Topologically Random Insertion of EmrE Supports aPathway for Evolution of Inverted Repeats inIon-coupled Transporters*□S

Received for publication, January 28, 2010, and in revised form, March 17, 2010 Published, JBC Papers in Press, March 22, 2010, DOI 10.1074/jbc.M110.108746

Iris Nasie1, Sonia Steiner-Mordoch1, Ayala Gold, and Shimon Schuldiner2

From the Department of Biological Chemistry, Alexander A. Silberman Institute of Life Sciences, Hebrew University of Jerusalem,91904 Jerusalem, Israel

Inverted repeats in ion-coupled transporters have evolvedindependently inmany unrelated families. It has been suggestedthat this inverted symmetry is an essential element of themech-anism that allows for the conformational transitions in trans-porters.We show here that small multidrug transporters offer amodel for the evolution of such repeats. This family includesboth homodimers and closely related heterodimers. In the for-mer, the topology determinants, evidently identical in each pro-tomer, are weak, and we show that for EmrE, an homodimerfrom Escherichia coli, the insertion into the membrane is ran-dom, and dimers are functional whether they insert into thecytoplasmic membrane with the N- and C-terminal domainsfacing the inside or the outside of the cell. Also, mutantsdesigned to insertwith biased topology are functional regardlessof the topology. In the case of EbrAB, a heterodimer homologuesupposed to interact antiparallel, we show that one of the sub-units, EbrB, can also function as a homodimer, most likely in aparallel mode. In addition, the EmrE homodimer can be forcedto an antiparallel topology by fusion of an additional transmem-brane segment. The simplicity of themechanismof coupling ionand substrate transport and the few requirements for substraterecognition provide the robustness necessary to tolerate such aunique and unprecedented ambiguity in the interaction of thesubunits and in the dimer topology relative to the membrane.The results suggest that the small multidrug transporters are atan evolutionary junction and provide a model for the evolutionof structure of transport proteins.

Evolution of large transporter genes is thought to havestarted from a small single one that duplicated, evolved inde-pendently, and then fused to the original one to generate a newgene coding for the large polytopic protein (1). Inverted repeatsin ion-coupled transporters have now been observed in trans-porters from many different families (2, 3). It has been sug-

gested that this inverted symmetry is an essential element of themechanism that allows for the conformational transitions intransporters (3).A possible mode for evolution of these inverted repeats has

been suggested based on the supposedly inverted topology ofEmrE, a small multidrug transporter from Escherichia coli. Aclaim for an antiparallel topology of themonomers in the EmrEhomodimer was made, but this is still a controversial issue and,in our view, is still inconsistent with the biochemical data. Thereasoning that the topology of the monomers in the EmrEdimer is parallel and Nin-Cin was supported by data from ourlaboratory that demonstrated the same topology for all pro-tomers in the intact cell and in membrane vesicles (4), by bio-chemical studies that established the equivalence of the resi-dues in the protomers (reviewed in Ref. 5 and 6), and byextensive cross-linking studies that suggested the proximity ofequivalent residues in the twomonomers (7).Moreover, dimerscross-linked at positions not compatible with antiparalleltopology were purified to homogeneity and shown to be fullyfunctional based on substrate binding and transport assays (8).To provide an approach that combines in vivo and in vitro stud-ies, we designed a series of genetic fusions (tandems) where themonomers are connected with the C terminus of the firstmonomer attaching to the N terminus of the second (tail tohead). This was achieved by means of defined linkers that arenot compatible with an antiparallel topology either becausethey are too hydrophilic or too short (9).The suggestion for an antiparallel topology of themonomers

was supported by a C-� model of the transmembrane regionconstructed by considering the evolutionary conservation pat-tern of each helix (10) and based on a reinterpretation of lowresolution electron densitymaps of two-dimensional crystals ofEmrE that showed quasi-symmetry in parts of the structure(11). Although possible, the model is one of many that can begenerated with the available data, and unfortunately, no exper-imental test was provided. An x-ray structure previouslyretracted (12) has been reanalyzed, and a C-� model of thecorrected structure with substrate is very similar to the modelproposed based on cryo-EM studies (13). The crystals used toderive the C-� model x-ray structure were obtained with pro-tein solubilizedwith detergents that inhibit activity by inducingmonomerization as shown not only by our data but from theresults of the authors as well (8, 13). Monomers arrange in thecrystal in a conformation that minimizes the energy needed for

* This work was supported, in whole or in part, by National Institutes of HealthGrant NS16708. This work was also supported by Grant 119/04 from theIsrael Science Foundation.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1 and Figs. S1–S5.

1 Both authors contributed equally to this work.2 Mathilda Marks-Kennedy Professor of Biochemistry at the Hebrew Univer-

sity of Jerusalem. To whom correspondence should be addressed: Dept. ofBiological Chemistry, Alexander Silberman Institute of Life Sciences,Hebrew University of Jerusalem, 91904 Jerusalem, Israel. Tel.: 972-2-6585992; Fax: 972-2-5634625; E-mail: shimon.schuldiner@huji.ac.il.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 20, pp. 15234 –15244, May 14, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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crystal formation and do not necessarily reflect the relativetopology in the membrane.In addition, genetic experiments were designed to support

the claim for an antiparallel topology. EmrE was fused to thetopology reporter proteins alkaline phosphatase and greenfluorescent protein, and the results showed that the topology ofthe EmrE fusion proteins in the membrane is sensitive to thedistribution of positive charges in the protein (14). Manipula-tion of the positive charges generated a set of mutants, somewith Nout-Cout (Cout) and others with Nin-Cin (Cin) apparenttopology (14, 15). The Cin and Cout mutants did not conferresistance to ethidium, and the authors concluded that this wasdue to the modified topology. Co-expression of the inactivemutants (Cin and Cout together) restored the ethidium resis-tance phenotype to the same level as seen with wild type EmrE(15). The suggested interpretation of this finding was that co-expression results in the generation of a functional, antiparallelheterodimer. However, because this conclusion is based solelyon the contention that theCin andCoutmutants are inactive, wedecided to re-evaluate these findings that were based only onthe phenotypic analysis.Here we show that small multidrug transporters (SMRs)3

display a remarkable plasticity regarding topology and mono-mer-monomer interactions. Thus, Cin and Cout mutants areshown to be functional provided they are expressed in a con-trolled mode and in a strain where the chromosomal wild typeemrE was inactivated. Moreover, the wild type protein, whenexpressed without any tag, inserts into the membrane in a ran-dom fashion generating a dual topology; approximately half ofthe dimers are Nin-Cin, and half are Nout-Cout. These findingsare in agreement with the generally accepted functional sym-metry of channels, transporters, and ion-coupled transporterswhere the direction of transport is dictated by the direction ofthe driving force acting on the transported substrates (except inspecial cases where there is a kinetic block). However, this is thefirst demonstration that the directionality of insertion of atransporter in vivo can be randomwithout affecting activity. Inthe same vein, a detailed analysis of EbrAB, a closely relatedantiparallel heterodimer from Bacillus subtilis, reveals that inaddition to the efficient interaction between the two pro-tomers, one of them can still interact productively with itself toform a functional homodimer, most likely parallel. Finally, inline with the above findings, the homodimeric EmrE can beforced to form an antiparallel dimer by fusion with an addi-tional transmembrane domain. These experiments demon-strate that SMR proteins are in an evolutionary junction andprovide an example of how protein fold can evolve while main-taining its function.

EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Mutagenesis—E. coli DH5�(Invitrogen), JM109 (Novagen), BW25113 (52), HMS174 (DES)(Novagen), and TA15 (53) strains were used throughout thiswork. BW25113 �emrE, �smr, �mdfA, �yjiO, and �acrB indi-vidual knockouts and combinations were prepared accordingtoDatsenko et al. (52) anddescribed byTal and Schuldiner (25).For specific labeling with [35S]methionine, BW25113 �emrE�mdfA cells were transformedwith the plasmid pGP1-2, whichcodes for the T7 polymerase under the inducible control of the� PL promoter (18). The plasmids used for gene expression ofEmrE Cin and Cout and EbrA and B, without any tags are pT7-7(16, 18) or pACYC184 (54) derivatives. Tagged EmrE is fused atthe C terminus to His6 residues and aMyc epitope as described(16). Transfer of EmrE constructs to pACYC184was performedusing NdeI and ClaI restriction sites. GpA-EmrE was con-structed by insertion at the BamHI site of E6EMH (9) of asequence coding for a nondimerizing mutant of human glyco-phorin A. The DNA sequence was manipulated so that the Nterminus of the second EmrE protomer (NP, the first methio-nine was removed) was connected to the C terminus (RSTPH)of the first EmrE protomer with a linker that codes for thesequence 1GSLEPEITLILFGVMALVIGTILLLLYGIRRLIGSG-AAS39. Tandems with linkers with variations of the abovesequence displayed similar phenotypes but were not studiedfurther (supplemental Table S1). A “negatively dominated” tan-demwas built substituting the essential glutamate 14 in the firstmonomer with glutamine (GpA-EmrE (E14Q)1).Resistance to Toxic Compounds—E. coli cells were trans-

formed with the indicated plasmids and were grown overnightat 37 °C in LB medium with the corresponding antibiotic. 5 �lof serial dilutions of the culture were spotted on LB plates con-taining 30 mM BisTris propane, pH 7.0, with or without theaddition of the indicated concentrations of the toxic com-pounds. When the pACYC184 plasmid was used, IPTG (200�M) was added for induction of expression. Growth was ana-lyzed after overnight incubation at 37 °C. Each experiment wasperformed at least twice.Binding Assays—[3H]Tetraphenylphosphonium (TPP�)

binding to His-tagged GpA-EmrE and GpA-EmrE (E14Q)1 wasassayed essentially as described (16). The amounts of purifiedproteins were determined on Coomassie-stained 16% TricineSDS-PAGEusing known amounts of the corresponding proteinfor calibration. The radioactivity was measured by liquid scin-tillation. All of the binding reactions were performed in dupli-cates, and in each experiment the values obtained in a controlreactionwith 25�MunlabeledTPP�were subtracted. All of theexperiments were repeated at least twice.Tagged Construct Topology Determination—HMS174 cells

expressing the appropriate single Cys mutant were incubatedwith the indicated concentrations of MTSES at 30 °C for 20min. After removal of the MTSES by centrifugation, the cellswere rinsed three times and lysed by incubation in a solutioncontaining sucrose (30%), Tris-Cl (30 mM, pH 8.0), 50 �g/mllysozyme, 10mM EDTA, and 1.5 �g/ml DNase. Themembranefractionwas collected and solubilized in 0.8%dodecyl-�-D-mal-toside/sodiumbuffer (150mMNaCl and 15mMTris-Cl, pH 7.5)

3 The abbreviations used are: SMR, small multidrug transporter; IPTG,isopropyl �-D-thiogalactopyranoside; BisTris, 2-[bis(2-hydroxyethyl)-amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MTSES, (2-sulfonatoethyl) methane-thiosulfonate; Mal-PEG, methyl-polyethylene glycol-maleimide-MR 5000;o-PDM, o-phenylenedimaleimide; TM, transmembrane domain; GpA, gly-cophorin A; TPP�, tetraphenylphosphonium.

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for 20 min at 25 °C, allowed to bind to nickel-nitrilotriaceticacid beads for 1 h at 4 °C and rinsed with sodium buffer supple-mented with 2.0% SDS and 6 M urea. N-Ethylmaleimide-fluo-rescein (Pierce) was added to a final concentration of 0.5mM for20min (at 30 °C). The reactionwas stopped by dilutionwith thesame buffer containing �-mercaptoethanol at a final concen-tration of 5 mM. The beads were then washed as describedabove. The protein was eluted from the beads using a buffercontaining 200 mM �-mercaptoethanol, 100 mM Tris-Cl, pH6.8, 4% SDS, 40% glycerol, 0.2% bromphenol blue (samplebuffer), and 450 mM imidazole and analyzed by 16% TricineSDS-PAGE. Fluorescence labeling analysis of the gel was doneusing a Fujifilm LAS-1000 imaging system and digitally ana-lyzedwith ImageGauge 3.46 Fujifilm software. The resultswerecalibrated to protein amount after staining of the same gelswithCoomassie staining, scanning, and quantitation using the samesoftware. Each experiment was performed at least three times.Untagged Construct Topology Determination—BW25113

�emrE �mdfA cells bearing pGP1-2 and EmrE and indi-cated mutants on pT7-7 were metabolically and specificallylabeled with [35S]methionine essentially as described in Refs.17 and 19. The cells were then incubated with MTSES andtreated as above except that membrane solubilization wasdone in 2% SDS, 6 M urea, and 15 mM Tris-Cl, pH 7.5, and 2.5mM Mal-PEG 5000 (Nektar Transforming Therapeutics,Huntsville, AL). The reaction was stopped after 1 h at 37 °Cby the addition of sample buffer. The proteins were analyzedusing 16% Tricine SDS-PAGE and visualized with a FujifilmFLA-3000 imaging system and digitally analyzed with ImageGauge 3.46 Fujifilm software. Each experiment was per-formed at least three times.Cross-linking with o-PDM—The membranes prepared from

cells expressing EmrEmutants selectively labeledwith [35S]me-thionine were solubilized as described above, and o-PDM wasadded to a final concentration of 1 mM. The reaction wasstopped after 1 h at 45 °C and 10min at 60 °C by the addition ofsample buffer. Samples containing �5500 dpm of labeled[35S]EmrE were analyzed on 16% Tricine SDS-PAGE and visu-alized with a Fujifilm FLA-3000 imaging system and digitallyanalyzed with Image Gauge 3.46 Fujifilm software. Each exper-iment was performed twice.

RESULTS

Tags and Reporters May Introduce Bias in TopologyDeterminations—The topological studies of EmrE in intactcells in our laboratory indicate that the protein assumes a Nin-Cin topology in the membrane (4). In our studies we used aconstruct in which the wild type gene was fused to a Mycepitope before a six-His tag at theC terminus (16). On the otherhand, studies using alkaline phosphatase and green fluorescentprotein fusions suggest that the fused wild type protein ismostly in aNout-Cout configuration (14).We show here that thediscrepancy in the topology of the wild type between the twogroups stems from the difference in the fusion tags used.To test the possible effect of tags, we designed a strategy to

assess the topology of untagged proteins that would obviate theneed to purify them. Single Cys residues were engineered at thesame positions in loops in Myc-His tagged at the C terminus

and in untagged proteins. Themutants were specifically labeledmetabolically with [35S]methionine as described previously(17–19). The cells were then challenged with the impermeantMTSES reagent, and unreacted thiols were assessed after solu-bilization and denaturation of the protein with Mal-PEG (20).Reaction with Mal-PEG resulted in a shift in the mobility thatcan be easily detected after SDS-PAGE analysis (Fig. 1,A and B,bottom panels, compare lanes with and without Mal-PEG). Intagged mutants, cysteine in loop 1 at position 22 (EmrE-K22C)but not at the C terminus, position 110 (EmrE-H110C), reacted

FIGURE 1. Tags introduce bias in topology of undecided proteins. Tagged(A) and untagged (B) EmrE K22C and EmrE H110C were metabolically labeledwith [35S]methionine in E. coli BW25113 �emrE�mdfA cells and treated withMTSES at the indicated concentrations as described under “Experimental Pro-cedures.” The unreacted thiols were estimated from the degree of reactionwith Mal-PEG. EmrE that reacted with Mal-PEG displays a higher apparent Mrin SDS-PAGE, and the ratios of reacted over nonreacted protein were calcu-lated. The bottom panels in A and B are samples of the types of changes at oneconcentration of MTSES: 0.3 and 0.1 mM, respectively. C, simplified flow chartof procedure. D, monomers of tagged and untagged EmrE K22C (not shown)and H110C are in a parallel topology relative to each other as judged from thefull cross-linking detected with o-PDM.

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quantitatively withMTSES in the intact cell, and therefore onlya fraction of�30%of the protein reactedwithMal-PEG (Fig. 1Aand see also Ref. 4). However, with untagged mutants bearingCys at the same positions, reactivity of both EmrE-K22C and-H110C was very similar and partial; �50–60% of the residuesare exposed in each case (Fig. 1B). These results suggest that theMyc-His tag is biasing the protein toward a Nin-Cin topology,whereas the untagged protein displays a dual topology, i.e.approximately half of the protein is Nin-Cin, whereas the otherhalf is Nout-Cout.The Monomers in the Dimer Are Parallel—The term dual

topology was previously used to describe the relative topologyof the monomers in the dimer (parallel or antiparallel). We useit here to describe the topology of the dimers relative to eachother. Previously we showed, using several approaches, that themonomers within each dimer are parallel, i.e. within a givendimer the topology of both monomers is identical (6–9).Because the results described above could also be interpreted asan indication of antiparallel topology and to distinguishbetween dual and antiparallel topology, we cross-linked thedimers with o-PDM (8), a rigid bifunctional cross-linking agentthat can react with Cys residues that are 7–11 Å apart (21). TheCys replacements, H110C at the C terminus and K22C in loop1, are too far apart to cross-link in an antiparallel structure(�35–45 Å). Despite this distance and as expected from a par-allel topology, efficient cross-linking was observed with o-PDMwhen either tagged or untagged EmrE was used (Fig. 1D, datafor H110C and supplemental Fig. S1, data for K22C as well).Cross-linking with o-PDM was already shown to result fromspecific interaction within the dimer rather than betweendimers (6–9, 22, 23). Further support is provided by an exper-iment where cross-linking between labeled protomers isobserved at the same levels in the presence or absence of excessunlabeled protein (supplemental Fig. S1). In these experiments,membranes with the 35S-labeled EmrE mutant were dilutedwith the same amount or a 4-fold excess ofmembranes express-ing the same mutant but unlabeled. The fact that the intensityof the cross-linked dimer is not affected by the presence of theunlabeled protein strengthens our contention that the o-PDM-mediated cross-linking is a result of a reaction between Cysresidues within the dimer.We conclude that insertion of untagged EmrE is a random

process that results in dual topology when one fraction of thedimer is Nin-Cin and another is Nout-Cout as expected from thevery weak topology determinants. However, within the dimer,the preferred relative topology is parallel. If antiparallel dimersexist without experimenter intervention, they are a smallminority that cannot be detected with the techniques we devel-oped.The variation in resultswithin the various constructs pro-vides a caveat about the bias that tags and reporters may intro-duce in topology determinations.Nin-Cin andNout-Cout EmrEConstructs Are Functional—The

above findings seem to contradict previous reports claimingthat expression of EmrE Cin or EmrE Cout individually did notconfer resistance to ethidium as shown for wild type EmrE (15).In that report, to push EmrE toward the Nin-Cin or Nout-Coutorientations, the (K�R) bias was systematically modified bysubstantialmutagenesis (Fig. 2A and Ref. 15).We generated the

same mutants and confirm the above results when theexpression was tested in the commonly used E. coli DH5�(supplemental Fig. S2). The published conclusion was basedonly on phenotypic characterization, a powerful tool that pro-vides definite answerswhen reflecting a positive trait. However,the experimentswere done in awild type strain harboring chro-mosomal emrE and without induction of expression because ofthe well known toxic effects of overexpression of membraneproteins. Therefore we carried out a more detailed study of thephenotype of EmrE Cin and Cout under controlled expressionconditions. We used the plasmid pACYC184 where a trc pro-moter controls the expression, and we grew the cells in thepresence of 200 �M of the inducer IPTG, conditions underwhich expression is heightened but not as high as to becometoxic (not shown). The increased and controlled expressionyielded weak but definite positive phenotypes in several com-mercially available E. coli strains such as JM109 and HMS174(supplemental Fig. S2). This is in contrast with the negativephenotypes obtained without induction (Ref. 15 andsupplemental Fig. S2, DH5�). In agreement with these findings,weak but distinct phenotypes were also reported in similarexperiments with induced BL21 cells (24).An evenmore dramatic factor influencing the phenotypewas

identified when using strains well defined genetically and func-tionally (Fig. 2B). In these strains we have inactivated one ormultiple multidrug transporters (25). Although not related inamino acid sequence, several multiple multidrug transportersare functionally related and even share some of the substrates.We show that the ability to confer resistance is independent ofthe sensitivity of the host cell because even in very sensitivestrains where the genes coding for the functionally relatedtransporters MdfA, yjiO, or AcrAB were inactivated, eitherindividually or together, the Cin and Cout mutants still provideonly a feeble resistance to ethidium and acriflavine as shown forthe other strains tested (data shown only for MdfA; Fig. 2B).Strikingly, however, when the chromosomal gene coding forEmrE is inactivated, the phenotype for the three substratestested is distinct and comparable with that shown by wild typeEmrE, somewhat weaker for Cout but very clear (Fig. 2B).

The results described above imply that the presence of EmrEis detrimental to the phenotypic expression of the Cin and Coutmutants. This may be due to a nonproductive interactionbetween wild type EmrE and the mutants. This reasoning isfurther supported by a series of experiments where negativedominance was observed when co-expressing the functionalCin and Cout mutants with E14C EmrE, an inactive mutant(supplemental Fig. S3). In these experiments we had to balancethe need for relatively high levels of expression of the Cin andCout mutants with low expression levels of E14C EmrE thatwere obtained from the constitutive leak of T7 promoter thatcontrol the plasmidic gene. The best results were obtained atlow (25 �M IPTG) or no induction when the phenotype con-ferred by the Cin and Cout mutants is not as strong as shownunder full induction (compare supplemental Fig. S3 with Fig.2B, bottom panel). Under these conditions the phenotype con-ferred by the Cin and Cout mutants is significantly impairedwhen co-expressed with the inactive E14C EmrE mutant(supplemental Fig. S3).

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We conclude that Cin and Cout mutants confer a robustresistance, provided they are expressed in a cell where the chro-mosomal gene coding for EmrE has been inactivated and theexpression levels are enhanced by induction. The results also

explain the variability in the pheno-type conferred by the Cin and Coutmutants in different E. coli strainsthat could be correlated withdifferent levels of expression ofchromosomal emrE in the variousstrains (Fig. 2, supplemental Fig. S2,and Ref. 24).When Is Heterodimerization Oblig-

atory?—The finding that expres-sion of chromosomal emrEmay havea deleterious effect on specificmutants led us also to investigatethe case of EbrAB, a heterodimerfrom the SMR family consideredobligatory solely on the basis of phe-notype (26, 27) (Fig. 3A). We con-firm previous findings that expres-sion of each one of the protomers,EbrA or EbrB, alone does not conferresistance to the toxicants whentested in a wild type strain such asDH5� (supplemental Fig. S4). Thesame phenotype is observed instrains where either mdfA, a genecoding for an MFS multidrugtransporter, or acrA, coding for asubunit of the major multidrugtransporter AcrAB, has been inacti-vated (Fig. 3B). However, EbrB butnot EbrA confers robust resistanceto ethidium and acriflavine (notshown) when expressed in any oneof three strains where the chromo-somal emrE gene was inactivatedalone or in combination with otherSMRs (�smr) or withmdfA (Fig. 3B,bottom panel). The results implythat EbrB has not completelycrossed the point where it can inter-act only in an antiparallelmodewithEbrA. It can also interact with itself(with a lower affinity), forming ahomodimer most likely with a par-allel topology. In the presence ofEmrE, it interacts in a nonproduc-tive way similar to what we showedabove for the EmrE Cin and Coutmutant proteins.Forcing aHomodimerwith Identi-

cal Protomers into an AntiparallelConfiguration—A wide range ofbiochemical experiments from ourlaboratory supports the contention

that the topology of the monomers in the native EmrE dimer isparallel and Nin-Cin (4–9). To provide an approach that com-bines in vivo and in vitro studies, we designed a series of geneticfusions (tandems) where the monomers are connected C ter-

FIGURE 2. A, schematic representation of the EmrE Cin and EmrE Cout mutations. The mutations were performedas described under “Experimental Procedures.” The sites were selected as in Ref. 15. The squares indicatepositive charges in the wild type EmrE and those remaining after mutagenesis; white circles indicate positivecharges added. B, phenotype of EmrE Cin and EmrE Cout. E. coli BW25113 �emrE, �mdfA, or �emrE�mdfAtransformed with pACYC184-EmrE (EmrE), pACYC184 (vector), or pACYC184 with each of the EmrE mutantswere grown overnight at 37 °C in LB medium containing chloramphenicol. 5 �l of serial dilutions of the culturewere spotted on LB plates containing 30 mM BisTris propane, pH 7.0, with 200 �M IPTG and with or without theaddition of ethidium (400 �g/ml in top and middle panels and 100 �g/ml in bottom panel), acriflavine (250, 100,and 25 �g/ml, respectively), and methyl viologen (0.6, 0.4, and 0.3 mM, respectively). Growth was analyzed afterovernight incubation at 37 °C. In control plates with no toxicants, growth of all strains was similar (not shown).When resistance was tested with the same strains without IPTG, the phenotypes were negative, and withdifferent host strains the results are variable (shown in Fig. S2), stressing again the inadequacy of a negativephenotype to determine the lack of activity of EmrE or similar proteins.

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minus of the first to N terminus of the secondmonomer (tail tohead). This was achieved by means of defined linkers that arenot compatible with an antiparallel topology either becausethey are too hydrophilic or too short (9). In these studieswe showed that all the constructs are functional in vivo becausethey confer resistance to EmrE substrates, and they catalyzeenergy-dependent ethidium efflux. In addition, the proteinswere purified and reconstituted into proteoliposomes andshown to catalyze transport with kinetic constants similar tothe wild type EmrE. Moreover, we showed that the functionalunit is the dimer itself and does not result from interdimericinteraction.Because of the high plasticity documented above, where the

dimers can insert in both topologies and the antiparallel het-erodimer can also function as a parallel homodimer, we askedwhether also in the EmrE homodimer there is a potential for anantiparallel interaction without introducing further mutations.For this purpose, we generated a series of tandem EmrE con-structs with nine transmembrane domains (TM) by geneticfusion of the monomers (N terminus of the second EmrE pro-

tomer to the C terminus of the firstone) with an additional transmem-brane segment in between them(Fig. 4A). The segment inserted wasbased on the sequence of a mutantof human glycophorin A (GpA) thatdoes not dimerize (28). Such afusion, with an odd number of TMs,would have the N and C termini atopposite sides of the membrane,contrasting with the previously gen-erated eight TM proteins withhydrophilic linkers of variablelength (9). Fusions with severalsequences of GpA and severallinkers were constructed (supple-mental Table S1), and they allyielded functional proteins. Onlythe first one will be described indepth and will be namedGpA-EmrE.The activity was tested in vivo by

assessment of the resistance it con-fers to E. coli cells. This wasachieved in solid medium contain-ing either ethidium (100 �g/ml),acriflavine (25 �g/ml), or methylviologen (0.3 mM) (Fig. 4B). Cellsexpressing either EmrE or GpA-EmrE displayed substantial andsimilar resistance to the threecompounds.To further characterize the pro-

tein activity and to rule out the pos-sibility that the transport observedin vivo is due to proteolytic prod-ucts, GpA-EmrE was purified andassayed for binding activity. GpA-

EmrE bound the high affinity substrate Tetraphenyl phospho-nium (TPP�) with a KD of 2.4 � 0.5 nM, very similar to that ofthe wild type protein or the tandem E6EMH (9, 29)(supplemental Fig. S5A). Purified GpA-EmrE reconstitutedinto proteoliposomes transports both substrates: themonovalent 1-methyl-4-phenylpyridinium and the divalentmethyl viologen (supplemental Fig. S5, B andC).GpA-EmrE Is the Functional Unit—The possibility that the

functional unit may be formed by the interaction of two dimerswas ruled out by several experimental approaches. First, weused negative dominance analysis. Specifically, replacement ofglutamate 14 in EmrE with an uncharged amino acid com-pletely abolishes binding, but the mutants can form mixeddimers with other functional EmrE protomers. The mixeddimers display at least a 20-fold lower affinity toward TPP�, aphenomenon resulting from negative dominance of the inac-tive mutant on the functional one (23, 30). In these experi-ments, increasing amounts of the well characterized mutantEmrE E14C were added to wild type EmrE and to GpA-EmrE,and subsequently the mixtures were subjected to the monomer

FIGURE 3. EbrAB, a heterodimer that can still function as a homodimer. A, schematic representation ofEbrAB. The squares represent the positive charges in each monomer. B, phenotype. E. coli BW25113 �acrA,�mdfA, �emrE, �smr, or �emrE�mdfA transformed with pACYC184-EmrE (EmrE), pACYC184 (vector), orpACYC184 with EbrA, EbrB, or both genes together were grown overnight at 37 °C in LB medium containingchloramphenicol. 5 �l of serial dilutions of the culture were spotted on LB plates containing 30 mM BisTrispropane, pH 7.0, with 200 �M IPTG and with or without the addition of ethidium (�acrA, 12.5 �g/ml; �mdfA,�emrE, and �smr, 400 �g/ml; and �emrE�mdfA, 100 �g/ml). Growth was analyzed after overnight incubationat 37 °C. In control plates with no toxicants, growth of all strains was similar (not shown).

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swapping procedure and assayed forTPP�binding activity (Fig.5A). The TPP� concentration used in this assay, 2.5 nM, equalstheKD of EmrE (9, 29) and GpA-EmrE. Therefore, inhibition ofbinding reflects the formation of mixed dimers because thecontribution of heterodimers EmrE/E14C is insignificantbecause of their lower affinity to TPP�. After monomer swap-ping with a 50-fold excess of the EmrE E14C mutant, the bind-ing activity of the wild type protein was inhibited by more than60% (Fig. 5A, circles). Under the same conditions, the bindingactivity of GpA-EmrE was hardly affected (Fig. 5A, triangles).To further substantiate the contention that GpA-EmrE is the

functional unit, wemutated one of the two essential glutamates(one in each protomer) that correspond to the membraneembedded glutamate 14 in wild type EmrE. GpA-EmrE-(E14Q)1 where one of the essential glutamates was changed toglutamine, confers only a very low but significant resistance toethidium and acriflavine (not shown) and displays bindingaffinity to TPP� (120 � 59 nM) almost 2 orders of magnitudelower than the wild type protein (9). GpA-EmrE-(E14Q)1reconstituted in proteoliposomes transports monovalent(1-methyl-4-phenylpyridinium) but not divalent (methyl violo-gen) substrates (supplemental Fig. S5, B and C). Transport ofthe monovalent 1-methyl-4-phenylpyridinium is inhibited byanother monovalent (TPP�) but not by a divalent (methylviologen) substrate (supplemental Fig. S5C). A parallel tandemprotein with eight TMs bearing only one Glu14 was also shownto display similar phenotype and affinities and lost the ability to

transport divalent substrates. The findings are explained by thefact that the mutated transporters bearing only one Glu14 resi-due exchange only 1H�/substrate as opposed to 2H�/substratefor the wild type proteins (9).GpA-EmrE Is Properly Packed—The glycophorin A linker

was designed to be long and hydrophobic enough to cross themembrane plane (supplemental Table S1 and Fig. 4A). Theresults described above demonstrate that the additional TMhas no significant effect on the activity of the protein and there-foremost likely does not affect the overall packing of the dimer,a prerequisite for function. However, because manipulations ofthis type may potentially affect integration of transmembranesegments and may induce generation of semi-inverted topolo-gies, we tested whether the insertion of the linker affected thepacking of the protomers in GpA-EmrE. The membranedomain of EmrE is completely resistant to a battery of pro-teases. After exposure of EmrE to either chymotrypsin or pro-teinase K, only the C-terminal tag is digested even at prolongedovernight digestions (9). When a parallel tandem with anexposed hydrophilic linker (E22EMH) is digested by three pro-teases: trypsin, chymotrypsin, and proteinase K (Ref. 9 and Fig.5B, lanes 5–8), the digestion results in the production of twopolypeptides with similar apparent mass (11–12 kDa). GpA-EmrE, on the other hand, is resistant to treatment with any ofthe three enzymes (Fig. 5B, lanes 1–4). Only the C-terminal tagis digested, producing a polypeptide with correspondinglylower apparent mass (22–24 kDa) as predicted from the sites ofdigestion of the three enzymes. The results support the conten-tion that both the glycophorin A linker and the rest of the pro-tein are well packed, embedded in the membrane, and pro-tected from digestion.The Protomers in GpA-EmrE Display Antiparallel Configura-

tion—To test whether the relative topology of the protomers inGpA-EmrE is as proposed, we assessed accessibility to imper-meant thiol reagents of single Cys residues engineered in Cys-less GpA-EmrE. As a control we used EmrE mutants with asingle residue engineered at positions previously shown asaccessible (K22C) or inaccessible (H110C) (4). After challeng-ing the cells with the impermeant MTSES reagent at the indi-cated concentrations, the membranes were prepared, and theproteins were purified. To assess reactivity with MTSES, freethiols were reacted after solubilization and denaturation withN-ethylmaleimide-fluorescein, and the fluorescence was esti-mated after SDS-PAGE. As previously shown, in EmrE, theaccessible residue K22C reacts with MTSES in intact cells, andtherefore only a small fraction (� 25%) of unreacted thiols isdetected (Fig. 6A). On the other hand when a mutant bearingresidue H110C is used, most (�80%) of the Cys residues areunreacted (Fig. 6A). The GpA-EmrEmutants used bear a singleCys residue engineered in the first loop (K22C) of either the firstor the second monomer. The K22C in the second monomer ispractically fully accessible to the impermeant MTSES reagentbecause only a small fraction (�15%) of unreacted thiols isdetected after exposure (Fig. 6B). On the other hand K22C inthe first monomer is only partly accessible, suggesting that themajority of the protein is organized with the first monomer in aNout configuration and the second in a Cin (Fig. 6B).

FIGURE 4. A, schematic representation of GpA-EmrE, an EmrE tandem withnine transmembrane helices. GpA-EmrE is shown schematically; transmem-brane helices are drawn as boxes. The N terminus of the second EmrE pro-tomer was connected to the C terminus of the first EmrE protomer with alinker derived from a mutant of human GpA (dark box) that does not dimerize(28) and six additional hydrophilic amino acids as described under “Experi-mental Procedures.” The Myc-His tag is fused to the C terminus of the secondmonomer. B, growth phenotype of cells expressing GpA-EmrE. E. coliBW25113 �emrE�mdfA cells transformed with either pT7-7-EmrE (EmrE),pT7-7 (vector), or pT7-7-GpA-EmrE (GpA-EmrE) were grown overnight at37 °C in LB medium with ampicillin. 5 �l of serial dilutions of the culture werespotted on the LB plates containing 30 mM BisTris propane, pH 7.0, with orwithout 100 �g/ml ethidium, 25 �g/ml acriflavine, or 0.3 mM methyl viologen.Growth was analyzed after overnight incubation at 37 °C.

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DISCUSSION

The results described in this paper document a unique plas-ticity in the interaction of protomers within the EmrE dimerand of the dimer relative to the membrane. Thus, dimers caninsert with either Nin-Cin or Nout-Cout topology andmonomers

that usually interact in a parallelmode can be forced to interact pro-ductively in an antiparallel mode. Inaddition, in the heterodimer EbrAB,EbrB can interact productively withEbrA or with itself.It was previously proposed that

antiparallel topology could be ob-tained by co-expression of inactiveEmrE mutants where the (K�R)bias of the EmrE protomers waschanged in opposite directions bymutagenesis (15). This conclusionnecessitates further experimentalsupport because the reassessmentof the EmrE mutants that weredesigned to insert into the mem-brane with the Nin-Cin or Nout-Couttopology shows that they are bothfunctional and capable of removingsubstrate from the cell. A similarconclusion was reached whenexpressing the same mutants inE. coli BL21 IPTG-induced cells andfollowing growth continuously inliquid medium (24). The resultsdescribed here emphasize the needfor caution in the analysis of nega-tive phenotypes. We have shownpreviously examples of negativephenotypes even though the proteinexpressed is active (31). The abilityof cells to carry a given function (inthis case growth in a toxic environ-ment) depends on the levels of theexpressed protein, its affinity to thesubstrate, and the rate at whichthe substrate can be removed rela-tive to the leaks, parameters thatmay well differ from strain to strain(25). Furthermore, the activity ofother transporters that share thesame substrate and the presence ofproteins that interfere with thefunction may confuse interpreta-tion (25). Herewe also show that themutants interact with the wild typeprotein produced by the chromo-somal emrE gene in a nonproduc-tive interaction that causes confu-sion in the interpretation of thephenotypes.In the SMR family of multidrug

transporters homodimers (e.g. EmrE, Hsmr, BPsmr, PAsmr,andTBsmr) and heterodimers (e.g. EbrAB) have been identified(26, 27, 32, 33). Membrane protein topology is largely governedby the positive-inside rule, i.e. loops rich in Lys andArg residuestend to orient toward the cytoplasm (34). Analysis of the (K�R)

FIGURE 5. A, GpA-EmrE does not take part in the intermolecular interactions with other EmrE molecules.Negative dominance of an inactive mutant (EmrE E14C) on EmrE activity and the lack of effect on GpA-EmrEactivity were demonstrated as follows. Increasing amounts of membranes containing the indicated amountsof inactive EmrE mutant E14C were solubilized in 1% dodecyl-�-D-maltoside/sodium buffer and mixed withsolubilized membranes containing 40 ng of EmrE (F) or GpA-EmrE (Œ). After incubation at 80 °C (10 min), themixture was transferred to 4 °C and assayed for [3H]TPP� binding as described under “Experimental Proce-dures.” B, probing the packing of GpA-EmrE. Membranes bearing 35S-radiolabeled GpA-EmrE (lanes 1– 4) andE22EMH (lanes 5– 8) were incubated with the corresponding proteases (T, trypsin; C, chymotrypsin; PK, protein-ase K) as described in Ref. 9. A cartoon is shown indicating the proteolysis sites of the different enzymes:E22EMH is digested by chymotrypsin (after Phe112 in the linker after monomer 1 and the corresponding residueafter monomer 2) and trypsin (after Lys119 as above) to produce two polypeptides with a similar molecular mass(11–12-kDa apparent mass in SDS-PAGE) as a result of the digestion of the linker. Proteinase K digests all thelinker and the tag. Under the conditions used, the released tag was not detectable; only a minor uncut andtag-less fraction of E22EMH is detectable. In the case of GpA-EmrE only the tag is digested producing apolypeptide of �22–24 kDa. The very minor low molecular weight bands observed are present also in theabsence of added proteases and may represent small amounts of unrelated proteins.

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bias suggests a very low bias for the homodimers and an anti-parallel arrangement of the protomers in the heterodimers (14).EmrE and EbrAB confer resistance to ethidium and, therefore,SMR homodimers and heterodimers with a supposed differentrelative topology of the protomers perform very similar, if notidentical, functions. Moreover, modification of the (K�R) biasof the heterodimer EbrAB has resulted in mutants of EbrA andEbrB that can function as homodimers (35). In other words, anumber ofmutations introduced by designmay revert the traitsacquired during evolution and change a heterodimer to a func-tional homodimer. Here we show that evenwithout any furthermutations, EbrB is capable of interacting with itself, providedthe chromosomal gene coding for EmrE is inactivated. Thefindings are in agreementwith amodel where EbrB can interactwith EbrA, EmrE, and itself in decreasing order of affinities. Theinteractionwith EmrE is a nonproductive one thatmay result inthe formation of a heterodimerwith low or no activity.We havepreviously shown that EmrE can interact with a variety ofhomologues forming heterodimers with various affinities (32).Antiparallel topology of a homodimer presents many

intriguing implications regarding biogenesis and insertion. Itwould be expected that topology determinants in identical pro-tomers would dictate a similar mode of insertion, resulting in aparallel arrangement. If a special mechanism that can direct

insertion only of antiparallel homo-dimers exists, it is unprecedentedand remains to be recognized. In thecase of a protein with ambiguoustopology determinants, one couldexpect a mixed population ofdimers with parallel (a fraction withNin-Cin as well as a fraction withNout-Cout topology) and antiparallelprotomers. However, even with arandom insertion, the proportion ofeach species would be determinedby the relative affinity of interaction.With the tools we have developed,wewere able to detect dual topologydimers but not antiparallel ones.Wesuggest that in the case of nativeEmrE, the parallel interaction is pre-ferred, but it can be imposed byproper manipulations as shownhere or by mutations accumulatedduring evolution. Usually it isassumed that a very accurate designof protein-protein interaction isessential for the function of an olig-omeric protein. However, proteindomain promiscuity seems to play amajor role in the creation and mod-ulation of molecular functionality,especially for signal transduction.Such domains connect componentsof signal transduction networksthrough specific protein-proteininteractions and delivering effectors

to the sites of their action (36, 37). In other cases, the composi-tion of oligomeric channels has been shown to be dependent onthe specific cell types with the same subunits able to generatehomo-oligomers as well as hetero-oligomers of varying compo-sition (38).We propose that the plasticity described here could provide

an example of how inverted repeats in modern large polytopictransporters may have developed. The interaction interfacesmust be appropriate for generation of a stable oligomer, and asubstrate binding cavitymust supply interaction points at givenand fixed locations. We suggest that the promiscuity amonginteracting protomers reported here is tolerated also function-ally because in EmrE, as in other proteins interacting with mul-tiple substrates, the size of the binding pocket must be largeenough to allow different molecules to reside in it in differentorientations and to establish interactions with different sets ofresidues on the pocket walls (39–41). Therefore, the minimalstructure where we find the necessary elements for substraterecognition and coupling may be provided by a dimer with theprotomers in either one of the possible topologies.In light of the plasticity described here, it will be important to

describe experiments to test the proposed models of EmrEstructure (10, 13). Both models have been obtained from lowresolution data, and in the case of the x-ray data derivedmodel,

FIGURE 6. Topology of GpA-EmrE. BW25113 �emrE�mdfA cells bearing (A) EmrE K22C (�) and EmrE H110C(●) or (B) GpA-EmrE K22C in the first (f) or the second (Œ) monomer were treated with MTSES at the indicatedconcentrations, and the unreacted thiols were estimated from the degree of reaction with N-ethylmaleimide-fluorescein as described under “Experimental Procedures.” A simplified flow chart is shown in the right panel.

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the crystals were obtained under conditions that inactivatethe protein because the detergents used disturb dimerization(8, 13).An additional aspect that contributes to the plasticity

described in this report stems from the generally accepted func-tional symmetry of channels, transporters, and ion-coupledtransporters where the direction of transport is dictated by thedirection of the driving force acting on the transported sub-strates. Nevertheless, this is the first demonstration that thedirectionality of insertion of a transporter in vivo can be ran-dom without affecting activity. We suggest that this is possiblebecause of the simplicity of the coupling mechanism of EmrEthat allows also for the flexibility needed to allow function in aparallel as well as in an antiparallel dimer. In the proposed cat-alytic mechanism of EmrE, the binding sites of substrate andprotons overlap, and occupancy is mutually exclusive. In otherwords, either molecule binds to the transporter only when thebinding site is not occupied by the other and induces a shift inthe equilibrium. The suggested mechanism necessitates thefine-tuning of the pKa of the glutamyl carboxyls at position 14,and indeed this pKa was estimated as between 7.5 to 8.0,depending on the technique used (29). In a protein with an Aspreplacement that displays a lower pKa (5.8–6.3), coupling ofthe two fluxes is impaired (42). Aromatic residues from threeTMs have also been identified as playing a role in proton andsubstrate binding (31, 43). We speculate that in the absence ofsubstrates, the carboxyls of Glu14, located approximately in themiddle of the binding cavity, are stabilized by interaction withprotons orwith at least part of aromatic residues contributed byTM1, TM2, and TM3. The aromatic residues provide an envi-ronment that may explain the unusually high pKa of these car-boxyls (44) and allow for interaction with the hydrophobic sub-strates as has been documented in other proteins that bindsimilar substrates (45, 46). Such a mechanism would be ratherinsensitive to the specific nature and distribution of the aro-matic residues around the carboxyls and could therefore pro-vide the necessary stepping stone for the evolution of the topo-logical inverted repeats observed in modern transporters.The evolutionary challenge of recognition and transport of a

wide spectrum of substrates may have selected for SMR het-erodimers that originated from gene duplication of the moreancient homodimers. After gene duplication, a relatively smallnumber of mutations in the hydrophilic domains may be suffi-cient to convert homodimers into heterodimers and vice versa.In this manner, one protein with an only slightly modifiedsequence may extend the range of the substrate specificity (5).The number of new combinations (and therefore new andwider spectrum of substrates) possible with heterodimers isundoubtedly higher than one can obtain with a homodimer (5).Topology evolution of larger proteins can now be envisagedstarting from the SMR heterodimers, which can then fuse andgive rise to larger proteins, some of them such as LacY, GlpT,and AcrB (39, 40, 47, 48) with domains in parallel orientationand others such as aquaporins, ClC channel, and a number oftransporters with two oppositely oriented membrane domains(2, 3, 49–51).

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Iris Nasie, Sonia Steiner-Mordoch, Ayala Gold and Shimon SchuldinerInverted Repeats in Ion-coupled Transporters

Topologically Random Insertion of EmrE Supports a Pathway for Evolution of

doi: 10.1074/jbc.M110.108746 originally published online March 22, 20102010, 285:15234-15244.J. Biol. Chem. 

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