Is ATP binding responsible for initiating drug translocation by the multidrug transporter ABCG2? Christopher A. McDevitt 1 , Emily Crowley 1 , Gemma Hobbs 1 , Kate J. Starr 2 , Ian D. Kerr 2 and Richard Callaghan 1 1 Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, UK 2 Centre for Biochemistry and Cell Biology, School of Biomedical Sciences, University of Nottingham, UK Resistance to chemotherapy presents a continuing and significant obstacle in the treatment of both solid tumours and haematological malignancies. One of the most prevalent primary cellular defence mechanisms against chemotherapeutic agents is the membrane- bound transporter [1]. The defining feature of these transporters is their ability to interact with a broad range of structurally unrelated compounds, a property that has led them to be described as ‘multidrug trans- porters’ [2–4]. The resistant phenotype is conferred by the reduction in cytoplasmic concentrations of chemo- therapeutic drugs to levels below that required for cytotoxicity. Resistance to chemotherapy has been attributed to the expression of three ‘multidrug trans- porters’, all members of the ATP binding cassette (ABC) superfamily, designated as ABCB1, ABCC1 and ABCG2. Specifically, ABCG2 has been implicated in clinical multidrug resistance in acute myeloid leu- kaemia [5–8]. However, although ABCB1 and ABCC1 have been extensively characterized, there are many unresolved issues relating to the basic biochemistry of ABCG2. ABCG2 is a 72 kDa integral membrane protein con- sisting of six transmembrane helices and an amino terminal nucleotide binding domain (NBD) [9–11]. It is described as being a ‘half-transporter’ as the canonical ABC transporter typically consists of two transmem- brane domains (TMDs) and two NBDs. Furthermore, the topological organization of ABCG2 is distinct from ABCB1 and ABCC1, as NBD is N-terminal to TMD [9]. To date, there are no high-resolution structures available for any of the eukaryotic ABC Keywords ABC transporter; chemotherapy; membrane protein; multidrug-resistance; power-stroke Correspondence R. Callaghan, Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK Fax: +44 1865 221 834 Tel: +44 1865 221 110 E-mail: richard.callaghan@ndcls.ox.ac.uk (Received 28 May 2008, revised 24 June 2008, accepted 27 June 2008) doi:10.1111/j.1742-4658.2008.06578.x ABCG2 confers resistance to cancer cells by mediating the ATP-dependent outward efflux of chemotherapeutic compounds. Recent studies have indi- cated that the protein contains a number of interconnected drug binding sites. The present investigation examines the coupling of drug binding to ATP hydrolysis. Initial drug binding to the protein requires a high-affinity interaction with the drug binding site, followed by transition and reorien- tation to the low-affinity state to enable dissociation at the extracellular face. [ 3 H]Daunomycin binding to the ABCG2 R482G isoform was examined in the nucleotide-bound and post-hydrolytic conformations. Binding of [ 3 H]daunomycin was displaced by ATP analogues, indicating transition to a low-affinity conformation prior to hydrolysis. The low-affinity state was observed to be retained immediately post-hydrolysis. Therefore, the dissoci- ation of phosphate and ⁄ or ADP is likely to be responsible for resetting of the transporter. The data indicate that, like ABCB1 and ABCC1, the ‘power stroke’ for translocation in ABCG2 R482G is the binding of nucleotide. Abbreviations ABC, ATP binding cassette; ATP-c-S, adenosine 5¢-[c-thio]-triphosphate; NBD, nucleotide binding domain; TMD, transmembrane domain; TNP-ATP, 2¢,3¢-O-(2,4,6-trinitrophenyl) adenosine 5¢-triphosphate. 4354 FEBS Journal 275 (2008) 4354–4362 ª 2008 The Authors Journal compilation ª 2008 FEBS transporters, although an 18 A ˚ structure of ABCG2 was obtained using electron microscopy [12]. This report indicated that soluble purified ABCG2 dis- played a propensity to form a higher order oligomer, a tetramer of dimers, which is consistent with the obser- vations of higher order oligomeric species in cell membranes [13]. Although the precise molecular composition remains controversial, there is a growing weight of evidence favouring a higher order structure [12–16]. ABCG2 displays distinct, but not exclusive, sub- strate specificity compared with other multidrug trans- porters. In particular, the protein confers resistance to the anticancer drugs mitoxantrone [17], methotrexate [18] and the camptothecins [19]. Although early cellu- lar studies failed to generate a consensus for the sub- strate profile, the discrepancies were attributed to a mutation generated during long-term selection in the presence of anticancer drugs. Selection in mitoxantrone produced R482G or R482T point mutations that pres- ent considerably broader substrate selectivity [20,21]. For example, the R482G isoform is a gain-of-function mutation which mediates the transport of doxorubicin, daunomycin and rhodamine 123, whereas it has a loss of function with respect to methotrexate transport. Recent investigations have demonstrated that ABCG2 R482G , like other multidrug transporters, con- tains more than one drug binding site. In addition, the binding sites are linked by both negative and positive heterotropic allostery. In a departure from the drug– protein interactions with ABCB1, the R482G isoform also contains multiple sites of interaction for a single drug (daunomycin), which can manifest as homotropic allostery [22]. The latter has been observed for the bacterial half-transporter LmrA, but not for any eukaryotic ABC protein [23]. The translocation of drugs across the plasma mem- brane requires that the drug binding event(s) in TMD is intrinsically coupled to the catalytic cycle within NBDs. The best evidence for an interaction between the two domains is the ability of numerous substrates and modulators of ABCG2 (and the R482G isoform) to stimulate the rate of ATP hydrolysis [21,24,25], albeit to a lesser degree than that commonly encoun- tered with ABCB1. The translocation event requires that the drug binding sites switch from the initial high-affinity, inward-facing configuration to an outward-facing, low-affinity configuration to facilitate dissociation [26]. Originally, the impetus for the switch in binding site affinity and orientation was thought to be the energy produced by nucleotide hydrolysis. In the case of ABCB1, this was revised through the obser- vations that nucleotide binding in the absence of hydrolysis could cause the conformational alteration (reviewed in [27,28]). The low-affinity conformation of drug binding sites in ABC multidrug efflux pumps is assumed to correspond to the outward-facing confor- mation. The energy produced by the hydrolysis of ATP is harnessed for the resetting of the transporter to the initial high-affinity, inward-facing configuration. Similar results were also obtained for ABCC1. Thus, the eukaryotic multidrug transporters are thought to mediate drug translocation through a ‘power stroke’ which is obtained by the binding of nucleotide. The focus of the present investigation was to ascer- tain whether the binding of nucleotide to ABCG2 R482G was the power stroke required to switch the configura- tion of the drug binding site(s). This hypothesis was examined using a direct measure of drug binding to the protein, which was trapped in both pre- and post- nucleotide hydrolytic conformations. Results Characteristics of drug binding to ABCG2 R482G - containing membranes The expression of ABCG2 R482G has previously been established in High-5 insect cells using recombinant baculovirus [22]. [ 3 H]Daunomycin (300–350 nm) bound to the membranes with a total binding capacity of 107 ± 13 pmolÆmg )1 , which was significantly reduced following the addition of a large molar excess of doxoru- bicin (30 lm). The remaining [ 3 H]daunomycin associ- ated with the membranes corresponded to nonspecific binding at sites other than the ABCG2 R482G protein. This fraction corresponded to 37 ± 8 pmolÆmg )1 , and therefore the specific binding component in the mem- branes was 70 pmolÆmg )1 . The dissociation constant for [ 3 H]daunomycin binding to ABCG2 R482G has previ- ously been estimated as 98 nm [22], and all subsequent binding assays in this study were conducted with 300– 350 nm of the radioligand. There was no detectable dis- placement of [ 3 H]daunomycin binding to membranes that did not express ABCG2 R482G (data not shown). A heterologous drug displacement assay was under- taken with ABCG2 R482G -containing membranes to characterize the potency of the drug–protein interaction. Figure 1A demonstrates that doxorubicin is able to dis- place 90 ± 2% of the specific binding component of [ 3 H]daunomycin. Moreover, the potency to displace [ 3 H]daunomycin binding is IC 50 = 1.73 ± 0.51 mm (n = 9), which is in good agreement with the value previously described [22]. Thus, High-5 insect cell membranes provide a specific method to examine the drug binding characteristics of ABCG2 R482G . C. A. McDevitt et al. The power stroke in ABCG2 FEBS Journal 275 (2008) 4354–4362 ª 2008 The Authors Journal compilation ª 2008 FEBS 4355 Characteristics of nucleotide binding to purified ABCG2 R482G Photolabelling of ABCG2 R482G by [a 32 P]azido-ATP was used to characterize the interaction of nucleotides with the transporter. As shown in Fig. 2A, [a 32 P]azido-ATP binds to ABCG2 R482G in a dose-dependent manner. Unfortunately, commercial preparations of the photo-active nucleotide do not attain sufficiently high concentrations to enable complete saturation of binding. However, the binding isotherm in Fig. 2A provides an estimate of the binding affinity for [a 32 P]azido-ATP as K D = 201 ± 80 lm. This affinity is similar to the value obtained for ATP binding to ABCB1 [29]. The ability of nucleotides to displace binding is shown in Fig. 2B, with values normalized to the amount bound in the absence of added nucleotide. Neither ADP nor AMP altered the photolabelling of [a 32 P]azido-ATP bound, whereas the ATP analogues adenosine 5¢-[c-thio]-triphosphate (ATP-c-S) and 2¢,3¢-O-(2,4,6-trinitrophenyl) adenosine Fig. 1. Heterologous displacement of [ 3 H]daunomycin binding to ABCG2 R482G by doxorubicin. (A) ABCG2 R482G -containing insect cell membranes (20 lg) were incubated with [ 3 H]daunomycin (300 nM) in the presence or absence of varying concentrations of doxorubicin (1 n M to 300 lM). Incubations were performed at 20 °C for a period of 120 min to ensure that equilibrium had been reached. Unbound [ 3 H]daunomycin was removed using a rapid filtration assay, and the amount of bound radioligand was determined by liquid scintillation counting. Values refer to the mean ± SEM of at least three inde- pendent membrane preparations, and the dose–response curve was fitted using nonlinear least-squares regression. (B) A series of nucleotides was examined for their propensity to displace the bind- ing of [ 3 H]daunomycin (300 nM) to ABCG2 R482G containing High-5 insect cell membranes (20 lg). The radioligand was incubated with the ABCG2 R482G -containing membranes in the presence of 10 mM nucleotide. The only exception was the ATP analogue TNP-ATP, which was used at a concentration of 0.6 m M. The amount of [ 3 H]daunomycin bound to the membranes in the absence of nucleo- tide was assigned a value of unity, and all other data were expressed as a fraction of this. Values correspond to the mean ± SEM of three independent membrane preparations. Fig. 2. The binding of nucleotides and analogues to ABCG2 R482G . (A) Purified ABCG2 R482G (0.25 lg) was photolabelled with [a 32 P]azido-ATP (3–300 lM) as described in Materials and methods. Labelled protein was visualized and quantified by autoradiography of SDS-PAGE analysis. The amount of bound protein was plotted as a function of nucleotide concentration, and the data were fitted with the Langmuir binding isotherm using nonlinear least-squares regression. (B) Photoaffinity labelling of purified ABCG2 R482G (0.25 lg) was undertaken using a fixed concentration (30 lM)of [a 32 P]azido-ATP in the presence or absence of ADP (10 mM), AMP (10 m M), ATP-c-S (10 mM) or TNP-ATP (1 mM). The intensity of labelling in the absence of excess nucleotide was assigned a value of unity. The power stroke in ABCG2 C. A. McDevitt et al. 4356 FEBS Journal 275 (2008) 4354–4362 ª 2008 The Authors Journal compilation ª 2008 FEBS 5¢-triphosphate (TNP-ATP) produced considerable reductions in the amount of bound nucleotide. Screening nucleotides for propensity to modify drug binding to ABCG2 R482G ABCG2 R482G -containing membranes were incubated with [ 3 H]daunomycin and a series of adenine nucleo- tides and three analogues to assess interactions. A fixed concentration of nucleotide (10 mm) was used, apart from TNP-ATP which was administered at 0.6 mm because of its higher potency. Figure 1B dem- onstrates the ability of the nucleotides to reduce the fraction of [ 3 H]daunomycin bound to ABCG2 R482G .In the presence of AMP, the binding of [ 3 H]daunomycin remained at 93 ± 4% (n =8, P > 0.05) of that obtained in the untreated control, and the addition of ADP produced a marginal decrease to 80 ± 5% (n =4, P > 0.05). The addition of ATP produced a statistically significant decrease (n =4, P < 0.05) in the amount of [ 3 H]daunomycin bound to a value of 59 ± 9%. The nonhydrolysable nucleotide, ATP-c-S, produced an even greater decrease to 59 ± 4% (n =8, P < 0.05). Despite the use of a considerably lower concentration (0.6 mm), the fluorescent and slowly hydrolysable analogue TNP-ATP reduced the binding to 35 ± 4% (n =6,P < 0.05). Binding of [ 3 H]daunomycin to ABCG2 R482G in a pre-hydrolysis configuration ATP, and its nonhydrolysable analogues ATP-c-S and TNP-ATP, reduced the degree of [ 3 H]daunomycin binding to ABCG2 R482G , thus warranting further examination of the effect of these nucleotide analogues. Figure 3 shows the effects of a range of ATP-c-S concentrations on the interaction of [ 3 H]dau- nomycin with ABCG2 R482G . At the highest concentra- tion of nucleotide, only approximately 20% of the radioligand was bound to the protein. The extent of binding was fitted with a dose–response curve, which generated a potency of IC 50 = 11.8 ± 1.6 mm for ATP-c-S. Similar analysis was undertaken for the slowly hydrolysable analogue TNP-ATP, as shown in Fig. 4. At a concentration of 2 mm, < 10% of the ini- tial binding of [ 3 H]daunomycin was observed. The potency of TNP-ATP to displace [ 3 H]daunomycin binding was characterized by IC 50 = 0.27 ± 0.02 mm, which is 44-fold greater than that of ATP-c-S. Both TNP-ATP and ATP-c-S cause a decrease in the extent of [ 3 H]daunomycin binding to ABCG2 R482G . Given the distinct sites for binding of nucleotides and drugs to the protein, this decrease occurs via a nega- tive allosteric mechanism. The addition of either nucle- otide analogue will effectively trap the protein in a conformation closely resembling the pre-hydrolytic state. The decrease in capacity for drug binding reflects a lower affinity interaction between [ 3 H]daunomycin and the protein immediately prior to ATP hydrolysis. Fig. 3. Heterologous displacement of [ 3 H]daunomycin binding to ABCG2 R482G by the nonhydrolysable nucleotide ATP-c-S. The effect of the nonhydrolysable ATP analogue ATP-c-S (100 l M to 20 mM) on [ 3 H]daunomycin (300 nM) binding to ABCG2 R482G was examined using High-5 cell membranes (20 lg). Incubations were undertaken at 20 °C for a period of 120 min, and the membrane-bound radioli- gand was harvested by vacuum filtration through a manifold. The general dose–response relationship was fitted to the data (mean ± SEM, n ‡ 3) using nonlinear least-squares regression. Fig. 4. Heterologous displacement of [ 3 H]daunomycin binding to ABCG2 R482G by TNP-ATP. [ 3 H]Daunomycin (300 nM) was incubated with ABCG2 R482G -containing High-5 insect cell membranes (20 lg) in the presence or absence of varying concentrations of the fluores- cent ATP analogue TNP-ATP (10 l M to 1.2 mM). Incubations were performed at 20 °C for a period of 120 min to ensure that equilib- rium had been reached. Unbound [ 3 H]daunomycin was removed using a rapid filtration assay, and the amount of bound radioligand was determined by liquid scintillation counting. Values refer to the mean ± SEM of at least three independent membrane prepara- tions, and the dose–response curve was fitted using nonlinear least-squares regression. C. A. McDevitt et al. The power stroke in ABCG2 FEBS Journal 275 (2008) 4354–4362 ª 2008 The Authors Journal compilation ª 2008 FEBS 4357 Binding of [ 3 H]daunomycin to ABCG2 R482G in a post-hydrolysis configuration Given that the [ 3 H]daunomycin binding site is switched to a low-affinity configuration on nucleotide binding, what is the consequence of ATP hydrolysis for the drug binding sites? To address this issue, ABCG2 R482G was trapped immediately post-hydroly- sis using sodium orthovanadate [21]. The metal oxo- anion vanadate serves as a transition state mimic, exploiting its chemical similarity to phosphate. Thus, ATP and vanadate generate an ADP-vanadate struc- ture mimicking the transition state for the hydrolysis of the c-phosphate of ATP [30]. Figure 5 demon- strates the effect of pre-incubation of ABCG2 R482G - containing membranes with 100 lm NaVO 3 and a series of ATP concentrations. The data show a 90% decrease in the amount of [ 3 H]daunomycin bound to the membranes, indicating that the capacity for sub- strate interaction is considerably reduced. The potency for vanadate trapping to reduce [ 3 H]dauno- mycin binding to ABCG2 R482G was 21.3 ± 3.3 mm of nucleotide. Therefore, the data demonstrate that ABCG2 R482G remains in a conformation that contains a low-affinity binding site for [ 3 H]daunomycin imme- diately post-nucleotide hydrolysis. Discussion A precise molecular mechanism for substrate translo- cation by any ABC protein remains unresolved, despite considerable investigation using varied approaches and recent high-resolution X-ray crystal structures. Investigations with multidrug transporters, involved in conferring drug resistance in cancer cells, have provided the most information. For two of the proteins, ABCB1 and ABCC1, it has been demon- strated that the binding of nucleotide imparts marked and essential conformational changes within TMDs. The present study provides the first evidence that nucle- otide binding per se also plays a role in the initiation of the drug translocation process for ABCG2, despite its structurally dissimilar architecture to the aforemen- tioned transporters. A radioligand binding approach was used in the investigations and has previously been evaluated for use with the ABCG2 R482G isoform [22]. The ‘gain-of-func- tion’ mutation confers resistance to the anthracycline daunomycin by transporting it out of the cytoplasm [31]. A previous study has indicated that there are two allosterically coupled binding sites for daunomycin, although it is unclear whether the coupling is between the two monomers in a transporter, or between distinct dimeric units [22]. Measurement of [ 3 H]daunomycin binding provides a useful insight into the pharmacology of the ABCG2 R482G isoform, as the binding site is in communication with those for different drug substrates. The initial nucleotide screen revealed that several nucleotide species were capable of modulating drug binding, thereby reaffirming the interdomain communi- cation reported for ABCG2. However, despite using relatively high concentrations, neither AMP nor ADP was capable of altering the drug–ABCG2 interaction. In the case of the monophosphate AMP, this was entirely expected as this nucleotide plays no role in the catalytic process of ABCG2, and was therefore a control for specificity of the interaction. The lack of effect of the diphosphate nucleotide indicates that, following inorganic phosphate release, the ADP-bound ABCG2 R482G isoform adopts a conformation capable of supporting the binding of [ 3 H]daunomycin. The triphosphate nucleotide ATP caused a consider- able decrease in the ability of ABCG2 R482G to bind [ 3 H]daunomycin. This decrease in drug binding was also observed in the presence of the ATP analogues ATP-c-S (nonhydrolysable) and TNP-ATP (slowly hydrolysable), although the magnitude of effect with the latter was more pronounced. The ATP analogues were preferred for subsequent investigations, as ATP is an inherently unstable or reactive compound in aqueous solutions, even at the reduced temperatures employed in radioligand binding assays. Detailed investigation revealed that [ 3 H]daunomycin binding by ABCG2 R482G was essentially abrogated in the presence Fig. 5. The binding of [ 3 H]daunomycin to vanadate-trapped ABCG2 R482G . ABCG2 R482G was trapped in the presence of sodium orthovanadate (100 l M) and a series of ATP concentrations (100 lM to 300 mM)at37°C for 30 min. The vanadate-trapped protein was then incubated with [ 3 H]daunomycin (300 nM) for 120 min at 20 °C. Bound and free radioligand were separated using a rapid filtration assay, and the former was detected using liquid scintillation count- ing. Values correspond to the mean ± SEM of at least three inde- pendent membrane preparations, and the dose–response curve was fitted using nonlinear least-squares regression. The power stroke in ABCG2 C. A. McDevitt et al. 4358 FEBS Journal 275 (2008) 4354–4362 ª 2008 The Authors Journal compilation ª 2008 FEBS of sufficient ATP-c-S or TNP-ATP. Thus, the ATP- loaded conformation of ABCG2 R482G ([E]Æ[ATP], where ‘E’ refers to ABCG2 R482G ) facilitates a negative heterotropic allosteric effect of NBDs on TMDs. This finding with ABCG2 R482G is entirely consistent with the observation for the interaction of [ 3 H]vinblastine with ABCB1 and for [ 3 H]estrone-sulfate binding to ABCC1 [32,33]. Such a decrease in the affinity or capacity of drug binding to ABCG2 R482G is likely to represent the outward-facing conformation of the transporter, as the presence of an inward-facing drug binding site with low affinity would preclude an effi- cient rate of translocation. A possible alternative explanation is that, although the binding of ATP reduces the drug binding site to low affinity, it does not generate an outward-facing conformation. How- ever, this would require that the drug binding site adopts an occluded inward-facing conformation to prevent dissociation, and that reorientation occurs fol- lowing harnessing of the energy from ATP hydrolysis. If one of the initial events in the nucleotide catalytic cycle is responsible for the decrease in affinity (and pre- sumably reorientation) of drug binding sites, what role do subsequent steps play in the translocation process? As mentioned above, the [E]Æ[ADP] conformation appears to have returned to high affinity, and the inter- vening steps in the catalytic cycle are responsible for the restoration of binding capacity. In order to main- tain ABCG2 R482G in a stable post-hydrolysis conforma- tion, we employed the vanadate trapping procedure. The data revealed that vanadate-trapped ABCG2 R482G protein ([E]Æ[ADP]Æ[Vi]) remained in a low-affinity [ 3 H]daunomycin binding conformation. By inference, therefore, the step in the catalytic cycle corresponding to the release of inorganic phosphate ([P i ]) is likely to correspond to the restoration of a high-affinity conformation for the transporter, which is supported by the restoration of high-affinity binding in the ADP- bound conformation. That this step of the catalytic cycle is associated with the greatest free energy change also makes it ideal for the mediation of drug binding site reorientation, although the binding data cannot unequivocally inform on the orientation of the sites, only their affinity for interaction with drugs. The data presented here suggest that the ABC- G2 R482G isoform undergoes the following sequence of conformational transitions: ½E H $½E L Á½ATP$½E L Á½ADP½P i  $½E H Á½ADP$½E H where [E] H and [E] L correspond to the high- and low- affinity conformations of ABCG2 R482G , respectively. The sequence, based on the measurement of drug– protein binding, indicates that the binding of ATP per se is the ‘power stroke’ for drug translocation, and that energy obtained from the hydrolysis process is used to reset the transporter. That ATP binding is responsible for the shift in binding affinity from high to low has now been demonstrated for all three eukaryotic multidrug efflux proteins in the ABC family. Materials and methods Materials [ 3 H]Daunomycin (0.185 TBq Ci Æ mmol )1 ) was purchased from Perkin Elmer LAS (Beaconsfield, UK) and Ready Protein + scintillation fluid was obtained from Beckman Coulter (High Wycombe, UK). Doxorubicin, sodium ortho- vanadate, ATP, ADP, AMP, ATP-c-S and TNP-ATP were purchased from Sigma (Poole, UK). GF ⁄ F filters were purchased from VWR International (Lutterworth, UK). Insect Xpress medium was obtained from Cambrex (Read- ing, UK) and Ex-cell 405 medium from JRH Biosciences (Andover, UK). Insect cell culture and membrane preparation The Trichoplusia ni (High-5) cell line was routinely used for the expression of ABCG2 R482G and maintained in shaking suspension cultures, as described previously [22]. High-5 cells at a density of approximately 3 · 10 6 cellsÆmL )1 were infected with recombinant baculovirus (approximately 1 · 10 8 plaque-forming unitsÆmL )1 ) at a multiplicity of infection of five. After 1 h of incubation with virus, the cells were diluted to a density of 1.5 · 10 6 cellsÆmL )1 and maintained in suspension for 3 days before harvesting by centrifugation (2000 g, 10 min). Crude membrane preparations were isolated as described previously [34], with the exception that buffers contained 20 mm Mops, pH 7.4, 200 mm NaCl and 0.25 m sucrose. Briefly, cells were ruptured with four rounds of nitrogen cavitation using 6500–10 000 kPa at 4 °C, with a 20 min incubation between rounds. Cell debris was removed by centrifugation at 2000 g for 10 min. Crude membranes were isolated by ultracentrifugation at 100 000 g for 60 min at 4 °C. Membranes were resuspended at protein concentra- tions of approximately 50 mgÆmL )1 in isolation buffer (0.25 m sucrose, 20 mm Mops, pH 7.4, containing a prote- ase inhibitor cocktail) and stored at )80 °C. Radioligand binding assay Radiolabelled drug binding assays were based on a previ- ously published technique used to investigate ABCB1 [35]. Membranes (20 lg) were incubated with a radiolabel, C. A. McDevitt et al. The power stroke in ABCG2 FEBS Journal 275 (2008) 4354–4362 ª 2008 The Authors Journal compilation ª 2008 FEBS 4359 [ 3 H]daunomycin, in a total volume of 100 lL in polypro- pylene test tubes for 120 min to ensure attainment of bind- ing equilibrium. The membranes, [ 3 H]daunomycin and any other drugs were incubated in hypotonic binding buffer, comprising 50 mm Tris ⁄ HCl, pH 7.4. Hypotonic buffer was used in binding assays to ensure no intraliposomal drug accumulation. Nonspecific binding to the filters or the lipid component of membranes was defined as the amount of [ 3 H]daunomycin bound in the presence of a large molar excess (30 lm) of doxorubicin. Drugs were added from con- centrated stocks in dimethylsulfoxide and the solvent con- centration was maintained at < 1% (v ⁄ v). Actual concentrations of [ 3 H]daunomycin added to the tubes were determined by liquid scintillation counting. Unbound ligand was separated from bound ligand through porous glass- fibre filters (GF ⁄ F) using rapid vacuum filtration on a 48-well manifold. The GF ⁄ F filters were pre-soaked in wash buffer supplemented with 0.1% (w ⁄ v) BSA for 10 min. Samples on the filters were rinsed twice with 10 mL of ice-cold wash buffer (50 mm Tris ⁄ HCl, pH 7.4, 20 mm MgSO 4 ). [ 3 H]Daunomycin bound to the filters was mea- sured by liquid scintillation counting using Ready Protein + scintillation fluid. Heterologous displacement assays used ABCG2 R482G - containing crude membranes incubated with a single con- centration of [ 3 H]daunomycin (300–350 nm)at20°C for 120 min. Doxorubicin was added over the concentration range 1 nm to 300 lm, obtained from the serial dilution of a concentrated stock in dimethylsulfoxide. All nucleotides and analogues were added from concentrated stocks in buf- fer containing 5 mm MgCl 2 , 100 mm Mops at pH 6.8. The NaVO 3 stock solution (100 mm) was treated as described previously by Goodno [36] to remove polymeric species. Membranes were incubated with 100 lm NaVO 3 in the presence of varying concentrations of MgATP (100 lm to 300 mm) in ATPase buffer (150 mm NH 4 Cl, 50 mm Tris ⁄ HCl, pH 7.4, 5 mm MgSO 4 ). The vanadate trapping of ABCG2 R482G was achieved at 37 °C for 30 min prior to the binding assay, according to a previously published procedure [21]. The amount of [ 3 H]daunomycin bound at each concen- tration of heterologous drug or nucleotide was expressed as a fraction of that obtained with radiolabel alone. The fraction bound was plotted as a function of added drug concentration, and nonlinear regression of the general dose–response relation (Eqn 1) was used to ascertain the potency (IC 50 ) and degree of displacement (F D ). Binding of [a 32 P]azido-ATP to purified ABCG2 R482G ABCG2 R482G was purified using immobilized metal affinity, anion exchange and gel filtration chromatography; full and extensive details have been described previously [22]. Binding of nucleotide to ABCG2 R482G was determined using photo- affinity labelling with [a 32 P]azido-ATP. Purified protein (0.25 lg) was incubated with [a 32 P]azido-ATP (3–300 lm)in the dark for 20 min in ATPase buffer (150 mm NH 4 Cl, 50 mm Tris, pH 7.4, 5 mm MgSO 4 , 0.02% NaN 3 )at4°C. At this temperature, ABCG2 does not generate measurable ATP hydrolysis. Samples were then irradiated with UV light (k = 265 nm, 100 W, 5 cm) for 8 min, and the samples were resolved by electrophoresis using 10% polyacrylamide gels. The gels were dried, and photolabelled protein was detected by autoradiography. Where displacement of nucleotide bind- ing was examined, the [a 32 P]azido-ATP concentration was fixed at 30 lm. Relative labelling intensities were determined using densitometric analysis of autoradiograms. Statistical analyses Heterologous displacement assays were analysed using the dose–response relationship shown below: B ¼ B min þ ðB max À B min Þ 1 þ 10 ½ðlogIC 50 ÀLÞ n  ð1Þ where B is the maximal [ 3 H]daunomycin binding, B max is the maximal binding, B min is the minimum binding, IC 50 is the concentration of drug that leads to half-maximal bind- ing of radiolabel (nm), n is the Hill slope factor and L is log 10 [ligand concentration (m)]. 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Is ATP binding responsible for initiating drug translocation by the multidrug transporter ABCG2? Christopher A. McDevitt 1 , Emily. that the binding of ATP per se is the ‘power stroke’ for drug translocation, and that energy obtained from the hydrolysis process is used to reset the transporter.