INTRODUCTION

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The membrane components of the maltose transport system of Escherichia coli catalyze the intracellular accumulation of malto-oligosaccharides at the expense of ATP hydrolysis (5). They comprise two integral membrane proteins, MalF and MalG, and two copies of a cytoplasmic ATP binding subunit, MalK (36). In addition to these components directly involved in substrate translocation, there is a periplasmic maltose binding protein (MBP) and a maltoporin (LamB) located in the outer membrane (5). This transporter is a member of the ATP binding cassette (ABC) superfamily, and the MalK sequence shares significant similarity with the sequences of several other prokaryotic and eukaryotic transport proteins that belong to this family.

The FGK2 complex has been extensively characterized both genetically and biochemically. In most instances, the transporter has been studied either in whole cells, in subcellular vesicles, or in some cases following purification and reconstitution, in proteoliposomes. In all cases, the transport and ATPase activities of the FGK2 complex have been shown to be strongly dependent on MBP. Efforts to study the molecular basis of these transport and ATPase activities have been limited by the need to reconstitute the transporter in proteoliposomes. It has been generally assumed that a detergent-soluble form of this and other ABC transporters would not be a useful model for studying their molecular properties. Indeed, several publications have reported that detergent solubilization rendered both the maltose and histidine transporters inactive (7, 21). An alternative approach has been to study the water-soluble ABC subunit (e.g., MalK or HisP) in isolation (28, 39). Although these studies have provided information about the responses of the proteins to nucleotides, they cannot address the effects of signaling by their respective periplasmic binding proteins.

In the work described here, we found evidence that, in contrast to these previous studies, the detergent-soluble form of the wild-type FGK2 transporter is active and retains important characteristics, such as the ability to respond to signaling by the periplasmic MBP. In addition, we provide a quantitative comparison of the activities of (i) the reconstituted membrane-bound form of the purified wild-type complex, (ii) the detergent-soluble form of the wild-type and uncoupled mutant complexes, and (iii) the isolated MalK subunit. We also report that this bacterial periplasmic binding protein-dependent ABC transporter responds to verapamil, a drug that is known to reverse the MDR phenotype by uncoupling ATP hydrolysis from drug efflux (15). These results indicate that the structural features required for verapamil binding and activity are broadly conserved among prokaryotic and eukaryotic ABC transporters with diverse functions.

	MATERIALS AND METHODS

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Strains and plasmids. All of the strains and plasmids used in this study are summarized in Table 1. Strain NT169 was used as a host for expression of the maltose complex, HS3309 was used as a host for expression of wild-type MBP and signaling-defective mutant MBP, and X90(DE3) (38) was used as a host for expression of the His₆-MalK protein.

Construction of plasmids pMR41 and pMR31. Plasmid pMR41 (ampicillin resistance [Amp^r]) contains both the gst-malG fusion and the malK genes individually under control of the Ptac promoter. To create the gst-malG fusion, a BamHI restriction site was introduced immediately upstream from the ATG of the malG coding sequence in plasmid pYSF12. From this plasmid, a BamHI-BsaAI fragment containing the entire malG coding sequence was ligated into plasmid pGEX-2T (Pharmacia Biotech), which had been digested with BamHI and SmaI, to create plasmid pCP13. A BamHI fragment derived from plasmid pMR11 (32) containing the wild-type malK gene under control of the Ptac promoter was introduced into pCP13 by first filling in the BamHI sticky ends and then ligating the blunt-end fragment into the EcoRV site within the lacI^q gene of plasmid pCP13.

Overproduction of MalK, MalF, and GST-MalG. NT169 cells were transformed with plasmids carrying the malF gene (pMR31) and the malK and gst-malG genes (pMR41). All genes were under control of the Ptac promoter. The bacteria were grown at 28°C to logarithmic phase in LB medium (10 g of tryptone per liter, 5 g of yeast extract per liter, 10 g of NaCl per liter) containing 100 µg of ampicillin per milliliter and 25 µg of chloramphenicol per ml. After induction with 1 mM isopropyl--D-thiogalactopyranoside (IPTG) for 4 h, the cells were pelleted and membranes were prepared as described elsewhere (9). Briefly, the cell pellet was resuspended in 20 mM Tris-HCl (pH 7)-5 mM MgCl₂-1 mM EDTA-50 mM NaCl-10 µg of phenylmethylsulfonyl fluoride per ml-1 mM dithiothreitol (DTT). The cells were passed twice through a French press at 16,000 lb/in², and the unbroken cells were removed by centrifugation at 3,000 × g for 10 min. The supernatant fraction was centrifuged at 100,000 × g for 1 h at 4°C. The pellet was resuspended in the same buffer to a protein concentration of 20 mg/ml and stored at 80°C.

Solubilization of membrane protein. Thawed membranes were solubilized in 20 mM Tris-HCl (pH 7)-5 mM MgCl₂-10 µg of phenylmethylsulfonyl fluoride per ml-1 mM DTT-20% glycerol-1.6% dodecyl maltoside to a final protein concentration of 4 mg/ml. After 30 min of incubation on ice, the solubilized membranes were centrifuged at 100,000 × g for 1 h at 4°C and the supernatant was removed for purification of the maltose transporter on an affinity column.

Affinity purification of the maltose transporter complex. A 2-ml volume of glutathione agarose (Pharmacia Biotech) resin was added to 10 ml of solubilized membrane supernatant and kept for 4 h at 4°C. The resin was transferred to a column and washed with 50 mM Tris-HCl (pH 8)-5 mM MgCl₂-1 mM DTT-10% glycerol-0.01% dodecyl maltoside. To cleave the glutathione S-transferase (GST) from MalG, biotinylated thrombin (Novagen) was added to the resin, the mixture was subjected to 18 h of incubation at 4°C, and then the complex was eluted from the column. The eluted complex was incubated with 50 µl of a streptavidin agarose suspension for 30 min and then centrifuged for 5 min at 1,000 × g through a spin filter centrifuge tube to remove the thrombin-containing beads. If thrombin was not used to cleave the GST-MalG hybrid, the GST-MalG-containing complex was eluted with the same buffer containing 10 mM glutathione.

Assay of ATP hydrolysis. ATP hydrolysis was measured as described elsewhere (19). Briefly, the purified complex (5 µg/ml) was added to a solution of 40 mM Tris-HCl (pH 7)-4 mM MgCl₂-5% glycerol-0.01% dodecyl maltoside. ATP was added to a final concentration of 4 mM, and the reaction mixture was incubated in a 37°C bath. Aliquots of 20 µl were removed after 0 to 30 min into 160 µl of 0.033% malachite green in 1 N HCl. After 1 min, the reaction was stopped by addition of 20 µl of 34% citric acid and the A₆₅₀ was read. When doing the assay in the presence of large amounts of protein, we used a variation of the method as described before (6). We established that the assay was linear over time and with respect to the concentration of the added complex up to 25 µg/ml. When added to ATPase assays, MBP was always saturated with maltose unless otherwise specified.

Overproduction and purification of MBP. MBP was purified by affinity chromatography as described before (24). Briefly, fresh transformants of HS3309 were grown for 18 h in Terrific Broth medium (12 g of tryptone per liter, 24 g of yeast extract per liter, 0.4% glycerol, 2.31 g of KH₂PO₄, 12.54 g of K₂HPO₄ per liter), and the periplasmic fraction was loaded onto an amylose column. After washing with 50 mM Tris-HCl (pH 7.5), the purified MBP was eluted with the same buffer containing 20 mM maltose. A Centricon 30 concentrator (Amicon) was used to concentrate the purified MBP and to wash out the excess maltose.

Expression and purification of His₆-MalK protein. Expression and purification of His₆-MalK was done as described before (30). Briefly, the his₆-malK gene (pCP97) was transformed into strain X90/(DE3), which was grown to logarithmic phase in Terrific Broth medium and induced for 1 h with 1 mM IPTG. The cells were pelleted, resuspended in 10 mM Tris-HCl (pH 8)-200 mM NaCl-10% glycerol and passed twice through a French press at 16,000 lb/in². The soluble fraction was recovered after a 30-min spin at 15,000 rpm in the ss-34 rotor of a Sorvall Re-5B centrifuge, added to Ni-nitrilotriacetic acid-agarose (QIAGEN), and incubated for 1 h at 4°C. The resin was transferred to a column and washed with 10 mM Tris-HCl (pH 8)-500 mM NaCl-20% glycerol. The His₆-MalK protein was eluted in the same buffer containing 250 mM imidazole.

Reconstitution of the purified complex into proteoliposomes. The dilution procedure described by Racker et al. (31) was used to reconstitute the purified maltose transporter into proteoliposomes. Dry, crude E. coli phospholipids (Avanti Polar Lipid, Inc.) were resuspended to a final concentration of 50 mg/ml in 50 mM Tris-HCl (pH 8)-2 mM 2-mercaptoethanol and stored at 80°C under nitrogen until use. Aliquots were thawed and sonicated to clarity in a bath sonicator.

	RESULTS

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Overproduction and purification of FGK2 complex. We used two compatible plasmids to express the transport complex. One plasmid, pMR31, encodes resistance to chloramphenicol and the MalF protein under control of the Ptac promoter on a P15A replicon. The other plasmid, pMR41, encodes ampicillin resistance, as well as the MalK protein and a hybrid GST-MalG protein, both under control of separate Ptac promoters. The GST-MalG hybrid protein retains the ability to participate in active transport. The fused gst-malG gene complements a malG null mutation (data not shown). In addition, the reconstituted complex containing the GST-MalG protein exhibited both transport and ATPase activities (C. Panagiotidis, unpublished results). These plasmids were introduced into strain NT169, which lacks the entire malB region and supplies the LacI repressor. The transformants were grown to logarithmic phase, and synthesis of the transport complex was induced by IPTG. Following harvesting of the cells and lysis in a French press, a crude membrane fraction was prepared and stored at 80°C. Aliquots of the membrane preparation were thawed and solubilized in buffer containing dodecyl maltoside. The detergent-soluble proteins were recovered following centrifugation and applied to a glutathione-agarose column. After extensive washing of the column, the transport complex was eluted either with glutathione or by incubation with the protease thrombin, which cleaves at a recognition site between the GST and MalG sequences. We routinely observed a small amount of MalK protein in the flowthrough of the glutathione agarose column. This is likely due to the presence of unassociated MalK subunits in the extract. The unassociated MalK may be the result of excess production of MalK subunits relative to MalF and GST-MalG subunits. Although we did not see any functional differences between the complex containing the GST-fused MalG protein and the thrombin-cleaved complex (data not shown), all of the experiments described in this report, unless stated otherwise, were done with the thrombin-cleaved FGK2 complex.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection of the purified FGK2 complex. The FGK2 complex was purified as described in Materials and Methods. The purified complex was then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane for immunoblotting with specific antibodies (Ab). (A) Coomassie staining of the gel. Lanes: 1, proteins eluted by glutathione; 2, proteins eluted by thrombin cleavage. (B) Immunoblotting of the purified complex with specific antibodies to GST-MalG, MalF, and MalK individually. Lanes: 1, proteins bound to the glutathione agarose resin; 2, proteins eluted by thrombin cleavage; 3, proteins retained on the column following thrombin cleavage. The values on the left are molecular weights (M.W.) in thousands.

ATP hydrolysis by the soluble FGK2 complex. Although the ATPase activity of the wild-type FGK2 complex has been studied in detail, there is little information about its ability to hydrolyze ATP in detergent solution. We found that the material eluted from the glutathione agarose column exhibited very high rates of ATP hydrolysis in the absence of MBP. In order to characterize this activity, we measured the rates of ATP hydrolysis at different ATP concentrations (Fig. 2). We found that the results followed a simple Michaelis-Menten type of kinetics. Double-reciprocal transformation of the data yielded values for K_m of 194 ± 10 µM and V_max of 1,411 ± 49 nmol/min/mg. These values are similar to those reported by others for the membrane-bound transporter when activated by liganded MBP (11). We did not observe the cooperativity with respect to ATP concentrations reported by Davidson et al. (7), presumably because, as they reported, there is no cooperativity at neutral pH. The ability of the purified maltose transporter to hydrolyze ATP in the absence of MBP was also observed when, instead of dodecyl maltoside, octyl glucoside was used to solubilize and purify the complex (data not shown).

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Saturation curve of ATP hydrolysis by the detergent-soluble complex. The purified FGK2 complex (final concentration, 5 µg/ml) was incubated in 40 mM Tris-HCl (pH 7)-4 mM MgCl2-5% glycerol-0.01% dodecyl maltoside and various concentrations of ATP. For each reaction, samples were removed at various times and the amount of inorganic phosphate was determined as described in Materials and Methods. The rates of inorganic phosphate production were linear over the course of the reaction. The apparent Km is 194 ± 10 µM, and the Vmax is 1,411 ± 49 nmol/min/mg. The inset is a plot of the first 10 data points.

View larger version (24K): [in this window] [in a new window]   FIG. 3.   The GST-fused FGK2 complex does not dissociate in detergent. A sample of the purified complex (50 µg), eluted by glutathione from the glutathione agarose column, was dialyzed, rebound to the column, and re-eluted. The proteins from both elutions were assayed for ATPase activity. (A) Sodium dodecyl sulfate-polyacrylamide gel of the first and second elutions. Lanes: 1, first elution; 2, second elution. (B) ATP hydrolysis rates of the complex.

MBP increases the activity of the purified maltose transporter. In whole cells, as well as in the membrane, the ATPase and transport activities of the maltose transporter are increased in the presence of MBP (11, 35). We have shown, however, that the detergent-purified complex can hydrolyze ATP in the absence of MBP. Nevertheless, the detergent-soluble form of the complex retains the ability to interact with MBP. When MBP containing maltose is added to the complex, there is an increase of about threefold in the rate of ATP hydrolysis (Fig. 4). This increase is MBP specific, since addition of a signaling-defective mutant MBP (17) resulted in a much smaller ATPase activity increase. In addition, maltose in the absence of MBP did not affect the rate of ATP hydrolysis (data not shown). The dependence of the ATPase activity on the concentration of MBP is similar to that reported both in vivo (22) and in membrane vesicles (23).

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Stimulation of the ATPase activity of the purified detergent-soluble complex by the wild type but not by a signaling-defective mutant MBP. The purified FGK2 complex was incubated in the absence and in the presence of different concentrations of maltose-saturated wild-type MBP or signaling-defective mutant MBP. The ATP hydrolysis rates of the mixtures were determined as described in legend to Fig. 2 in the presence of 4 mM ATP. Symbols: , wild-type MBP; , signaling-defective mutant MBP.

View larger version (15K): [in this window] [in a new window]   FIG. 5.   MBP increases the Vmax of the ATPase activity but does not change the apparent Km for ATP. The rate of ATP hydrolysis by the soluble FGK2 complex was measured at different ATP concentrations (as described in the legend to Fig. 2) in the absence and in the presence of 50 µM maltose-saturated wild-type MBP. Symbols: , soluble FGK2 complex; , soluble FGK2 complex in the presence of 50 µM MBP. In the absence of MBP, the apparent Km is 154 ± 22 µM and the Vmax is 1,733 ± 148 nmol/min/mg, whereas in the presence of 50 µM MBP, the apparent Km is 143 ± 24 µM and the Vmax is 4,697 ± 1,186 nmol/min/mg.

The ATPase activity of the reconstituted FGK2 complex is MBP dependent. Several studies have shown that when the wild-type FGK2 complex is reconstituted from detergent solution, its ATPase activity is dependent on the presence of liganded MBP (7, 8). In order to test the hypothesis that either the presence of the GST moiety fused to MalG or some other aspect of the protocol that we used to prepare the detergent-soluble complex resulted in a permanent change in the properties of the FGK2 complex, we studied the ATPase activity of the soluble intact complex following reconstitution in proteoliposomes. We reconstituted the detergent-soluble purified FGK2 complexes with E. coli phospholipids into proteoliposomes. We compared the ATPase activities of complexes reconstituted with and without liganded MBP. If the GST moiety or other effects of the detergent irreversibly changed the dependence of the complex on MBP, we would expect that even following reconstitution into proteoliposomes, the FGK2 complex would exhibit high rates of ATP hydrolysis with only two- to threefold stimulation by liganded MBP. As shown in Fig. 6, the activity of the FGK2 complexes reconstituted with liganded MBP is more than 12 times as high as the activity of the complexes reconstituted in the absence of liganded MBP (3,026 versus 229 nmol/min/mg). These results are indistinguishable from those reported by others for the MBP dependence of ATP hydrolysis by the FGK2 complex (11). These results also indicate that the changes produced by dodecyl maltoside that result in activation of the ATPase are reversible upon reconstitution into proteoliposomes.

View larger version (18K): [in this window] [in a new window]   FIG. 6.   The ATPase activity of the reconstituted FGK2 complex is MBP dependent. The purified FGK2 complex was reconstituted into proteoliposomes (as described in Materials and Methods) in the absence and in the presence of wild-type MBP. The reconstituted complex (final protein concentration, 3 µg/ml) was incubated in 40 mM Tris-HCl (pH 7)-4 mM MgCl2-5% glycerol-4 mM ATP. For each reaction, samples were removed at various times and the amount of inorganic phosphate was determined as described in Materials and Methods. Symbols: , reconstituted FGK2 purified complex; , reconstituted FGK2 purified complex with wild-type MBP.

Purified MalK protein is less active than the intact complex and is not activated by MBP. It was shown that the MalK subunit isolated from either Salmonella typhimurium (39) or E. coli (27) displays ATPase activity. Although we presented evidence that the ability of the soluble FGK2 complex to hydrolyze ATP is not due to free MalK subunits that had dissociated from the MalF and MalG subunits, we considered the possibility that detergent or MBP is able to stimulate the ATPase activity of the isolated MalK subunit. Finding that detergent stimulates the free MalK subunit would mean that the effect of the detergent is attributable to this subunit of the complex, and finding that MBP is able to stimulate the ATPase activity of the isolated MalK subunit would support the hypothesis that the ABC subunits are exposed to the periplasm and directly interact with the ligand-binding proteins (2). In contrast, finding that the activity of the isolated MalK subunit is not stimulated by the MBP would support the idea that the binding protein transmits a stimulatory signal via the integral membrane proteins that, in turn, activates ATP hydrolysis by the MalK subunits that form part of the complex.

View larger version (14K): [in this window] [in a new window]   FIG. 7.   Purified His6-MalK is not stimulated by MBP. The purified FGK2 complex and the purified His6-MalK protein were incubated in the absence and in the presence of different concentrations of maltose-saturated MBP. The ATP hydrolysis rates of the mixtures were determined as described in legend to Fig. 2. Symbols: , detergent-soluble FGK2 complex; , His6-MalK.

FGK2 complex is inhibited by Na-azide and PCMBS. Most ABC transporters, including the FGK2 complex, are sensitive to inhibition by SH-specific compounds, as well as azide, which is known to covalently modify nucleotide binding sites. In order to find out if the intact detergent-soluble complex resembles the membrane-bound complex with respect to sensitivity to these compounds, we examined the effects of a polar, membrane-impermeant mercurial, p-chloromercuribenzosulfonic acid (PCMBS), and azide on the ATPase activity of the intact complex. We focused on PCMBS because preliminary results indicate that the wild-type complex is insensitive to PCMBS in whole cells but is rapidly inactivated at low PCMBS concentrations in everted membrane vesicles (C. Panagiotidis and M. Reyes, unpublished data). When the detergent-soluble complex was treated with various concentrations of PCMBS, the ATPase activity was inhibited by >95% (data not shown). Half-maximal inhibition was observed at 25 to 50 µM. Sodium azide also inhibited the activity of the purified complex by >90%, with half-maximal inhibition at 10 µM. Vanadate, although known to inhibit the activity of several ABC transporters, including the membrane-bound FGK2 complex at acidic pH, had no effect on the ATPase activity of the detergent-soluble complex (data not shown). The inability of vanadate to inhibit the MBP-independent F500GK2 complex at neutral pH has been previously reported (7). Vanadate may only inhibit specific conformations of ABC transporters achieved when ADP is bound at the nucleotide binding sites.

Verapamil increases the ATPase activity of the purified FGK2 complex and lowers its affinity for ATP. Verapamil is a known modulator of the activity of P-glycoprotein (15). In the presence of 10 to 50 µM verapamil, the ATPase activity of P-glycoprotein is increased by about 50 to 150% (3, 20, 29). Verapamil is a competitive inhibitor of P-glycoprotein (14). Recent work suggests that verapamil binds P-glycoprotein with different affinities, probably at two different binding sites (13, 33). Mutations that alter residues in the hydrophobic linker region change the sensitivity of the P-glycoprotein to verapamil and related compounds (3, 4). The same stimulation was observed with the bacterial protein LmrA from Lactococcus lactis, which shares a high degree of sequence similarity with P-glycoprotein (37). Because the bacterial importers including the FGK2 complex also have sequence similarity to the other members of the ABC transporter family, especially surrounding the ATP binding regions, including the hydrophobic linker region, we wanted to find out if verapamil would have a similar effect on the ATPase activity of the FGK2 complex. When we assayed ATP hydrolysis by the soluble intact complex in the presence of increasing concentrations of verapamil, we observed a 70 to 100% increase in the ATPase activity of the complex at 1 to 2.5 mM (Fig. 8), concentrations that are approximately 100-fold higher than that required to stimulate the activity of P-glycoprotein.

View larger version (17K): [in this window] [in a new window]   FIG. 8.   Concentration-dependent stimulation of detergent-soluble FGK2 complex ATPase activity by verapamil. The purified FGK2 complex was assayed for ATP hydrolysis activity (as described in the legend to Fig. 2) with 4 mM ATP in the absence and in the presence of increasing concentrations of verapamil.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

ABC transporters comprise the largest single group of paralogous proteins. Although members of this family perform a variety of transport functions in diverse cell types, the transporters must share common mechanisms determined by the signature ABC sequence and similarity in overall structural organization (16). Efforts to understand the molecular basis of transport and the defects in medically important family members, such as the P-glycoprotein responsible for multiple drug resistance in tumors and the CFTR protein, which malfunctions in individuals with cystic fibrosis, are complicated by the complex nature of the cells and tissues in which they function. In contrast, bacterial ABC transporters offer the opportunity to study the mechanism of transport in simple unicellular organism that are amenable to both biochemical and genetic analyses. Characterization of the maltose and histidine transporters has taken advantage of the ability to both purify the transporters and study them genetically but is limited by the necessity to study their activities following reconstitution in proteoliposomes. This has been based on the observations that the detergent-soluble forms of the transporters are inactive.

Both the wild-type maltose and histidine transporters have been reported to be inactive in detergent solution. Davidson et al. studied a mutant form of FGK2, F500GK2, that, in contrast to the wild-type complex, hydrolyzes ATP in the absence of MBP. They reported that the mutant complex was able to hydrolyze ATP in detergent solution and concluded that the mutations responsible for the uncoupled phenotype were also responsible for the ability to retain activity in detergent solution (7). Our results are different and clearly show that both the wild-type and F500GK2 complexes are active in detergent solution. Indeed, the complexes differ in their responses to MBP signaling, with the wild type stimulated and the mutant inhibited by MBP. These results are consistent with studies that reported the activity of several eukaryotic ABC transporters in detergent solution (1, 12, 25, 34).

In Table 3, we provide a summary of the ATPase activities of the different forms of the FGK2 transporter, including a comparison with the purified isolated MalK subunit. In agreement with the results of others (27, 39), we found that the isolated MalK subunit exhibited low activity that was not affected by MBP. In addition, the membrane-bound form of the wild-type transporter showed the same low level of activity that was stimulated 15-fold by MBP. Detergent solution of the wild-type transporter resulted in an eightfold increase in specific activity, that could be further increased by MBP. In contrast, as reported for membrane preparations (11), the F500GK2 complex was more active than the wild type in detergent solution but was inhibited by MBP. These results indicate that the purified soluble FGK2 complex is an accurate model for studying signal transmission from MBP to the MalK subunit via the MalFG subunits.

In addition to these results, we found that verapamil, a compound that reverses the MDR phenotype and activates the ATPase activity of the P-glycoprotein (15), also activates the ATPase activity of the intact complex. We also found that, as was reported for P-glycoprotein (34), verapamil lowers the affinity of the complex for ATP. There is some information about the sites in the P-glycoprotein that may be important for binding of and/or stimulation by verapamil. Alterations of sequences N terminal to the Walker B motif appear to alter the ability of verapamil to activate the P-glycoprotein ATPase (3, 4). It should also be possible to study the effects of malK mutations on the ability of verapamil to stimulate FGK2 complex ATPase activity.

With the recent appearance of the three-dimensional structure of a related ABC transporter subunit, HisP (18), it is possible to speculate about how the membrane components and ligands control the ability of MalK to hydrolyze ATP. Although the free MalK subunit exhibits ATPase activity, it is not stimulated by MBP. There must be an intimate association between the MalK subunits and the MalF and MalG subunits that can serve to activate ATP hydrolysis (26). Data presented here suggest that the activation by MBP is due not to an increase in the affinity of the MalK subunit for ATP but to a change that increases the rate of hydrolysis. Although we do not understand the molecular basis of the activation of ATP hydrolysis by detergent, we speculate that the interaction of the detergent molecules with the membrane-spanning segments of the MalF and MalG proteins somehow promotes conformational changes that are transmitted to the MalK subunit. The ability to study these phenomena in solution with a combination of genetic and biophysical methods may provide more direct information about these proposed changes.