©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
EmrE, an Escherichia coli 12-kDa Multidrug Transporter, Exchanges Toxic Cations and H and Is Soluble in Organic Solvents (*)

(Received for publication, December 9, 1994)

Hagit Yerushalmi Mario Lebendiker Shimon Schuldiner (§)

From the Division of Microbial and Molecular Ecology, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The smallest membrane protein shown to catalyze ion-coupled transport is documented in this report. A gene coding for a small 110-amino acid membrane protein (emrE or mvrC) has been previously identified and cloned and shown to render Escherichia coli cells resistant to methyl viologen and to ethidium. In this report, it is shown that the resistance is due to extrusion of the toxic compounds in a process that requires a proton electrochemical gradient rather than ATP. For this purpose, cells in which the unc gene was inactivated were used so that the interconversion between the proton gradient and ATP is not possible, and the effect of agents, which specifically affect either of them, was tested on transport of ethidium in the intact cell. In addition, EmrE has been overexpressed and metabolically labeled with [S]methionine. Strikingly, the protein can be quantitatively extracted with a mixture of organic solvents such as chloroform:methanol and is practically pure after this extraction. Moreover, after addition of E. coli lipids to the chloroform:methanol extract, EmrE has been reconstituted in proteoliposomes loaded with ammonium chloride. Upon dilution of the proteoliposomes in ammonium-free medium, a pH gradient was formed that drove transport of ethidium and methyl viologen into the proteoliposome. Both substrates compete with each other and exchange with previously transported solute. EmrE is a multidrug transporter of a novel type, and, because of its size and its solubility properties, it provides a unique model to study structure-function aspects of transport reactions in ion-coupled processes.


INTRODUCTION

Toxic compounds are part of the natural environment in which living cells dwell, and development of strategies for life in this environment is a necessary trait for survival. Resistance to a wide range of cytotoxic compounds is a common phenomenon observed in many organisms throughout the evolutionary scale. Removal of toxic substances by transport is one of the strategies that have evolved. Multidrug transporters can actively remove a wide variety of substrates in an energy-dependent process and decrease the concentration of the offending compounds near their target. The proteins responsible for performing this task have been found in many organisms from bacteria to man. From analysis of their structure and properties, three families can be distinguished. One of them (ABC type) is best known for the P-glycoprotein in its various forms, which confers multidrug resistance to cancerous cells (Doige and Ames, 1993; Endicott and Ling, 1989; Gottesman and Pastan, 1993; Gros et al., 1986; Higgins, 1992). This family includes also many bacterial transport proteins. The ABC transporters utilize ATP to actively transport a wide variety of compounds. In some of the bacterial transporters, several subunits are required for activity, but in the P-glycoprotein and many others, one large polypeptide seems to be sufficient to perform all of the functions. The other family (TEXANs) includes proteins that render fungi and bacteria resistant to antibiotic treatment (Lewis, 1994; Neyfakh et al., 1991; Nikaido, 1994; Paulsen and Skurray, 1993). The TEXANs are antiporters, 400-500 amino acids long, which exchange various drugs with one or more protons and utilize in this way the proton electrochemical gradient to actively transport a variety of substrates. Proteins in mammals, located in intracellular organelles, which usually transport neurotransmitters such as dopamine, adrenaline, serotonin, and acetylcholine, also behave as multidrug transporters and belong to the TEXAN family (Schuldiner, 1994; Schuldiner et al., 1995).

Very little attention has been paid to bacterial proteins of the third family (MiniTEXANs) (Grinius et al., 1992). They are very small, about 100 amino acids long, and, by analogy to the other known systems, it has been hypothesized that they confer resistance to several antibiotics presumably also by active extrusion. Being the smallest putative ion-coupled transporters known, they roused our attention since they could provide a simple model to study.

A variety of genes coding for MiniTEXANs has been identified in several bacteria based on their ability to confer resistance to several drugs (Grinius et al., 1992). One of these genes, emrE or mvrC, is an Escherichia coli gene, which has been identified and cloned on the basis of its ability to confer resistance to ethidium (Purewal, 1991) and to methyl viologen (paraquat) (Morimyo et al., 1992). Previous studies have suggested the existence of an efflux system for toxic cationic compounds in E. coli (Midgley, 1987), and, because of the hydrophobic nature of the predicted polypeptide, it was suggested that EmrE is indeed an efflux system (Purewal, 1991). However, no direct evidence for this suggestion has yet been presented, and the nature of the energy required for extrusion has not been studied.

The predicted sequence of EmrE suggests that it is a highly hydrophobic 12-kDa protein (Fig. 1). A hydropathic analysis of the sequence reveals the presence of four putative transmembrane segments with only one charged residue (Glu) in the putative transmembrane domain and a total of eight charged amino acids throughout the protein. A protein similar in size and number of charges, which has been studied in detail by many groups, is subunit c, a small membrane-bound subunit of the H-ATPase (Fillingame, 1992). Subunit c is also known as the proteolipid because of its solubility in organic solvents. Since subunit c is purified by extraction in a mixture of chloroform and methanol, it was tempting to test a similar extraction procedure for EmrE.


Figure 1: Model of the secondary structure of EmrE. The model shown is based on the predictions of the hydropathic profile as calculated according to Engelman et al.(1986). Putative transmembrane segments are shown in boxes connected by hydrophilic segments. The charged residues are shaded.



In this report, we describe studies in whole cells that provide evidence that EmrE is an antiporter that exchanges ethidium, methyl viologen, and a series of other quaternary compounds with one or more protons. In addition, EmrE displays unique properties that make it soluble in a mixture of organic solvents such as chloroform:methanol. EmrE was overproduced so that it was practically the only protein extractable in the above solvent mixture. The protein thus purified was reconstituted in proteoliposomes, and it catalyzed DeltapH-driven uptake of ethidium and [^14C]methyl viologen. Uptake of both substrates was inhibited by a series of toxic cations, and preaccumulated solute exchanged with unlabeled excess substrate.


EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids

E. coli JM109 (Yanish-Perron et al., 1985), TA15 (Goldberg et al., 1987), and TA15Deltaunc carrying the deletion unc702 (Harel-Bronstein et al., 1995) were used throughout this work.

emrE was cloned by polymerase chain reaction using primers based on the published sequence of the gene (Morimyo et al., 1992; Purewal, 1991). The sense primer (CGGAATTCATATGAACCCTTATATTTATCTTG) overlapped with the first ATG in the coding area of the gene and included two restriction sites for cloning (NdeI and EcoRI). The reverse primer (CCGAATTCAAGCTTAATGTGGTGTGCTTCGTGAC) overlapped with the first termination codon after the open reading frame and included sites for the restriction enzymes HindIII and EcoRI. The polymerase chain reaction was performed using genomic DNA from E. coli EP432 (Pinner et al., 1992), a K12 derivative, as a template. The reaction yielded a 333-base pair segment that was purified, digested with the proper enzymes, and cloned into both pT7-7 (Tabor and Richardson, 1985) predigested with NdeI and HindIII and into pKK223-3 (Pharmacia Biotech Inc.) predigested with EcoRI and HindIII. The plasmids obtained were named pT7-32 and pKK56, respectively.

Overexpression and Specific Labeling of EmrE

pT7-32, which contains the T7 polymerase promoter 10 and the translation start site for the T7 gene 10 protein, was used for labeling EmrE with [S]methionine essentially as previously described (Pinner et al., 1992). pT7-32 was transformed into TA15 carrying pGP1-2 (Tabor and Richardson, 1985). Transformants were grown at 30 °C in minimal medium supplemented with thiamine (2.5 µg/ml), ampicillin and kanamycin (50 µg/ml), and 0.5% glucose to a cell density of 0.6 A. The temperature was then increased to 42 °C to induce the T7 polymerase; 15 min later, rifampicin (200 µg/ml) was added, and incubation continued for an additional 10 min. Then, the culture was shifted back to 30 °C for 40 min. [S]Methionine (specific activity, 1350 Ci/mmol) was added to the cell suspension (10 µCi/ml), and incubation continued for an additional 40 min. Cells were collected by centrifugation and washed with a solution containing 20 mM Tris-Cl, pH 7.5, and 150 mM NaCl and sonicated 3 times for 10 s using a probe-type sonicator. Undisrupted cells were removed by centrifugation, and the membranes were then collected by further centrifugation at 213,500 times g for 20 min. The membrane pellet was resuspended in the above buffer, frozen in liquid air, and stored at -70 °C.

For overexpression, E. coli JM109/pKK56 was grown in minimal medium A with 0.5% glycerol, thiamine, and ampicillin as above. When the culture reached an A = 0.5, isopropyl-1-thio-beta-D-galactopyranoside was added to 0.5 mM; 2 h later, the cells were chilled on ice and harvested by centrifugation. Membranes were prepared by disrupting the cells using a French pressure procedure (Rosen, 1986), except that the buffer used was 10 mM Tris-Cl, pH 7.5, 250 mM sucrose, 150 mM choline chloride, 0.5 mM dithiothreitol, 2.5 mM MgSO(4), and 15 µg/ml DNaseI. After ultracentrifugation, membranes were resuspended at 10 mg of protein/ml, frozen in liquid air, and stored at -70 °C.

Resistance to Toxic Compounds

For testing resistance to toxic compounds, cells were grown on solid media supplemented with different concentrations of the compound or alternatively in media in which filter paper disks with the corresponding compound were placed on the disk. Zones of inhibition were measured after 24 h of incubation at 37 °C.

Extraction of EmrE and Reconstitution

To follow EmrE, membranes containing [S]methionine-labeled protein and overexpressed protein were routinely mixed to yield approximately 1200 cpm/µg of membrane protein. For extraction, membranes in a volume of 10 µl (100 µg of membrane protein) were mixed with 150 µl of a mixture of chloroform:methanol (1:1) and incubated for 20 min on ice. For phase separation, 30 µl of water were added, and the suspension was centrifuged. The upper phase was removed, and the lower phase was dried under Argon and resuspended appropriately. For analysis in SDS-PAGE, (^1)the sample was resuspended in sample buffer. SDS-PAGE analysis was in 16% gels as described (Schagger and Jagow, 1987).

For reconstitution, the volumes were increased (600 µl of membranes and 5 mg of protein/ml were extracted with 4.5 ml of organic solvent), and E. coli phospholipids (160 µl of 50 mg/ml) were added prior to extraction. The dried extract was resuspended in a solution containing 0.15 M NH(4)Cl, 0.015 M Tris-Cl, pH 7.5, 1 mM dithiothreitol, and 1 µg of plasmid DNA. The suspension was frozen in liquid air and kept at -70 °C. Before the assay, the proteoliposome suspension was thawed at 37 °C and sonicated in a bath-type sonicator for a few seconds until clear. In proteoliposomes prepared for assay of [^14C]methyl viologen, no DNA was added. Control experiments showed no effect of DNA on transport of the radioactive label.

Transport of [^14C]methyl viologen into proteoliposomes was assayed by dilution of 3 µl of the ammonium chloride containing proteoliposomes into 200 µl of an ammonium-free medium containing 42 µM [^14C]methyl viologen (77 nCi), 140 mM KCl, 10 mM tricine, and 5 mM MgCl(2) (pH 8). At given times, the reaction was stopped by dilution with 2 ml of the same ice-cold solution, filtered through Schleicher and Schull filters (0.2 µm), and washed with an additional 2-ml solution. The radioactivity on the filters was estimated by liquid scintillation.

Transport of ethidium into proteoliposomes was assayed by dilution of 10-15 µl of proteoliposomes to 2.5 ml of a medium as above except that ethidium (1 µg/ml) replaced the methyl viologen. Fluorescence was measured in a Perkin-Elmer fluorimeter (Luminescence Spectrometer LS-5) with exciting light at 545 nm and emission at 610 nm.

Transport of Ethidium in Whole Cells

Cells grown in LB medium to A of 0.4 were collected by centrifugation and resuspended to A of 0.1 in minimal medium A (Davies and Mingioli, 1950) supplemented with 0.05% NaCl, 1 mM MgSO(4), 0.1 mM CaCl(2), and 0.36% glucose. CCCP (40 µM) was added for 5 min at 25 °C. Ethidium was added to 1 µg/ml, and fluorescence was recorded as above. For efflux experiments, incubation with CCCP and ethidium was for 60 min at 37 °C; cells were collected by centrifugation and quickly resuspended in medium containing the same ethidium concentration with or without CCCP; the fluorescence was monitored as above. When unc cells were used, the resuspension medium (25 mM bis-Tris, pH 7.5, 0.1% NH(4)Cl, 0.05% NaCl, 1 mM MgSO(4), 0.1 mM CaCl(2), and 0.36% glucose) contained no phosphate to allow testing the effect of arsenate.

Protein determination was according to Bradford(1976).

Phospholipids were prepared from E. coli as described (Viitanen, et al., 1986).


RESULTS

Cells Carrying EmrE Are Resistant to Multiple Drugs

It is quite often found that resistance to toxic compounds conferred by transporters shows a very broad spectrum. This phenomenon has very far reaching clinical implications and deserves attention. EmrE (E. coli multidrug resistance E) was cloned independently by two groups: in one case as a gene that confers resistance to ethidium and named ethidium resistance protein and ethidium efflux protein (Purewal, 1991; Purewal et al., 1990) and in the other as conferring resistance to methyl viologen, a powerful superoxide radical propagator and named MvrC (Morimyo et al., 1992). A strain carrying a mutation (acrA) that results in increased susceptibility to a range of toxic compounds was used to estimate the ability of emrE to confer cross-resistance. Mutant cells carrying emrE were cross-resistant to several monovalent cations, including euflavine, proflavine, safranine O, quinaldine red, and pyronin Y (Purewal et al., 1990). In Table 1, wild type K12 cells carrying plasmids with (pT7-32) or without (pT7-7) emrE were compared. Cells expressing emrE display a higher resistance to ethidium, methyl viologen, erythromycin, sulfadiazine, and tetraphenyl phosphonium. EmrE-expressing cells show also a significant level of resistance to tetracycline, albeit lower than that conferred by the various Tet genes. Thus, while EmrE allows growth at 2 µg/ml tetracycline (Table 1), cells carrying pBR322 grow at concentrations at least 10 times higher.



Extrusion of Ethidium Requires Delta

By analogy to other systems, in previous studies it has been suggested that the resistance conferred by EmrE is due to a modification of the membrane permeability or to some type of pumping activity. We explored this assumption by studying the ability of EmrE to transport ethidium in whole cells. Lambert and Le Pecq(1984) developed an assay to study accessibility of nucleic acids to ethidium in E. coli cells. Addition of uncouplers was shown to induce a slow but very massive entry of ethidium, which was manifested by an increase in its fluorescence due to binding to nucleic acids. In Fig. 2A, results of such an experiment were reproduced, both in control cells and in cells expressing emrE. Addition of CCCP (40 µM) to energized cells in the presence of ethidium induced a large increase in the fluorescence of the latter. The levels of fluorescence at equilibrium (about 40-50 min, not shown) were practically identical. However, the rate of entry of ethidium after addition of CCCP was about twice higher in cells expressing emrE (t = 25 min in the control as compared with t = 12 min in the overexpressing cells), suggesting that under these conditions, EmrE is the main entry pathway upon deenergization of the cell. In addition, a dramatic difference was observed when the converse experiment was performed. Cells treated with CCCP were incubated for 60 min at 37 °C to allow for equilibration, collected by centrifugation and rapidly resuspended in a medium containing the same concentration of ethidium but devoid of uncoupler (Fig. 2B). Upon removal of the uncoupler in the overexpressing cells, a rapid decrease in the fluorescence is observed even before the tracing begins and is completed in less than 2 min. This decrease in the fluorescence, which represents removal of ethidium from the cell against a concentration gradient, was completely prevented when the cells were resuspended in a medium with CCCP. The rate of extrusion in the control cells is much slower and reaches the new equilibrium only after about 20 min. The results described demonstrate that EmrE overexpression was accompanied by an increased rate of ethidium efflux in an energy-dependent reaction.


Figure 2: Energy-dependent transport of ethidium in whole cells. E. coli JM109/pT7-7 and E. coli JM109/pT7-32 grown in LB medium to A = 0.4 were collected by centrifugation and resuspended in minimal salt medium as described under ``Experimental Procedures.'' A, cells treated with CCCP (40 µM) prior to the addition of ethidium (1 µg/ml). If CCCP was omitted, no time-dependent increase in fluorescence was observed. B, cells incubated for 60 min at 37 °C with CCCP and ethidium as in A were centrifuged and quickly resuspended in medium containing the same ethidium concentration with (+CCCP) or without CCCP. C and D, E. coli TA15 Deltaunc/pT7-32 were treated as in A except that the following additions were made: in C, no additions or 10 mM KCN (+KCN); in D, 40 µM CCCP (+CCCP) or 12.5 mM arsenate (+ARS) and then 40 µM CCCP.



It is difficult to identify the driving force for active transport in a strain possessing the H-ATPase since the main forms of cell energy (oxidation energy, Delta, and ATP) can be interconverted by this enzyme. To demonstrate the nature of the energy source required for ethidium extrusion, we used a strain carrying a deletion in the unc operon so that the F(1)-F(0) H-ATPase was inactive, and it was then possible to manipulate independently both Delta and ATP. In these experiments, the effect of various agents on ethidium entry into the cell was tested. As in Fig. 2A, ethidium entry is due to failure of the extrusion machinery to remove it from the cell. The results of such an experiment are summarized in Fig. 2, C and D. Addition of CCCP and cyanide, two agents that inhibit Delta but did not affect ATP levels in this strain under these conditions (data not shown), induced ethidium entry into the cell as judged from the increase in its fluorescence. On the other hand, arsenate, an agent that decreased dramatically the ATP level (data not shown) without any effect on Delta, had no effect on ethidium uptake either. The data presented strongly supported the contention that EmrE is an H-ethidium antiporter, which utilizes the Delta generated by the bacterial primary pumps to extrude toxic cations and reduce in this way their concentration in the cytoplasm.

Overexpression and Identification of EmrE: EmrE Is Soluble in Chloroform:Methanol

To identify EmrE, we used pT7-32, a derivative of pT7-7 in which emrE is expressed from a T7 polymerase promoter, so that we were able to specifically label its product with [S]methionine in the presence of rifampicin, an inhibitor of the host but not of the phage RNA polymerase, as described by Tabor and Richardson(1985). Thus, in membranes prepared from a strain carrying pT7-32 and resolved by SDS-PAGE, a radioactive polypeptide was detected, which displays an apparent molecular mass of 9 kDa (Fig. 3, laneG). No labeling was detected when membranes were prepared from cells carrying pT7-7 with no insert (data not shown). Upon staining, no obvious difference could be discerned in membranes prepared from either the pT7-32 cells or those carrying pT7-7 (Fig. 3, lanesA and C).


Figure 3: Labeling of EmrE with [S]methionine and solubilization with chloroform-methanol. Membranes (15-20 µg of protein) from E. coli JM109 cells carrying pT7-32 (A), pKK56 (B), pT7-7 (C), or pKK223-3 (D) were solubilized with sample buffer and analyzed by SDS-PAGE. In parallel, chloroform:methanol extracts from pT7-32 (E) and pKK56 (F) were analyzed. In lanesG and H, the material shown in lanesA and E was also analyzed for radioactivity by phosphor autoradiography (Fuji Film Co. Ltd).



As described above, there are some similarities in the overall properties of EmrE and the subunit c of the F(1)-F(0)-H-ATPase (Fillingame, 1992). Both are small polypeptides (11.9 and 8.2 kDa, respectively) with a relatively high percentage of hydrophobic amino acids typical of intrinsic membrane proteins and a small number of charges. Subunit c was also called the proteolipid because it was extracted with solvents used to extract membrane lipids and it was purified after extraction with these solvents. When membranes in which EmrE was labeled with [S]methionine were similarly extracted with a mixture of chloroform and methanol (1:1), more than 85% of the radioactivity was detected in the organic phase. When the organic phase was analyzed in SDS-PAGE and the radioactivity was assessed by imaging, again a single polypeptide was observed, identical to the one detected in the intact membrane (Fig. 3, laneH). As expected, most other proteins are not extracted by organic solvents as evident from the pattern of the polypeptide composition analyzed by SDS-PAGE and visualized by staining with Coomassie Blue (Fig. 3, laneE). Practically no other proteins are observed, while a faint band corresponding to EmrE can now be clearly detected. These results are even more dramatic when EmrE was overexpressed using another vector, pKK223-3, in which the gene was fused to the strong and highly regulated tac promoter (plasmid pKK56). When membranes prepared from cells induced by the addition of isopropyl-1-thio-beta-D-galactopyranoside were extracted as described above, a strong band corresponding to EmrE was observed (Fig. 3, laneF). This band was absent in extracts from membranes prepared from cells that were transformed with plasmid with no insert (not shown).

Purified EmrE Is Functional after Reconstitution into Liposomes

The finding that EmrE is soluble in chloroform:methanol provides a potentially very powerful technical tool for many purposes such as structural analysis. Much of this depends on whether the original conformation of the protein is maintained in the solvent. One of the ways to test whether the protein was grossly denatured in the treatment relies on assay of the activity after solubilization, i.e. reconstitution into proteoliposomes and test of transport activity. The approach we used was to add E. coli phospholipids to the organic solvent extract, evaporate the solvent, and resuspend the protein and the lipid in a buffer containing ammonium chloride. The suspension was then frozen and sonicated so that a population of relatively uniform size, single layer proteoliposomes was formed. The driving force imposed was a pH gradient generated by the ammonium diffusion gradient obtained upon dilution of the proteoliposomes prepared in 0.15 M NH(4)Cl into media in which the ammonium was replaced by KCl (Taglicht et al., 1991). EmrE-catalyzed transport in proteoliposomes was documented with two substrates, namely ethidium and [^14C]methyl viologen. To measure ethidium uptake, we took advantage of the effect of DNA on the quantum yield of its fluorescence. Proteoliposomes were prepared in the presence of DNA, and the fluorescence was monitored. Addition of ethidium to a cuvette containing proteoliposomes caused a large and biphasic increase in the fluorescence; a practically instantaneous increase was followed by a slower reaction (Fig. 4A). We assume that the fast increase is due to binding to the DNA present in the extraliposomal space. The slow phase was due to accumulation of ethidium in the inside of the liposome since addition of the ionophore nigericin, which collapses the transmembrane pH gradient, or methyl viologen, which competes with ethidium as shown below, completely inhibits the fluorescence increase corresponding to this phase (Fig. 4, B and C). In addition, when control liposomes (no protein) were added to the cuvette, only the rapid increase in fluorescence was observed, which was due to binding of ethidium to the DNA present in the extraliposomal space (Fig. 4D). Ethidium accumulated in the proteoliposomes was released by collapse of the pH gradient upon addition of nigericin (Fig. 4A). The results demonstrate that ethidium is transported in a process that is dependent on EmrE and driven by DeltapH but does not allow for a detailed analysis of transport.


Figure 4: Transport of ethidium into proteoliposomes reconstituted with EmrE. A, proteoliposomes (Lipo, 5 µg of protein) reconstituted with EmrE in ammonium chloride medium were diluted into an ammonium-free medium as described under ``Experimental Procedures.'' Ethidium (Eth) was added to 1 µg/ml. Nigericin (nig) was added to 4 µM to collapse the pH gradient. B, as in A except that the liposomes were added last. C, as in A except that methyl viologen (MV) was added to 1 mM prior to the liposomes. D, the liposomes used contained no EmrE.



A more quantitative documentation of EmrE-catalyzed transport was obtained by following accumulation of another substrate, [^14C]methyl viologen. In these experiments, the pH gradient is generated as above, and a time-dependent accumulation of [^14C]methyl viologen was observed in proteoliposomes reconstituted with EmrE (Fig. 5A, opensquares). Accumulation was linear during the first 5 min, reached a maximal level, and slowly leaked back to the medium (Fig. 5B, opensquares) as expected from the transient nature of the driving force. Transport was almost completely abolished when nigericin was added to the suspension so that no pH gradient was formed (Fig. 5A, filledsquares). No accumulation over the nigericin levels was detectable when liposomes reconstituted without EmrE were diluted to the reaction medium (Fig. 5A, filledcircles). The gradient of [^14C]methyl viologen that the proteoliposomes maintain was about 100 (in > out). Thus, about 10% of the total radioactivity accumulated in the proteoliposomes while, based on measurements in similar systems (Taglicht et al., 1993), their internal volume represented only 0.1% of the total. The accumulated substrate was free in solution rather than irreversibly bound to internal structures since it could be rapidly released after equilibrium was reached; at the plateau level, addition of nigericin (Fig. 5B, filledsquares) caused a rapid release of all of the preaccumulated substrate within less than a minute. Also, addition of an excess (1 mM) of unlabeled methyl viologen, which exchanged with the intraliposomal isotope (filledcircles), caused the release of all of the radioactivity within 5-10 min.


Figure 5: Transport of [^14C]methyl viologen into proteoliposomes reconstituted with EmrE. A, proteoliposomes (1 µg of protein) reconstituted with EmrE in ammonium chloride medium were diluted into an ammonium-free medium containing [^14C]methyl viologen as described under ``Experimental Procedures'' (box-box). In parallel series, nigericin was added to 15 µM (-), or liposomes with no EmrE were used in the assay (bullet-bullet). B, experiment was as in A except that after 7.5 min, 15 µM nigericin (-) or 1 mM methyl viologen (bullet-bullet) was added. To the control series, no additions were made (box-box). C, initial rates of uptake (1 min) were measured at the indicated concentrations of methyl viologen. The straightline was obtained by linear regression. Each experimental point is the average value of duplicates.



The kinetics of uptake were further analyzed by monitoring the initial rate of uptake at various concentrations of methyl viologen. The initial rate of uptake increased with the concentration of the substrate and then saturated (not shown). When the results of such an experiment were plotted according to Lineweaver-Burk (Fig. 5C), an apparent K(m) of 247 µM and a V(max) of 1572 nmol/min/mg protein were calculated from the linear regression of the plot.

To test the substrate specificity of EmrE, the ability of several drugs to inhibit the initial rate of [^14C]methyl viologen uptake was measured, and the results are shown in Fig. 6. Of the compounds tested, the most potent was the lipophilic cation TPP with an IC of 8 nM, while erythromycin had no effect up to 100 µM. The other inhibitors were, in order of potency, ethidium (2.5 µM) > acriflavine (8 µM) > tetracycline= reserpine (25 µM) = MPP (30 µM).


Figure 6: Transport of [^14C]methyl viologen is inhibited by a variety of drugs. Initial rates of uptake (1-3 min) were measured at the indicated concentrations of the following compounds: TPP (bullet-bullet), ethidium (circle-circle), acriflavine (-), reserpine (box-box), tetracycline (-), erythromycin (-), and MPP (-). Each experimental point is the average value of duplicates.




DISCUSSION

The results described in this report demonstrate that EmrE, a 110-amino acid polypeptide, is an antiporter that exchanges H with toxic cations such as ethidium and methyl viologen. The studies with whole cells confirmed that expression of EmrE confers resistance to a variety of toxic cations most likely due to the increased ability of the cell to remove them from the cell and therefore lower their concentration in the cytoplasm. This assumption was supported by the demonstration that active removal of ethidium is markedly faster in cells overexpressing EmrE. In addition, the use of a Deltaunc strain, which cannot interconvert phosphate bond energy with the electrochemical gradient of protons, allowed to establish conditions in which the source of energy available for transport could be controlled. The studies with this strain demonstrated that EmrE uses the electrochemical gradient of protons to extrude ethidium. We concluded at this stage that most likely the basis of the resistance to the toxicity of the other compounds tested is also due to their active extrusion by the same mechanism.

The studies in the intact cells were extended and confirmed by purification and reconstitution of EmrE. The reconstituted protein catalyzed the accumulation of ethidium and methyl viologen in a process dependent on DeltapH generated by an ammonium diffusion gradient. These studies directly demonstrated that EmrE functions as an antiporter that exchanges one or more H with toxic cations. Transport of methyl viologen and ethidium was demonstrated directly, while transport of the other cations was inferred from their ability to inhibit the accumulation of [^14C]methyl viologen. As predicted from its ability to confer resistance to a variety of drugs, EmrE also showed a wide range of specificity. Transport was inhibited very potently by TPP and also by ethidium, acriflavine, reserpine, tetracycline, and MPP. As for the first three drugs, EmrE was already shown to confer resistance to them (Midgley, 1987; Purewal et al., 1990). Tetracycline was tested because of the partial but significant resistance observed in this study (Table 1). The anti-hypertensive reserpine is a potent inhibitor (K(i) in the subnanomolar range) of vesicular monoamine transporters, a protein from the TEXAN family of multidrug transporters (Schuldiner, 1994; Schuldiner et al., 1995). It also inhibits BMR, the Bacillus subtilis multidrug transporter, the closest bacterial homolog of the vesicular monoamine transporters. BMR is able to efflux out of bacterial cells structurally diverse compounds such as ethidium, TPP, and acridine and is inhibited by reserpine at the low micromolar range (Ahmed et al., 1993; Neyfakh et al., 1991). Interestingly, sensitivity to reserpine is characteristic also of the mammalian multidrug efflux transporter, P-glycoprotein (Pearce et al., 1989). EmrE displays the lowest sensitivity to reserpine, with an IC of 25 µM. The neurotoxin MPP, an agent that causes Parkinsonism in model systems, is a phenyl-pyridinium with structural similarity to the dipyridinium methyl viologen and is also a substrate of the vesicular monoamine transporter (Liu et al., 1992). It inhibits EmrE with an IC of 30 µM. Although a more comprehensive survey is needed, it seems that there is a significant overlap in the specificity range of the various multidrug transporters known.

A unique feature of EmrE is its size; a 110-amino acid protein with four putative transmembrane segments catalyzes the same activities that have been previously observed in proteins three to five times larger. The fact that the small proteins of this family are transporters has been hypothesized before but without experimental support (Grinius et al., 1992). Also, several putative transporters with only one transmembrane segment have been cloned in recent years based on expression cloning in oocytes (Wells and Hediger, 1992). However, the overall size of these proteins is high, and the possibility that they may influence transport indirectly has not been ruled out. In this report we directly demonstrated that purified EmrE catalyzes the H-cation exchange activity, and no other polypeptides are required for this activity. The following question can then be raised: why are the other transporters so large when the minimal subunit necessary is at the most 110 amino acids? One possibility is that EmrE functions as a homooligomer to form at least the 12 transmembrane segments usually observed in other transporters. On the other hand, it is also possible that only some domains of the larger proteins are needed for transport, and the rest functions for regulation of the activity and/or interaction with other proteins.

Another very unique feature of EmrE, is its solubility in chloroform:methanol, which provides us with a powerful and unique tool for purification and characterization. Since only a handful of minor proteins is soluble in these organic solvents, after overproduction of EmrE it was possible to extract it highly purified. Most importantly, EmrE was active after this treatment, suggesting that the extraction did not induce major irreversible changes in its structure. The reconstituted protein has an apparent K(m) for methyl viologen of 247 µM and a V(max) of 1572 nmol/min/mg protein. The latter represents a turnover number of 14 min, which is in the same order of magnitude of many ion-coupled transporters (Taglicht et al., 1991). No quantitative data is available on the native protein, but the specificity range of the protein is practically identical to the range of resistance reported. The solubility of EmrE and subunit c in organic solvents does not reside in a specially high proportion of hydrophobic amino acids in both proteins; the percentage in both proteins is 49 and 44%, respectively, which is not different from other classical ion-coupled transporters, which is between 41 and 49.6% in seven H-coupled bacterial transporters analyzed based on the sequences available from the data bank. (^2)On the other hand, the percent of charged amino acids is 7.2 and 9.6%, respectively, while in the 12 transmembrane helix transporters scanned as above, it is between 11 and 14.8%. Another important difference is the number of net charges, which is only +1 in EmrE and -2 in subunit c from E. coli. The range in the other transporters is between -7 and +7, with the lowest absolute number of net charges in the group analyzed being 4. It is therefore suggested that the existence of unpaired charges on the protein represent the main energy barrier for solubilization in the highly hydrophobic milieu of the organic solvents. This has been extensively discussed in the case of charge insertion in membranes (Engelman et al., 1986) and has also received some experimental support in experiments in which neutralization of charges of membrane proteins with H or Ca rendered them soluble in organic solvents (Gitler, 1977).

Multidrug resistance is a major concern in medical and agricultural diseases. In medicine, the emergence of resistance to multiple drugs is a major obstacle in the treatment of several tumors as well as many infectious diseases. In agriculture, the control of resistance of plant pathogens is of major economic importance. However, besides being an economical and health-threatening phenomenon, it seems that the great diversity of transporters that play a role in drug resistance points to evolution of these systems as a necessary trait for survival in a variety of adverse environments. Outside the protected atmosphere of the laboratory, all organisms have to cope with a large variety of toxic compounds. Some of these compounds are produced by living organisms as part of their fight for survival, while others are toxic wastes, either natural or man made. Obviously, only those organisms that have developed through evolution the ability to cope with a wide variety of compounds, have been able to survive. EmrE is a representative of one of the strategies that evolved, most likely to provide protection from the toxic compounds. Because of their small size and the mechanistic similarity with the TEXANs, we propose to call this family MiniTEXANs. As described here, the MiniTEXANSs seem to be a unique, effective, and very exciting family of proteins. Because of their size and properties, they may provide a very useful model to understand structure-function aspects of transport reactions in ion-coupled processes.

Note Added in Proof-After submission of this article, L. L. Grinius and E. B. Goldberg ((1994) J. Biol. Chem.269, 29998-30004) reported the purification of Smr, a protein from Staphylococcus aureus homologous to Emr.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 972-2-585992; Fax: 972-2-634625; shimons{at}vms.huji.ac.il.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; TPP, tetraphenylphosphonium; MPP, 1-methyl-4-phenylpyridinium; CCCP, carbonyl cyanide m-chlorophenylhydrazone; Delta, proton electrochemical gradient.

(^2)
The sequences analyzed were: Bmr, the Bacillus multidrug transporter (accession number P33449[GenBank]); Lac permease (A03418[GenBank]); MelB, thiomethylgalactoside permease (A03421[GenBank]); NhaA and NhaB, Na/H antiporters (P13738 [GenBank]and P27377[GenBank]); PutP, proline permease (L01152[GenBank]); and TetC, tetracycline antiporter (A03508[GenBank]).


REFERENCES

  1. Ahmed, M., Borsch, C., Neyfakh, A., and Schuldiner, S. (1993) J. Biol. Chem. 268, 11086-11089 [Abstract/Free Full Text]
  2. Bradford, W. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  3. Davies, B., and Mingioli, E. (1950) J. Bacteriol. 60, 17-28
  4. Doige, C. A., and Ames, G. F. L. (1993) Annu. Rev. Microbiol. 47, 291-319 [CrossRef][Medline] [Order article via Infotrieve]
  5. Endicott, J., and Ling, V. (1989) Annu. Rev. Biochem. 58, 137-171 [CrossRef][Medline] [Order article via Infotrieve]
  6. Engelman, D., Steits, T., and Goldman, A. (1986) Annu. Rev. Biophys. Chem. 15, 321-353 [CrossRef][Medline] [Order article via Infotrieve]
  7. Fillingame, R. (1992) J. Bioenerg. Biomembr. 24, 485-491 [Medline] [Order article via Infotrieve]
  8. Gitler, C. (1977) Biochem. Biophys. Res. Commun. 74, 178-182 [Medline] [Order article via Infotrieve]
  9. Goldberg, E. B., Arbel, T., Chen, J., Karpel, R., Mackie, G. A., Schuldiner, S., and Padan, E. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2615-2619 [Abstract]
  10. Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427 [CrossRef][Medline] [Order article via Infotrieve]
  11. Grinius, L., Dreguniene, G., Goldberg, E. B., Liao, C. H., and Projan, S. J. (1992) Plasmid 27, 119-129 [Medline] [Order article via Infotrieve]
  12. Gros, P., Croop, J., and Housman, D. (1986) Cell 47, 371-380 [Medline] [Order article via Infotrieve]
  13. Harel-Bronstein, M., Dibrov, P., Olami, Y., Pinner, E., Schuldiner, S., and Padan, E. (1995) J. Biol. Chem. 270, 3816-3822 [Abstract/Free Full Text]
  14. Higgins, C. (1992) Annu. Rev. Cell Biol. 8, 67-113 [CrossRef]
  15. Lambert, B., and Le Pecq, J. B. (1984) Biochemistry 23, 166-176 [Medline] [Order article via Infotrieve]
  16. Lewis, K. (1994) Trends Biochem. Sci. 19, 119-123 [CrossRef][Medline] [Order article via Infotrieve]
  17. Liu, Y., Peter, D., Roghani, A., Schuldiner, S., Prive, G., Eisenberg, D., Brecha, N., and Edwards, R. (1992) Cell 70, 539-551 [Medline] [Order article via Infotrieve]
  18. Midgley, M. (1987) Microbiol. Sci. 4, 125-127 [Medline] [Order article via Infotrieve]
  19. Morimyo, M., Hongo, E., Hama-Inaba, H., and Machida, I. (1992) Nucleic Acids Res. 20, 3159-3165 [Abstract]
  20. Neyfakh, A., Bidnenko, V., and Chen, L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4781-4785 [Abstract]
  21. Nikaido, H. (1994) Science 264, 382-388 [Medline] [Order article via Infotrieve]
  22. Paulsen, I., and Skurray, R. (1993) Gene (Amst.) 124, 1-11 [CrossRef][Medline] [Order article via Infotrieve]
  23. Pearce, H. L., Safa, A. R., Bach, N. J., Winter, M. A., Cirtain, M. C., and Beck, W. T. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5128-5132 [Abstract]
  24. Pinner, E., Padan, E., and Schuldiner, S. (1992) J. Biol. Chem. 267, 11064-11068 [Abstract/Free Full Text]
  25. Purewal, A. S. (1991) FEMS Microbiol. Lett. 82, 229-232
  26. Purewal, A. S., Jones, I. G., and Midgley, M. (1990) FEMS Microbiol. Lett. 68, 73-76
  27. Rosen, B. (1986) Methods Enzymol. 125, 328-336 [Medline] [Order article via Infotrieve]
  28. Schagger, H., and Jagow, G. V. (1987) Anal. Biochem. 166, 368-379 [Medline] [Order article via Infotrieve]
  29. Schuldiner, S. (1994) J. Neurochem. 62, 2067-2078 [Medline] [Order article via Infotrieve]
  30. Schuldiner, S., Shirvan, A., and Linial, M. (1995) Physiol. Rev. , in press
  31. Tabor, S., and Richardson, C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1074-1078 [Abstract]
  32. Taglicht, D., Padan, E., and Schuldiner, S. (1991) J. Biol. Chem. 266, 11289-11294 [Abstract/Free Full Text]
  33. Taglicht, D., Padan, E., and Schuldiner, S. (1993) J. Biol. Chem. 268, 5382-5387 [Abstract/Free Full Text]
  34. Viitanen, P., Newman, M. J., Foster, L., Wilson, T. H., and Kaback, H. R. (1986) Methods Enzymol. 125, 429-452 [Medline] [Order article via Infotrieve]
  35. Wells, R., and Hediger, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5596-5600 [Abstract]
  36. Yanish-Perron, C., Viera, J., and Messing, J. (1985) Gene (Amst.) 33, 103-199 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.