Functional Reconstitution of Substrate Transport by Purified Multidrug Resistance Protein MRP1 (ABCC1) in Phospholipid Vesicles*

The 190-kDa multidrug resistance protein MRP1 (ABCC1) is a polytopic transmembrane protein belonging to the ATP-binding cassette transporter superfamily. In addition to conferring resistance to various antineoplastic agents, MRP1 is a transporter of conjugated organic anions, including the cysteinyl leukotriene C4 (LTC4). We previously characterized the ATPase activity of reconstituted immunoaffinity-purified native MRP1 and showed it could be stimulated by its organic anion substrates (Mao, Q., Leslie, E. M., Deeley, R. G., and Cole, S. P. C. (1999) Biochim. Biophys. Acta 1461, 69–82). Here we show that purified reconstituted MRP1 is also capable of active transport of its substrates. Thus LTC4 uptake by MRP1 proteoliposomes was osmotically sensitive and could be inhibited by two MRP1-specific monoclonal antibodies. LTC4 uptake was also markedly reduced by the competitive inhibitor,S-decyl-glutathione, as well as by the MRP1 substrates 17β-estradiol 17-β-(d-glucuronide), oxidized glutathione, and vincristine in the presence of reduced glutathione. The K m for ATP and LTC4 were 357 ± 184 μm and 366 ± 38 nm, respectively, and 2.14 ± 0.75 μm for 17β-estradiol 17-β-(d-glucuronide). Transport of vincristine required the presence of both ATP and GSH. Conversely, GSH transport was stimulated by vincristine and verapamil. Our data represent the first reconstitution of transport competent purified native MRP1 and confirm that MRP1 is an efflux pump, which can transport conjugated organic anions and co-transport vincristine together with GSH.

The 190-kDa multidrug resistance protein MRP1 (ABCC1) is a polytopic transmembrane protein belonging to the ATP-binding cassette transporter superfamily. In addition to conferring resistance to various antineoplastic agents, MRP1 is a transporter of conjugated organic anions, including the cysteinyl leukotriene C 4 (LTC 4 ). We previously characterized the ATPase activity of reconstituted immunoaffinity-purified native MRP1 and showed it could be stimulated by its organic anion substrates (Mao, Q., Leslie, E. M., Deeley, R. G., and Cole, S. P. C. (1999) Biochim. Biophys. Acta 1461, 69 -82). Here we show that purified reconstituted MRP1 is also capable of active transport of its substrates. Thus LTC 4 uptake by MRP1 proteoliposomes was osmotically sensitive and could be inhibited by two MRP1-specific monoclonal antibodies. LTC 4 uptake was also markedly reduced by the competitive inhibitor, S-decyl-glutathione, as well as by the MRP1 substrates 17␤-estradiol 17-␤-(D-glucuronide), oxidized glutathione, and vincristine in the presence of reduced glutathione. The K m for ATP and LTC 4 were 357 ؎ 184 M and 366 ؎ 38 nM, respectively, and 2.14 ؎ 0.75 M for 17␤-estradiol 17-␤-(Dglucuronide). Transport of vincristine required the presence of both ATP and GSH. Conversely, GSH transport was stimulated by vincristine and verapamil. Our data represent the first reconstitution of transport competent purified native MRP1 and confirm that MRP1 is an efflux pump, which can transport conjugated organic anions and co-transport vincristine together with GSH.
Resistance of tumors to multiple structurally unrelated anticancer drugs is a major obstacle to successful cancer chemotherapy. Increased expression in tumor cells of plasma membrane proteins such as the MDR1 P-glycoprotein (ABCB1) or the multidrug resistance protein MRP1 1 (ABCC1) is often associated with such multidrug resistance (1,2). P-glycoprotein and MRP1 both belong to the large ATP-binding cassette (ABC) superfamily of membrane transport proteins but share less than 20% amino acid identity (3,4). Despite this limited sequence similarity, both proteins confer resistance on cancer cells by reducing intracellular drug concentrations in an ATPdependent fashion (1,2). P-glycoprotein, like many ABC proteins, contains two hydrophobic membrane spanning domains (MSDs) each followed by a nucleotide binding domain (NBD). On the other hand, in addition to a four-domain core structure, MRP1 and several of its more closely related proteins contain a third NH 2 -proximal MSD (MSD1) of approximately 200 amino acids with an extracytosolic NH 2 terminus (2,4,5). The precise function of MSD1 is presently unknown, although certain modifications of MSD1 and/or the intracellular loop linking it to MSD2 by truncation or deletion can inactivate MRP1 (6,7). Another distinctive structural feature of MRP1 and its related proteins is that the sequence similarity between their two NBDs is significantly lower than between the NBDs of Pglycoprotein and related protein transporters (2,4). This relative dissimilarity in the MRP-related proteins may reflect the non-equivalent functions of their two NBDs, as we and others have recently demonstrated for MRP1 (8 -10).
MRP1 and its five homologs MRP2-6 (20), together with the sulfonylurea receptor (ABCC8) proteins (21) and the cystic fibrosis transmembrane conductance regulator (22), form one of the largest human ABC transport protein subfamilies described to date. The physiological functions of these MRP subfamily members are diverse, and although several of them are known to transport conjugated organic anions (20), the substrates transported by others are less certain (20,(23)(24)(25)(26)(27)(28)(29)(30). Both MRP1 and MRP2 (ABCC2) have been purified, and the reconstituted proteins have been shown to possess constitutive ATPase activities that can be stimulated by their substrates (31)(32)(33). Results from these and other studies are consistent with the concept that MRP1 and MRP2 are organic anion and drug efflux pumps that transport their substrates concomitant with ATP hydrolysis. However, direct substrate transport by purified reconstituted MRP1 or MRP2 has not yet been reported.
In the present study, we have further characterized MRP1 by demonstrating ATP-dependent uptake of LTC 4 and E 2 17␤G by proteoliposomes containing purified MRP1. The kinetic parameters of substrate transport by the MRP1 proteoliposomes were found to be comparable with those obtained previously in transport studies using MRP1-enriched plasma membrane vesicles. We also present evidence that vincristine and GSH are co-transported by the MRP1 proteoliposomes. These results represent the first functional reconstitution of substrate transport in a lipid vesicle system containing purified MRP1.
Cell Culture and Plasma Membrane Preparation-The doxorubicinselected, MRP1-overexpressing multidrug-resistant H69AR small cell lung cancer cell line was maintained as described previously (4,37). Plasma membranes enriched in MRP1 were prepared from H69AR cells as described (32) and stored at Ϫ80°C before use.
Solubilization of MRP1-MRP1 from H69AR plasma membranes was solubilized as before (32), and the process was modified slightly as follows. 5 mg of plasma membranes was resuspended in 10 ml of buffer A (5 mg ml Ϫ1 CHAPS and protease inhibitor mixture in phosphatebuffered saline, pH 7.4). Thus, the final protein concentration and the ratio of detergent to protein were approximately 0.5 mg ml Ϫ1 and 10:1 (w/w), respectively. The mixture was incubated on ice for 1 h with gentle and frequent vortexing. The sample was then centrifuged at 100,000 ϫ g for 30 min at 4°C, and the supernatant containing solubilized MRP1 was immediately subjected to immunoaffinity chromatography.
Purification of MRP1-Purified mAb QCRL-1 was cross-linked to CNBr-activated Sepharose 4B resin as described (32). The resin was then washed with 25 ml of 0.1 M sodium acetate, pH 4.0, 25 ml of 0.1 M glycine, pH 2.7, and 25 ml of buffer B (50 mM Tris-HCl, pH 7.4, 20% glycerol, 0.5 M NaCl, 5 mM CHAPS, and protease inhibitor mixture), all by brief centrifugation (30 s, 100 ϫ g, each time). Solubilized MRP1 (10 ml) was mixed with the washed resin and incubated for 3 h at 4°C with gentle shaking. The resin with bound protein was packed into a 15-ml spin column (Bio-Rad) and washed 10 times with 50 ml of buffer B as above. The heptapeptide (8 mg) corresponding to the QCRL-1 epitope (SSYSGDI) (35) was dissolved in 2 ml of buffer B and mixed with the resin. The mixture was then incubated overnight at 4°C with gentle shaking. MRP1 was eluted, and the immunoaffinity column regenerated as described previously (32).
Reconstitution of Purified MRP1 into Phospholipid Vesicles-Purified MRP1 was reconstituted into proteoliposomes using rapid detergent removal by gel filtration as follows. Bovine brain lipid extract was dissolved in chloroform, dried under a stream of argon, pumped under vacuum for 2 h at room temperature, and then resuspended at a concentration of 20 mg ml Ϫ1 in reconstitution buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM DTT, 1 mM MgCl 2 ). The lipid suspension was sonicated under argon for 30 min in a bath-type sonicator at room temperature and then stored in aliquots at Ϫ80°C until use. For reconstitution, 3.8 mg of bovine brain lipid and 220 l of 30% (w/v) CHAPS were added in an Eppendorf tube and vortexed 30 s at room temperature. 10 g of purified MRP1 was then added to the lipid/ detergent solution, bringing the final volume to approximately 1 ml. The solution was mixed gently, incubated on ice for 1 h, and passed through a Sephadex G-50 (1 ϫ 25 cm) column. Turbid fractions were collected and proteoliposomes harvested by centrifugation at 145,000 ϫ g for 1 h at 4°C. The pellet was resuspended in 100 l of transport buffer (50 mM Tris-HCl, pH 7.5, 250 mM sucrose) for measurement of LTC 4 transport. For [ 3 H]E 2 17␤G transport, the pellet was resuspended in 20 l of buffer and for [ 3 H]VCR and [ 3 H]GSH transport in 10 l of buffer. Proteoliposomes were homogenized by passing five times through a 27.5-gauge needle and stored in aliquots at Ϫ80°C until use. Recovery of MRP1 in the proteoliposomes was typically 30 -50%. Control liposomes were prepared as described for the proteoliposomes except that MRP1 was omitted.
SDS-Polyacrylamide Gel Electrophoresis, Silver Staining, and Protein Determination-Electrophoresis was performed using 7.5% SDSpolyacrylamide minigels in a Bio-Rad Mini Protein II electrophoresis cell, and proteins were stained with alkaline silver (38). Protein concentrations in the plasma membrane preparations were determined by the Bio-Rad protein assay, and proteins in detergent solutions were quantitated using a modified Lowry assay (39), both with bovine serum albumin as standard.
Assay of ATPase Activity-The effect of different lipids on ATPase activity of purified MRP1 was determined essentially as described (32). Typically, 0.5-1.5 g of purified MRP1 was incubated at 37°C in 0.1 ml of assay buffer containing 50 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 for 4 h. The ATP concentration used was 2.5 mM. Lipids were resuspended in assay buffer by sonication and added to the reaction mixture over a range of concentrations (0.25-2 mg ml Ϫ1 ). Reactions were stopped by adding 33.3 l of 18% SDS, and the amount of inorganic phosphate was determined immediately as described (40,41).
Preparation of Spin Columns for Transport Assays-Latex-free 1-ml syringes plugged with glass wool were filled with Sephadex G-50 Fine resin (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with transport buffer. Syringes were then placed in 13-ϫ 100-mm test tubes and centrifuged for 5 min at 2500 rpm (Sorvall RT6000D). The syringes were filled until the bed volume reached approximately 1 ml by repeated fills and centrifugations and subjected to a final spin for 10 min at 2500 rpm to ensure that the resin was uniformly packed and that excess buffer was removed.
Transport of Substrates into MRP1 Proteoliposomes-ATP-dependent transport of MRP1 substrates was measured using spin column gel filtration as follows. Frozen proteoliposomes, typically containing 1-2 g of protein, were thawed on ice and then mixed with 3 H-labeled substrates in transport buffer such that the final reaction mixture contained substrate, 10 mM MgCl 2 , 4 mM AMP, or 4 mM ATP (with 10 mM creatine phosphate and 100 g ml Ϫ1 creatine kinase) in a final volume of 70 l. [ 3 H]LTC 4 (100 nCi) was added at an initial concentration of 50 nM, and transport was measured at 37°C. In certain experiments, Fab fragments of mAbs QCRL-1, QCRL-3, and QCRL-4, and inhibitors were also included in the transport mixture. At various times, 20 l of proteoliposomes were removed, diluted into 80 l of ice-cold transport buffer, and immediately subjected to purification by spin column gel filtration (400 ϫ g, 3 min) at room temperature. More than 90% of the proteoliposomes passed through the column in the first fraction, and the free radiolabeled substrates [

RESULTS
Purification of Native MRP1-We previously described a differential two-step procedure for solubilization of native MRP1 from H69AR plasma membranes (32). In the present study, we found that more than 90% of MRP1 could be solubilized with 0.5% (w/v) CHAPS after incubation for 1 h at 4°C at a membrane protein concentration of approximately 0.5 mg ml Ϫ1 in phosphate-buffered saline (results not shown). We subsequently used this simpler one-step procedure for protein solubilization prior to immunoaffinity chromatography (32). With this modified approach, approximately 200 g of MRP1 could be purified from 5 mg of plasma membranes, a yield comparable with that obtained previously (32). The purity of MRP1 was at least 80%, as judged by densitometry of a silverstained SDS-polyacrylamide gel (Fig. 1, lanes 1-3). This esti-mate of purity is very conservative and analysis by electron microscopy indicates that the MRP1 preparations are homogeneous and suitable for single particle image analysis. 2 In addition, at a loading of 1 g per lane, no additional proteins were detectable on a Coomassie Blue-stained gel (not shown).
Effect of Lipids on MRP1 ATPase Activity-To determine the effect of different lipid environments on MRP1, various purified and synthetic phospholipids, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, were examined individually, and in a limited number of combinations, for their ability to stimulate MRP1 ATPase activity. None of the individual lipids, nor the combinations tested, significantly enhanced MRP1 ATPase activity relative to basal ATPase activity in the absence of lipid. Indeed, several synthetic phospholipids such as 1,2-dimyristoyl-snglycero-3-phosphatidylcholine (sodium salt) and 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine had a moderate inhibitory effect (data not shown). In contrast, bovine brain lipids, which are comprised of a mixture of phospholipids containing a minimum of 40% phosphatidylethanolamine, stimulated MRP1 ATPase activity 2.5-fold above basal activity at a concentration of 2 mg ml Ϫ1 lipid (data not shown). Purified brain phosphatidylserine (which contains different phosphatidylserine molecules with varied fatty acid content) also stimulated MRP1 ATPase activity by approximately 2.5-fold. Because of the ready availability of the bovine brain lipids, they were used routinely in subsequent reconstitution experiments.
Preparation of MRP1 Proteoliposomes-Proteoliposomes were formed by rapid removal of detergent by gel filtration. Bovine brain lipids were used in approximately 100,000-fold molar excess to MRP1, assuming an average lipid molecular mass of 700 Da. Typically, 30 -50% of MRP1 was recovered in the proteoliposome preparations isolated by gel filtration, compared with the 20 -30% recovery obtained previously using rapid dilution (32). A gel of purified MRP1 in the proteoliposomes stained with alkaline silver is shown in Fig. 1 (lanes  4 -6). CHAPS was used during both purification and reconstitution, because it permitted formation of homogeneously large proteoliposome populations that were well suited for transport measurements (42). MRP1 proteoliposomes stained with uranyl acetate were determined to have diameters of 150 -200 nm by electron microscopy (not shown).
[ 3 H]LTC 4 Uptake by MRP1 Proteoliposomes-In early experiments using nitrocellulose filters (pore size 0.22 m) to measure LTC 4 uptake, we observed that a large proportion of the proteoliposomes passed through the filters. To avoid this problem, a spin column method was used to separate proteoliposomes from free [ 3 H]LTC 4 (43,44). [ 3 H]LTC 4 uptake was measured at 37°C, and the membrane potential was not experimentally manipulated; except for Mg 2ϩ , no other cations were present in the transport mixture. ATP hydrolysis was required for transport, because [ 3 H]LTC 4 uptake by the proteoliposomes was significantly less in the presence of AMP-PCP, a non-hydrolyzable analog of ATP ( Fig. 2A). ATP-dependent [ 3 H]LTC 4 uptake by control liposomes without MRP1 was very low (Fig. 2B) 1. Purified and reconstituted MRP1 resolved on a polyacrylamide gel and stained with alkaline silver. Lanes 1-3, purified MRP1, 0.15 g, 0.3 g, 0.6 g of protein, respectively; lanes 4 -6, purified reconstituted MRP1, 0.1 g, 0.2 g, 0.4 g of protein, respectively. The 7.5% polyacrylamide gel was stained with alkaline silver. system (Fig. 2B). [ 3 H]LTC 4 uptake did not reach equilibrium (steady state) by 10 min, suggesting that there is a continued inward pumping of LTC 4

by MRP1 and that the outward passive diffusion of LTC 4 back across the membrane is low.
To confirm that the ATP-dependent [ 3 H]LTC 4 uptake by the proteoliposomes truly represents transport into the vesicle lumen, rather than surface or intramembrane binding of substrate, the effect of changes in osmolarity on uptake was examined. As shown in Fig. 2C, [ 3 H]LTC 4 uptake decreased as the concentration of sucrose in the transport buffer increased, indicating that ATP-dependent [ 3 H]LTC 4 uptake by the proteoliposomes is osmotically sensitive, as expected for a true transport process.

Inhibition of [ 3 H]LTC 4 Transport by S-decyl-GSH and Various
Substrates of MRP1-To further characterize LTC 4 transport mediated by MRP1 proteoliposomes, the effects of the competitive inhibitor S-decyl-GSH and several other compounds known to be MRP1 substrates were examined; the results are summarized in Table I.

[ 3 H]LTC 4 transport was inhibited by approximately 90% by 1 M S-decyl-GSH. This finding is consistent with our previous observation that Sdecyl-GSH is a potent inhibitor of [ 3 H]LTC 4 uptake by MRP1
enriched plasma membrane vesicles (K i 116 nM) (13). LTC 4 uptake was also inhibited by the MRP1 substrates GSSG and E 2 17␤G by approximately 70% at 2 mM and 100 M, respectively. We have previously shown that the chemotherapeutic agent VCR is co-transported with GSH in MRP1-enriched plasma membrane vesicles (13,17). In the MRP1 proteoliposome system, VCR alone was a very poor inhibitor of LTC 4 uptake with only approximately 20% inhibition observed at 100 M. Similarly, GSH alone at 1 mM inhibited LTC 4 uptake by less than 20%, indicating that GSH itself is a poor inhibitor of LTC 4 uptake. However, VCR (100 M) and GSH (1 or 3 mM) together reduced LTC 4 transport by approximately 55%.

Inhibition of [ 3 H]LTC 4 Uptake by mAbs QCRL-3 and QCRL-4 -
The MRP1-specific mAbs QCRL-3 and QCRL-4 recognize intracellular conformation-dependent epitopes localized to the first and second nucleotide binding domains of MRP1, respectively (36). Both the intact antibodies and their Fab fragments have been shown previously to inhibit MRP1 transport activity in plasma membrane vesicles (13-15, 17, 18, 36, 45), whereas mAb QCRL-1, which recognizes a linear epitope (35), does not. We found that [ 3 H]LTC 4 uptake by the MRP1 proteoliposomes was also strongly inhibited by the Fab fragments of QCRL-3 (Fig. 3A) and QCRL-4 ( Fig. 3B) with IC 50 values of approximately 1 g ml Ϫ1 . In contrast, the Fab fragment of mAb QCRL-1 had no effect on LTC 4 transport in the MRP1 proteoliposomes even at a concentration of 20 g ml Ϫ1 .

Kinetic Parameters of [ 3 H]LTC 4 Transport in MRP1
Proteoliposomes-Kinetic parameters of LTC 4 transport by MRP1enriched plasma membrane vesicles have been reported previously (11,13) and for comparison, similar studies were carried out with the MRP1 proteoliposomes. According to a non-linear regression analysis of the Michaelis-Menten plot of the data points, the apparent K m for ATP was 200 Ϯ 80 M. According to Lineweaver-Burk analysis (Fig. 4), the apparent K m for ATP was 357 Ϯ 184 M. Similarly, the apparent K m for LTC 4 was 366 Ϯ 38 nM (Fig. 5A). These values are somewhat higher than those we obtained previously in transport studies using MRP1enriched plasma membrane vesicles (K m for LTC 4 , 97-105 nM; K m for ATP, 70 M) (11,13) and those obtained in studies of substrate-stimulated MRP1 ATPase activity (K m for ATP, 104 M) (32). However, the K m for ATP is comparable to that for substrate-stimulated MRP1 and MRP2 ATPase activities in Sf9 insect cell membranes (400 M) reported by Bakos et al. (46). The V max for LTC 4 transport in the MRP1 proteoliposomes was 125 Ϯ 12 pmol min Ϫ1 mg Ϫ1 MRP1 (Fig. 5A).
Kinetic   for up to 10 min at 37°C (data not shown). Uptake was measured at several E 2 17␤G concentrations (0 -25 M), and a Lineweaver-Burk analysis yielded an apparent K m of 2.14 Ϯ 0.75 M for this substrate (Fig. 5B). This value is very similar to that for E 2 17␤G transport in MRP1-enriched plasma membrane vesicles (K m 1.5-2.5 M) (14,16). The V max for E 2 17␤G transport was 42 Ϯ 7 pmol min Ϫ1 mg Ϫ1 MRP1 (Fig. 5B).
To test whether VCR is co-transported together with GSH (17), we examined GSH transport in the MRP1 proteoliposomes. [ 3 H]GSH uptake assays were carried out as described for [ 3 H]VCR, except that the incubation time was 20 min and the initial substrate concentration was 100 M. Under these conditions, [ 3 H]GSH was transported into the proteoliposomes at approximately 0.6 nmol (20 min) Ϫ1 mg Ϫ1 MRP1, and this activity was stimulated approximately 2-fold by the addition of 100 M VCR (Fig. 7). [ 3 H]GSH uptake was stimulated even further (approximately 3-fold) to 1.87 Ϯ 0.19 nmol (20 min) Ϫ1 mg Ϫ1 MRP1 by 100 M verapamil (Fig. 7), consistent with our previous results using MRP1-enriched plasma membrane vesicles (45). DISCUSSION The ability to functionally reconstitute purified ABC proteins represents a major step toward understanding the mechanistic relationships between ATP binding and hydrolysis and coupling of these events to translocation of substrates across lipid membranes. It is also a necessary prerequisite for detailed structural studies of these proteins using techniques such as electron microscopy and image analysis as reported for P-glycoprotein (47). Although purification of ABC proteins in an active form has proven a challenging task, as might be expected for any large integral membrane protein, success has been achieved in recent years with a variety of experimental strategies (31-33, 48 -52).
The intrinsic ATPase activities reported for the various ABC proteins appear to be quite variable with values ranging from a low of 1.3 nmol min Ϫ1 mg Ϫ1 for ABCR (ABCA4) (the photoreceptor protein involved in Stargardt macular dystrophy (48)), to 50 nmol min Ϫ1 mg Ϫ1 for cystic fibrosis transmembrane conductance regulator (49), to a high of approximately 1 mol min Ϫ1 mg Ϫ1 for P-glycoprotein (50). The intrinsic ATPase activity of immunoaffinity purified native MRP1 is considerably lower than that of P-glycoprotein at approximately 5-10 nmol min Ϫ1 mg Ϫ1 MRP1 (32). However, half-maximal stimulation of MRP1 ATPase activity by several conjugated organic anion substrates was observed at concentrations that correspond well with K m values obtained in transport studies using membrane vesicles. This suggested to us that MRP1 might be a more highly coupled transporter than P-glycoprotein (32). On the other hand, Chang et al. (31) reported that purified histidine- tagged recombinant MRP1 exhibited a high ATPase activity (460 nmol min Ϫ1 mg Ϫ1 ) comparable to that of P-glycoprotein. The basis for the apparent discrepancy between our findings and those of Chang et al. (31) is unknown. However, it is well established that the lipid environment in which a membrane protein is reconstituted can affect its interaction with substrates and thus modulate its activity. For example, it has been shown that the thickness of the lipid bilayer, its physical phase, and the lipid headgroup structure can all be important for the function of the Ca 2ϩ -ATPase in the sarcoplasmic reticulum of skeletal muscle (53). More pertinent to the present study is the observation that certain lipids significantly influence the characteristics of purified P-glycoprotein ATPase activity and the apparent coupling between its drug-binding and catalytic sites (52, 54 -56). Among the natural and synthetic lipids tested in the present study, the greatest stimulation of basal ATPase activity of purified CHAPS-solubilized MRP1 was observed with bovine brain lipid extract and purified brain phosphatidylserine. No single synthetic lipid significantly stimulated MRP1 ATPase activity, a finding that contrasts with comparable studies of hamster P-glycoprotein, where addition of a single synthetic lipid (dipalmitoylphosphatidylethanolamine) led to a 3-fold stimulation of its basal ATPase activity (52). On the other hand, the ATPase activity of human ABCR could not be stimulated by its substrate, all-trans-retinal, when the protein was reconstituted in vesicles composed of single synthetic lipids (57). Thus, overall, there appears to be considerable variability among the ABC transporters with respect to their response to their lipid environment. Our data suggest that the low basal ATPase activity of native MRP1 may be less influenced by its lipid environment than P-glycoprotein ATPase activity and that a combination of various phospholipids constitutes the optimal environment for MRP1. Whether or not MRP1 binding of its conjugated or unconjugated substrates is also less dependent on lipid environment than has been shown for P-glycoprotein (56) remains to be determined. In this regard, it is of interest that certain lipid analogs have been reported to be substrates of MRP1 (58,59).
The results shown here demonstrate for the first time transport of LTC 4 , E 2 17␤G, VCR, and GSH by purified MRP1 in an artificial lipid bilayer system. The relatively high hydrophilicity of the MRP1 organic anion substrates limits their ability to penetrate lipid membranes, which facilitates analysis of their transport kinetics in this system. The sensitivity of LTC 4 transport to changes in osmolarity confirmed that the ATP-dependent uptake of this substrate by MRP1 proteoliposomes involved true transport into the vesicle lumen. This conclusion was further supported by our findings that the K m values for LTC 4 and E 2 17␤G were comparable with those reported previously in membrane vesicle transport studies (11,13,14,16), and the K m for ATP was similar to that reported in studies of MRP1 ATPase activity (32,46). LTC 4 transport by MRP1 proteoliposomes was strongly inhibited by Fab fragments of the MRP1specific mAbs QCRL-3 and QCRL-4, again in agreement with earlier membrane vesicle studies (36). Finally, LTC 4 transport was inhibited by compounds shown previously to be competitive inhibitors and/or substrates of MRP1. Collectively, these data provide strong support for the conclusion that MRP1 acts as an energy-driven efflux pump that appears not to require additional components for its basal activity.
ATP-dependent transport of VCR was also observed in the MRP1 proteoliposomes but only when GSH was present. That GSH but not 2-mercaptoethanol increased VCR uptake (Fig.  6B) is consistent with our earlier conclusion that it is not the sulfhydryl-reducing capacity of GSH that is responsible for the increased uptake of unconjugated drug substrates but some other interaction of this tripeptide with MRP1 (13,17). GSH was also transported by the proteoliposomes, and its uptake was stimulated approximately 2-fold by VCR (Fig. 7). Taken together, these results provide further support to our previous suggestion that VCR and GSH are co-transported by MRP1 (17). Kinetic analyses of VCR and GSH co-transport were not possible, because the quantity of purified MRP1 required for such studies greatly exceeded that which is readily available at present. However, GSH transport in the MRP1 proteoliposomes was also stimulated by verapamil and to an even greater degree than VCR, an observation that is again consistent with our recent studies on drug-stimulated MRP1-mediated GSH transport in membrane vesicles (45). We have postulated that the greater ability of verapamil to stimulate GSH uptake is related to the greater lipophilicity of this compound compared with VCR. Consistent with this suggestion we observed during a recent examination of 20 dithiane and dithiane tetraoxide analogs of verapamil that the relative lipophilicity of an analog was the strongest predictor of its ability to stimulate GSH uptake by MRP1. 3 The purification of native (32) and histidine-tagged recombinant MRP1 (31) and MRP2 (33) and an analysis of their respective basal and substrate-stimulatable ATPase activities represent an important step toward our understanding of how substrate transport by members of the MRP subfamily is coupled to ATP binding and hydrolysis and how it compares with other ABC transporters. Hagmann et al. (33) recently reported that liposomes containing purified histidine-tagged recombinant MRP2 exhibited low level basal ATPase activity (25 nmol min Ϫ1 mg Ϫ1 MRP2) that could be stimulated by GSSG, GSH, and S-decyl-GSH. This intrinsic ATPase activity is significantly lower than that reported by Chang et al. (31) for recombinant MRP1 but somewhat higher than that reported for native MRP1 by our group (32). However, the K m (ATP) of MRP2 was estimated to be 2.1 mM (33), which is more than six times higher than that obtained for native MRP1 (32) (and this study) but comparable with that reported for recombinant MRP1 by Chang et al. (31). On the other hand, the K m (ATP) of MRP2 enriched in membrane vesicles is approximately 400 M (46). Unfortunately, the ability of LTC 4 and E 2 17␤G to stimulate the ATPase activity of purified MRP2 was not tested, although these organic anions are known to be MRP2 substrates (20,33,46). Thus, it is not possible at present to compare the substrate stimulatable ATPase activities of these two highly related proteins with each other, or with other ABC transporters such as P-glycoprotein. Moreover, comparison of the transport properties of the native MRP1 as described here with those of recombinant MRP1 (31) and MRP2 (33) awaits the demonstration that the latter proteins are transport-competent in a proteoliposome system.
In summary, we have improved our system for purification of MRP1 and demonstrated its functional reconstitution with respect to both ATPase activity and transport of its conjugated and unconjugated substrates. This system will be useful for studies aimed at understanding how translocation of MRP1 substrates is coupled to ATP hydrolysis and how (and if) this process can be influenced by other cellular components.