hydrolytic step, as envisaged in an alternating sites model of transport, is very difficult to reconcile with the strong negative effect on transport of mutations that enhance ATPase activity of NBD1, such as the D793E mutation, and the relatively minor effect of mutations at the same location that eliminate the negative side chain at the position of Asp793. Login to comment