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In Vitro Monomer Swapping in EmrE, a Multidrug Transporter from Escherichia coli, Reveals That the Oligomer Is the Functional Unit*

  • Dvir Rotem
    Affiliations
    Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel
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  • Neta Sal-man
    Affiliations
    Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel
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  • Shimon Schuldiner
    Correspondence
    To whom correspondence should be addressed: Tel.: 972-2-6585992; Fax: 972-2-5634625
    Affiliations
    Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel
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  • Author Footnotes
    * This work was supported by grants from the Deutsche-Israeli Program (Federal Ministry of Education, Science and Research-BMBF-International Bureau at the German Aerospace Center Technology), Grant NS16708 from the National Institutes of Health, and Grant 463/00 from the Israel Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:December 21, 2001DOI:https://doi.org/10.1074/jbc.M108229200
      EmrE is a small multidrug transporter, 110 amino acids long that extrudes various drugs in exchange with protons, thereby rendering Escherichia coli cells resistant to these compounds. Negative dominance studies and radiolabeled substrate-binding studies suggested that EmrE functions as an oligomer. Projection structure of two-dimensional crystals of the protein revealed an asymmetric dimer. To identify the functional unit of EmrE, a novel approach was developed. In this method, quantitative monomer swapping is induced in detergent-solubilized EmrE by exposure to 80 °C, a treatment that does not impair transport activity. Oligomer formation is highly specific as judged by several criteria, among them the fact that 35S-EmrE can be “pulled out” from a mixture prepared from generally labeled cells. Using this technique, we show that inactive mutant subunits are functionally complemented when mixed with wild type subunits. The hetero-oligomers thus formed display a decreased affinity to substrates. In addition, sulfhydryl reagents inhibit the above hetero-oligomer even though Cys residues are present only in the inactive monomer. It is concluded that, in EmrE, the oligomer is the functional unit.
      TPP+
      tetraphenylphosphonium
      35S-EmrE
      EmrE labeled with [35S]methionine
      DM
      n-dodecyl-β-maltoside
      PAGE
      polyacrylamide gel electrophoresis
      NEM
      N-ethylmaleimide
      EmrE-His
      EmrE fused to Myc/His epitope
      Transporters are responsible for creating and maintaining the different composition of the cell interior relative to the exterior in both prokaryotic and eukaryotic cells. This is also the case for the solutes' gradients across internal organelles. Their functioning is therefore highly relevant to maintenance of proper cell homeostasis, and they are targets of action of many drugs. In many cases, they are also responsible for failure of treatment of tumors and infectious diseases because of transporter-mediated multiple drug resistance (
      • Nikaido H.
      ,
      • Gottesman M.M.
      • Pastan I.
      ).
      The multidrug transporter EmrE, a protein from Escherichia coli, provides a unique experimental paradigm for the study of these transporters (
      • Schuldiner S.
      • Granot D.
      • Mordoch S.S.
      • Ninio S.
      • Rotem D.
      • Soskin M.
      • Tate C.G.
      • Yerushalmi H.
      ,
      • Schuldiner S.
      • Granot D.
      • Steiner S.
      • Ninio S.
      • Rotem D.
      • Soskin M.
      • Yerushalmi H.
      ). It is a small multidrug transporter, 110 amino acids long, that extrudes various drugs in exchange with protons, thereby rendering bacteria resistant to these compounds (
      • Schuldiner S.
      • Granot D.
      • Mordoch S.S.
      • Ninio S.
      • Rotem D.
      • Soskin M.
      • Tate C.G.
      • Yerushalmi H.
      ,
      • Schuldiner S.
      • Granot D.
      • Steiner S.
      • Ninio S.
      • Rotem D.
      • Soskin M.
      • Yerushalmi H.
      ). The protein has been characterized, purified, and reconstituted in a functional form (
      • Yerushalmi H.
      • Lebendiker M.
      • Schuldiner S.
      ). Hydropathic analysis of the EmrE sequence predicts four α-helical transmembrane segments. This model is experimentally supported by Fourier transform infrared spectroscopy studies that confirm the high α-helicity of the protein and by high resolution heteronuclear NMR analysis of the protein structure (
      • Arkin I.
      • Russ W.
      • Lebendiker M.
      • Schuldiner S.
      ,
      • Schwaiger M.
      • Lebendiker M.
      • Yerushalmi H.
      • Coles M.
      • Groger A.
      • Schwarz C.
      • Schuldiner S.
      • Kessler H.
      ). The transmembrane segments of EmrE are tightly packed in the membrane without any continuous aqueous domain, as was shown by cysteine scanning experiments (
      • Steiner Mordoch S.
      • Granot D.
      • Lebendiker M.
      • Schuldiner S.
      ). These results suggest the existence of a hydrophobic pathway through which the substrates are translocated. EmrE has only one membrane-embedded charged residue, Glu-14, which is conserved in more than 50 homologous proteins and was shown to be part of a binding site common to protons and substrates (
      • Ninio S.
      • Rotem D.
      • Schuldiner S.
      ). The occupancy of this site by H+ and substrate is mutually exclusive and provides the basis of the simplest coupling for two fluxes (
      • Yerushalmi H.
      • Schuldiner S.
      ,
      • Yerushalmi H.
      • Schuldiner S.
      ).
      In vivo and in vitro negative dominance studies have been performed to examine the oligomeric state of the protein (
      • Yerushalmi H.
      • Lebendiker M.
      • Schuldiner S.
      ). Co-expression of wild type and non-functional mutants of EmrE resulted in a reduction in the resistance conferred by the transporter. In addition, co-reconstitution of purified non-functional mutants of EmrE with wild type EmrE in proteoliposomes inhibited the wild type transport activity in a dose-dependent manner (
      • Yerushalmi H.
      • Lebendiker M.
      • Schuldiner S.
      ). The results suggested that this inhibition is due to the formation of mixed oligomers in which the presence of nonfunctional subunits cause inactivation. The oligomeric nature of EmrE is further supported by the finding that detergent-solubilized purified EmrE binds between 0.25 and 0.3 mol of the substrate TPP+1 per mol of protein. These data suggest that an oligomeric EmrE complex may form a single TPP+-binding pocket (
      • Muth T.R.
      • Schuldiner S.
      ). Moreover, EmrE was crystallized in two dimensions, and the projection structure reveals an asymmetric dimer (
      • Tate C.G.
      • Kunji E.R.S.
      • Lebendiker M.
      • Schuldiner S.
      ).
      To further study the oligomeric nature of EmrE, a novel approach was developed. In this method, quantitative monomer swapping is induced in detergent-solubilized EmrE by exposure to 80 °C, a treatment that does not impair transport activity. Oligomer formation is highly specific, as judged by several criteria, among them the fact that the35S-EmrE can be pulled out from a mixture prepared from generally labeled cells. Inactive mutant subunits are functionally complemented when mixed with wild type subunits. In addition, sulfhydryl reagents inhibit the above hetero-oligomer even though Cys residues are present only in the inactive monomer. It is concluded that, in EmrE, the oligomer is the functional unit.

      DISCUSSION

      The oligomeric state of ion-coupled transporters has been investigated in a number of cases. The approaches used are based on analysis of the particle size of the protein in the membrane (
      • Friesen R.H.
      • Knol J.
      • Poolman B.
      ,
      • Eskandari S.
      • Kreman M.
      • Kavanaugh M.P.
      • Wright E.M.
      • Zampighi G.A.
      ) or on detergent-solubilized preparations (see for example Refs.
      • Schroers A.
      • Burkovski A.
      • Wohlrab H.
      • Kramer R.
      and
      • Kilic F.
      • Rudnick G.
      ). Evidence for oligomer formation has been presented in many instances, and the functional relevance of the oligomerization has been documented in a few cases (
      • Gerchman Y.
      • Rimon A.
      • Venturi M.
      • Padan E.
      ,
      • Schroers A.
      • Burkovski A.
      • Wohlrab H.
      • Kramer R.
      ,
      • Kilic F.
      • Rudnick G.
      ,
      • Veenhoff L.M.
      • Heuberger E.H.
      • Poolman B.
      ).
      The results presented here describe a novel in vitro system for the study of oligomerization and its functional implications. EmrE oligomers are stable in the detergent-solubilized preparation and dissociate only after exposure to high temperature or strong denaturing reagents such as the detergent SDS. An experimental protocol for generating mixed oligomers was designed based on an initial dissociation step induced by exposure to high temperature for short periods and random and rapid association of the monomers in solution at a lower temperature. The exposure to high temperature had little effect on either substrate binding or ΔpH-driven uptake in proteoliposomes. EmrE provides, therefore, a unique experimental paradigm not only because of its size but also because of its stability to denaturing agents such as temperature, organic solvents (
      • Yerushalmi H.
      • Lebendiker M.
      • Schuldiner S.
      ), and SDS-urea (
      • Schuldiner S.
      • Granot D.
      • Mordoch S.S.
      • Ninio S.
      • Rotem D.
      • Soskin M.
      • Tate C.G.
      • Yerushalmi H.
      ).
      The results demonstrate that EmrE forms homo-oligomers since35S-EmrE binds to Ni-NTA beads only through formation of oligomers with EmrE-His. No other proteins seem to be required for this process because oligomerization is detected also when purified EmrE-His is used. In addition, competition experiments were performed with unlabeled untagged wild type EmrE showing an apparent affinity of interaction in the 100–200 nm range. Oligomer formation appears to be exquisitely specific as no formation is detected with unrelated tagged membrane proteins such as NhaA-His and even with other Smr proteins from E. coli that show a distinct homology to EmrE. The lack of interaction of EmrE with other Smr proteins fromE. coli supports the contention that EmrE is functionalin vivo only as a homo-oligomer. In the case of Smr proteins from other organisms, it has been suggested that they can function as hetero-oligomers based on a synergistic effect on resistance phenotype when two proteins are co-expressed (
      • Lee A.
      • Mao W.
      • Warren M.S.
      • Mistry A.
      • Hoshino K.
      • Okumura R.
      • Ishida H.
      • Lomovskaya O.
      ,
      • Jack D.L.
      • Storms M.L.
      • Tchieu J.H.
      • Paulsen I.T.
      • Saier Jr., M.H.
      ).
      We have recently shown that the basic oligomeric structure detected in two-dimensional crystals of EmrE is a dimer (
      • Tate C.G.
      • Kunji E.R.S.
      • Lebendiker M.
      • Schuldiner S.
      ), but it is possible that the functional unit of the protein is a higher degree oligomer formed by two or more dimers. Previous evidence from negative dominance experiments and from ligand binding measurement to purified EmrE were consistent with a trimeric structure but did not rule out the possibility that EmrE functions as a dimer or a tetramer (
      • Yerushalmi H.
      • Lebendiker M.
      • Schuldiner S.
      ,
      • Muth T.R.
      • Schuldiner S.
      ). The experiments described here demonstrate that in the mixed oligomers the inhibitory effect of E14C is on the substrate binding site. The kinetic analysis of this effect revealed three distinct species based on their binding properties: one similar to wild type, one with a 20-fold lower affinity, and a nonfunctional one. These findings suggest that hetero-oligomers with one Cys residue at position 14 bind TPP+ with different kinetic properties. To further characterize this population and to try to detect hetero-oligomers with potentially different affinities, we designed a more sensitive protocol. In these experiments, the tagged protein is the inactive E14C mutant, while the untagged is the wild type. The functional complementation detected reveals only one functional species with a low affinity identical to the one measured in the negative dominance experiments. In our most recent model, it was suggested that the carboxyl moieties at position 14 are in close proximity and form a charge cluster (
      • Yerushalmi H.
      • Schuldiner S.
      ,
      • Yerushalmi H.
      • Schuldiner S.
      ), the negative charge being essential for substrate binding. The results described above suggest that replacing one of the charges in this site has a marked effect on the affinity toward the substrate, while replacing more than one, yields a non-functional protein.
      That the binding site is contributed by each of the subunits was demonstrated also by experiments in which a hetero-oligomer with Cys residues only in the inactive monomer was challenged with NEM, a sulfhydryl reagent. NEM inhibited the activity of this hetero-oligomer even though it does not have any effect on the activity of the wild type. Therefore, the inhibitory effect described must be due to an interaction with the Cys residue at position 14 in the inactive subunit. NEM inhibits the activity by lowering even further the affinity (90 nm), and it may be doing so by modifying the environment around the cluster. This novel finding allows insertion of various compounds to further probe the binding site.
      The novel method described here for generation of mixed oligomers has provided significant information on the functional size of the protein. It also allows generation of hetero-oligomers with desired compositions, and it could be of general use with other proteins as well. In addition, as was shown here, we have developed an unexpected means to gain access to the vicinity of the binding site.

      Acknowledgments

      We thank Michal Sharoni for performing some of the experiments.

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