Parallel topology of genetically fused EmrE homodimers
2007; Springer Nature; Volume: 27; Issue: 1 Linguagem: Inglês
10.1038/sj.emboj.7601951
ISSN1460-2075
AutoresSonia Steiner‐Mordoch, Misha Soskine, Dalia Solomon, Dvir Rotem, Ayala Gold, Michal Yechieli, Yoav Adam, Shimon Schuldiner,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle6 December 2007free access Parallel topology of genetically fused EmrE homodimers Sonia Steiner-Mordoch Sonia Steiner-Mordoch Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Misha Soskine Misha Soskine Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Dalia Solomon Dalia Solomon Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Dvir Rotem Dvir Rotem Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, IsraelPresent address: Department of Chemistry, Oxford University, Oxford, UK Search for more papers by this author Ayala Gold Ayala Gold Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Michal Yechieli Michal Yechieli Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Yoav Adam Yoav Adam Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Shimon Schuldiner Corresponding Author Shimon Schuldiner Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Sonia Steiner-Mordoch Sonia Steiner-Mordoch Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Misha Soskine Misha Soskine Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Dalia Solomon Dalia Solomon Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Dvir Rotem Dvir Rotem Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, IsraelPresent address: Department of Chemistry, Oxford University, Oxford, UK Search for more papers by this author Ayala Gold Ayala Gold Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Michal Yechieli Michal Yechieli Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Yoav Adam Yoav Adam Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Shimon Schuldiner Corresponding Author Shimon Schuldiner Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Author Information Sonia Steiner-Mordoch1,‡, Misha Soskine1,‡, Dalia Solomon1, Dvir Rotem1, Ayala Gold1, Michal Yechieli1, Yoav Adam1 and Shimon Schuldiner 1 1Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel ‡These authors contributed equally to this work *Corresponding author. Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Silberman Bldg 3-345, Jerusalem, Givat Ram 91904, Israel. Tel.: +972 2 658 5992; Fax: +972 2 563 4625; E-mail: [email protected] The EMBO Journal (2008)27:17-26https://doi.org/10.1038/sj.emboj.7601951 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info EmrE is a small H+-coupled multidrug transporter in Escherichia coli. Claims have been made for an antiparallel topology of this homodimeric protein. However, our own biochemical studies performed with detergent-solubilized purified protein support a parallel topology of the protomers. We developed an alternative approach to constrain the relative topology of the protomers within the dimer so that their activity can be assayed also in vivo before biochemical handling. Tandem EmrE was built with two identical monomers genetically fused tail to head (C-terminus of the first to N-terminus of the second monomer) with hydrophilic linkers of varying length. All the constructs conferred resistance to ethidium by actively removing it from the cytoplasm. The purified proteins bound substrate and transported methyl viologen into proteoliposomes by a proton-dependent mechanism. A tandem where one of the essential glutamates was replaced with glutamine transported only monovalent substrates and displayed a modified stoichiometry. The results support a parallel topology of the protomers in the functional dimer. The implications regarding insertion and evolution of membrane proteins are discussed. Introduction EmrE belongs to the small multidrug resistance (SMR) family (Paulsen et al, 1996; Bay et al, 2007) of multidrug transporters and is a unique experimental paradigm for biochemical and biophysical studies of membrane-based, ion-coupled transporters due to its size, stability and its retention of function when solubilized in detergent (Yerushalmi et al, 1995; Yerushalmi and Schuldiner, 2000b; Tate et al, 2001). Study of this small, 110-residue multidrug transporter of Escherichia coli has provided valuable information for the understanding of the coupling mechanism of ion-coupled transporters (Yerushalmi and Schuldiner, 2000a, 2000b; Soskine et al, 2004; Adam et al, 2007). However, the structural information that became available for this protein in recent years has been in conflict with the existing biochemical knowledge (Ma and Chang, 2004; Pornillos et al, 2005). Even though the two X-ray crystallography papers for the protein have recently been retracted (Chang et al, 2006), their publication sparked a controversy regarding the relative topology of the protomers in the functional dimer and this controversy is still ongoing (Fleishman et al, 2006; Soskine et al, 2006; Rapp et al, 2007). The claim for an antiparallel topology was supported by a reinterpretation of the electron density maps of 2D crystals of EmrE that showed that parts of the structure are related by quasi-symmetry (Ubarretxena-Belandia et al, 2003). A Cα-model of the transmembrane region was constructed by considering the evolutionary conservation pattern of each helix (Fleishman et al, 2006). Much of the biochemical data on EmrE seem to agree with this model. Von Heijne and co-workers designed experiments to support the concept of dual topology. They showed that the topology of the EmrE fusion proteins in the membrane is sensitive to the distribution of positive charges in the protein (Rapp et al, 2006). In addition, manipulation of the positive charges generates a set of mutants, some with Cout, others with Cin apparent topology (Rapp et al, 2006, 2007). The Cin and Cout mutants, bearing three mutations each, do not confer resistance to ethidium. The authors conclude that this is due to the modified topology. However, it was not yet tested whether the introduction of positive charges may affect homodimerization or recognition of the positive substrates. Coexpression of the inactive mutants restores the ethidium bromide resistance phenotype to the same level as seen for wild-type EmrE (Rapp et al, 2007). A possible interpretation of this finding is that coexpression results in the generation of a functional, antiparallel heterodimer. However, a more direct biochemical analysis of the mutants is needed to support it. In addition, six mutations (three in each monomer) may have indeed pushed EmrE to form a hetero-oligomer. Whether the protein that has not been mutated can form functional homodimers with an antiparallel topology still remains to be directly demonstrated. Moreover, the existence of homodimers with antiparallel orientation would pose a problem for the insertion machinery of membrane proteins. Identical protomers with Cout and Cin topologies would insert exactly at a 1:1 ratio to prevent the existence of unpaired units. How this ratio is controlled and how the assembly of such a dimer is achieved is very difficult to envisage with our present knowledge. Since an apparent antiparallel topology of a homodimer has many intriguing implications regarding biogenesis, insertion and evolution of ion-coupled transporters, the topic has already attracted much attention (Bowie, 2006; Pornillos and Chang, 2006; Rapp et al, 2006). Our own biochemical studies support a symmetrical relationship between the monomers in the EmrE homodimer (reviewed in Schuldiner, 2007). These studies were performed with the protein after solubilization and purification. To provide an approach that combines in vivo and in vitro studies, we designed a series of genetic fusions where the monomers are connected tail to head (C-terminus of the first to N-terminus of the second monomer) by means of defined linkers that are not compatible with an antiparallel topology either because they are too hydrophilic or too short (Figure 1). The fused dimers (tandems) confer resistance to ethidium by actively removing it from the cytoplasm. The purified proteins bound the high-affinity substrate tetraphenylphosphonium (TPP+) and upon reconstitution transported methyl viologen (MV2+) into proteoliposomes by a proton-dependent mechanism. The contention that the tandem is the functional unit is supported by negative dominance studies. In these studies we also show that a single mutation changes the stoichiometry of the transporter and its ability to recognize divalent substrates. Figure 1.Design of the EmrE tandems. Construction of EmrE to EmrE fusions is shown schematically; location of essential carboxyl residue E14 is shown; transmembrane helices are drawn as boxes. The N-terminus of the second EmrE protomer is two amino acids long (NP, the first methionine was removed) and was connected to the C-terminus (RSTPH) of the first EmrE protomer with a variety of linker sequences (displayed above). Linker sequences were chosen in a manner that enforces the parallel orientation of the two EmrE protomers (with respect to the membrane plane) within each tandem polypeptide chain. This was achieved either by very hydrophilic linker (E22EMH) or by linkers that are too short to cross the membrane plane (E2EMH, E4EMH, E6EMH). A ‘negatively dominated’ tandem was built using the six-amino-acid linker and substituting the essential glutamate 14 in the first monomer with glutamine (Q6EMH). Download figure Download PowerPoint Results Genetically fused dimers of EmrE (Tandem EmrE) confer resistance to ethidium by actively removing it from the cytoplasm Tandem EmrE was constructed by genetic fusion of the monomers tail to head with linkers of various lengths (Figure 1). The N-terminus of the second EmrE protomer is two amino acids long (NP, the first methionine was removed) and was connected to the C-terminus (RSTPH) of the first EmrE protomer with a variety of linker sequences (Figure 1). Linker sequences were chosen in a manner that enforces the parallel orientation of the two EmrE protomers (with respect to the membrane plane) within each tandem polypeptide chain. This was achieved either by very hydrophilic linker (E22EMH) or by linkers that are too short to cross the membrane plane (E2EMH, E4EMH, E6EMH). The activity of each of the tandem proteins has been tested both in vivo and in vitro. In vivo the resistance conferred by each of the proteins was assessed by testing the ability of cells expressing them to grow under otherwise non-permissive conditions. This was achieved in solid media containing ethidium (100 μg/ml) in which 5 μl of logarithmic dilutions of an overnight culture were spotted. Cells carrying the vector plasmid without any insert cannot grow in this medium at any of the dilutions tested (Figure 2A, pT7-7). Cells expressing either EmrE or the various tandems were able to grow at each of the dilutions. While all the tandems conferred resistance to ethidium, those with shorter linkers were slightly less effective. At the high dilutions, the single colonies observed displayed a similar size indicating similar growth rates (Figure 2A). As expected, all the cells grew to a similar degree in control plates containing only ampicillin (Figure 2A, control). Figure 2.EmrE tandems are functional transporters in vivo. (A) Growth phenotype of cells expressing tandems. E. coli DH5α cells transformed with either pT7-7-EmrE (EmrE), pT7-7 (vector) or with the indicated tandems were grown overnight at 37°C in LB–ampicillin medium. A 5-μl volume of 10−3–10−6 dilutions of the culture was spotted on the LB–ampicillin plates containing 30 mM BisTris propane, pH 7.0, with or without 100 μg/ml ethidium bromide. Growth was analyzed after overnight incubation at 37°C. (B) Ethidium efflux activity of the tandems. E. coli HMS 174 cells harboring the plasmid pT7-7 containing EmrE or tandems with varying linker length, or pT7-7 alone were grown in minimal medium A to mid-logarithmic phase and then induced with 0.5 mM IPTG for 2 h. The cells were then transferred to minimal medium containing ethidium bromide and CCCP, as described under Materials and methods. After 60 min of incubation at 37°C, the cells were washed quickly in CCCP-free medium and monitored for fluorescence (excitation at 525 nm, emission at 585 nm). Glucose (20 mM) was added to initiate the active efflux of ethidium. (C) Comparison of the expression levels of the EmrE and tandems in HMS 174 cells after induction. His-tagged proteins were purified as described under Materials and methods and analyzed by SDS–PAGE. Download figure Download PowerPoint To further evaluate the ability of the various constructs to actively remove ethidium from the cytoplasm, we assayed active transport of ethidium in intact cells. In these studies, after induction of expression of the various constructs, cells were starved in the presence of ethidium by incubation in the absence of an energy source and in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). After removal of the uncoupler, transport was started by addition of glucose. Although ethidium is present both in the medium and the cells, the fluorescence observed originates almost exclusively from the intracellular ethidium bound to nucleic acid. Upon addition of glucose, a quick decrease in the fluorescence is observed, which represents removal of ethidium from the cell against its concentration gradient (Figure 2B). This decrease was completely prevented when the cells were resuspended in a medium with CCCP (not shown). The rate of extrusion in the cells bearing wild-type EmrE or any of the four tandems tested was very similar and reached very similar equilibrium after about 2 min. In cells transformed with empty vector, removal of ethidium is much slower and is most likely mediated by multidrug transporters other than EmrE. Expression levels of the tandems were assessed after purification and were found to be very similar (Figure 2C). No proteolytic products were detectable and a visible impurity in all cells is due to the metal-binding protein YodA (David et al, 2002, 2003). Taken as a whole the results described demonstrate that all the tandems are capable of active extrusion of ethidium. Tandem EmrE binds the high-affinity substrate TPP+ and transports MV2+ into proteoliposomes The tandem proteins were purified in order to further characterize their activity and to rule out the possibility that the transport observed in vivo is due to proteolytic products. The results of the binding experiments are summarized in Figure 3A. All the EmrE tandem fusions bound TPP+, although to somewhat lesser degrees than the wild-type EmrE due to some decrease of the affinities from 2 to 4–7 nM (Figure 3C). Tandem EmrE contains two essential glutamates (one in each protomer) that correspond to the membrane-embedded glutamate 14 in wild-type EmrE. EmrE tandem where one of the essential glutamates was substituted by glutamine (Q6EMH) displays a profoundly lower binding affinity to TPP+ (59 nM; Figure 3C). The results are in agreement with previous findings where it was shown that both Glu14 within the EmrE dimer are required for assembly of the high-affinity substrate-binding site (Rotem et al, 2001). Figure 3.Substrate binding and transport activity of the tandems. (A) [3H]TPP+-binding activity of the purified tandems. EmrE and tandems were purified as described under Materials and methods and assayed for the ability to bind the high-affinity substrate TPP+ with 2.5 nM of [3H]TPP+ in 0.08% DDM Na buffer. (B) [14C]MV2+ uptake activity of the reconstituted tandems. EmrE (filled circles), E22EMH (filled diamonds), E6EMH (hollow squares), E4EMH (hollow circles) and Q6EMH (hollow diamonds) were purified, reconstituted and assayed as described under Materials and methods. (C) TPP+ binding affinities of the tandems used in this study, assayed by [3H]TPP+ binding and determined as previously described (Rotem et al, 2001). Download figure Download PowerPoint To further test the functionality of the purified tandem proteins, we reconstituted them into proteoliposomes and assayed their ability to catalyze proton gradient-driven uptake of MV2+. Upon generation of a pH gradient, the rates of uptake and the level of accumulation of MV2+ detected in proteoliposomes reconstituted with either one of the ‘wild-type’ tandems were very similar to each other and even slightly higher than the rates observed for wild-type EmrE proteoliposomes (Figure 3B). These findings allow us to conclude that tandem EmrE is a functional transporter. Probing packing and topology of the protomers in tandem EmrE The linkers were designed to be either short enough or hydrophilic enough in order not to cross the membrane plane (Figure 1). The results described above demonstrate that the linkers have no significant effect on the activity of the protein and therefore most likely do not affect the overall packing of the dimer, a prerequisite for function. However, since manipulation of charge bias (Gafvelin and von Heijne, 1994) or lipid composition (Bogdanov et al, 2002; Zhang et al, 2003) may affect the integration of transmembrane segments and may induce generation of semi-inverted topologies, we tested whether the insertion of the linkers affected the packing and the relative topology of the protomers in tandem EmrE. The membrane domain of EmrE is completely resistant to a battery of proteases, including proteinase K. After exposure of EmrE to either chymotrypsin or proteinase K, only the C-terminal tag is digested (Figure 4A, lanes 1–3), even at prolonged overnight digestions. When E22EMH is treated with either one of the enzymes, the linker and the tag are digested by the protease treatment (Figure 4A, lanes 4–6) supporting the contention that the packing in the membrane is undistinguishable from that of EmrE and that the linker is completely exposed to the protease. As expected, only proteinase K digests E6EMH since there are no sites for either one of the other enzymes (not shown). In addition, to support the contention that the protomers in E22EMH are in a parallel orientation, we treated the protein with hexamethylene diisocyanate (HMDC), a reagent previously shown to react with Lys22 in the first loop of EmrE (Soskine et al, 2002). The cross-linking is dependent on the proximity of the two monomers, since it is not observed after SDS treatment that induces dissociation of the dimer (Soskine et al, 2002). The results of this experiment are shown in Figure 4B. EmrE (lanes 4–6) and E22EMH (lanes 1–3) were digested with proteinase K as above so that the tags and the linker were digested and yielded a construct shorter than the tagged EmrE (lanes 5 and 2, respectively). After digestion, cross-linking resulted in both cases in the production of a polypeptide with an apparent molecular mass of about 22 kDa, smaller than the tagged dimer (lanes 3 and 6). These experiments support the contention that the relative topology of the protomers in tandem EmrE is parallel and that a dimer with parallel topology is functional. Figure 4.Probing packing and topology of the protomers in E22EMH. (A) Membranes bearing radiolabeled EmrE-His tagged (EmrE, lanes 1–3) and E22EMH (lanes 4–6) were incubated with the corresponding proteases as described under Materials and methods. In EmrE, the MycHis tag is digested, producing a polypeptide of about 10 kDa (lanes 2 and 3); E22EMH is digested to produce polypeptides with a similar molecular mass as a result of the digestion of the tag and the linker. (B) After digestion with proteinase K, both proteins were treated with the cross-linker HMDC as described under Materials and methods. In both cases, cross-linking is very effective (lanes 3 and 6), producing a polypeptide slightly smaller that the original tandem because the tag has been digested. Download figure Download PowerPoint Probing interdimeric interaction by ‘monomer swapping’ with E14C, an inactive EmrE mutant EmrE dimers solubilized with DDM (n-dodecyl-β-maltoside) may be reversibly dissociated by a short heat treatment. If a mixture of two mutant proteins is subjected to this procedure, mixed dimers are formed upon cooling (‘monomer swapping’) (Rotem et al, 2001; Sharoni et al, 2005). Replacement of Glu14 with an uncharged amino acid completely abolishes binding, but the mutants can form mixed dimers with other functional EmrE protomers. The mixed dimers display more than 20-fold lower affinity toward TPP+, a phenomenon defined as a negative dominant effect of the inactive mutant on the functional one (Rotem et al, 2001; Elbaz et al, 2004). We used this experimental paradigm to test the contention that the functional unit is within the tandem rather than derived from interdimeric interaction. Increasing amounts of the well-characterized mutant EmrE E14C were added to wild-type EmrE and to two tandems with the longest linkers, E22EMH and E6EMH, and the mixtures were subjected to the monomer swapping procedure and assayed for TPP+-binding activity. The TPP+ concentration used in this assay, 2.5 nM, equals the KD of EmrE. Therefore, inhibition of binding reflects the formation of mixed dimmers, since the contribution of heterodimers EmrE/E14C is insignificant due to their lower affinity (Figure 5). After monomer swapping with a 50-fold excess of the E14C mutant, the binding activity of the wild-type protein was inhibited by more than 80% (Figure 5, closed squares). Under the same conditions, binding activity of both tandems is only barely affected (∼15%; Figure 5, closed circles, open squares). Figure 5.The tandem as the functional unit: it does not take part in the intermolecular interactions with other EmrE molecules. Increasing amounts of the membranes containing the indicated amounts of inactive EmrE mutant E14C were solubilized in 1% DDM Na-buffer and mixed with the solubilized membranes containing ∼40 ng of EmrE (WT), E22EMH or E6EMH. After incubation at 80°C (10 min), the mixture was transferred to 4°C and assayed for [3H]TPP+ binding as described under Materials and methods. Inset: about 100 ng of His-tagged EmrE (1), E22EMH (2) and E6EMH (3) were mixed with [35S]methionine-labeled, cysteine-less untagged EmrE solubilized as above, mixture was treated as described under Materials and methods, after pull down with Ni-NTA, radiolabeled untagged protein was analyzed by SDS–PAGE and visualized with a Fujifilm FLA-3000 imaging system. Download figure Download PowerPoint Interdimeric interaction that does not result in functional changes was also ruled out in a parallel experiment where unlabeled tagged proteins were used as ‘bait’ to pull down untagged radiolabeled wild-type protein (Figure 5, inset). The generation of heterodimers between tagged and radiolabeled untagged EmrE is evidenced by the fact that after the swapping procedure, most of the untagged protein is associated with the Ni–NTA (Ni2+–nitrilotriacetic acid) beads. Neither E22EMH nor E6EMH pull down more than one-tenth of the radiolabeled protein. Tandem EmrE with a single Glu14: modified stoichiometry and specificity EmrE transports monovalent and divalent substrates with the same stoichiometry of 2H+/substrate. Thus, transport of monovalent substrates involves charge movement (i.e., electrogenic), whereas transport of divalent substrate does not. Both protons are released from Glu residues at position 14. Heterodimers were generated by monomer swapping to test whether two glutamyl residues are required for substrate recognition and binding (Rotem et al, 2001). Heterodimers with only one Glu residue bind substrate with a lower affinity (Rotem et al, 2001). Previously, reconstitution of these heterodimers could not yield reliable results, since our protocol includes a step where the proteins are treated with octyl glucoside, a detergent that increases the fraction of monomeric protein and allows reshuffling of the preformed heterodimers (Soskine et al, 2006). Therefore, to further study this question, a tandem was generated in which one of the Glu14 was replaced with glutamine (Q6EMH). The tandem also allowed testing the effect of such replacement in vivo. It confers decreased but significant resistance to ethidium (Figure 2A). In addition, it actively removes ethidium albeit at rates slower than those of E6EMH or any of the other tandems (Figures 2B and 6A). Furthermore, the level of removal as judged from the equilibrium value is lower than that achieved by the other tandems. The results demonstrate that a tandem protein with only one carboxyl is capable of catalyzing the transport of ethidium. The lower levels of removal and resistance may be due to the different stoichiometry dictated by the fact that since there is only one Glu14 per dimer, we may expect that only 1H+ is exchanged per substrate molecule rather than two for the protein with two carboxyls. To test this contention, we purified Q6EMH and characterized its activity in vitro. As expected from monomer swapping experiments, the purified protein binds TPP+ (Figure 3A) with an affinity significantly lower than the wild-type or the other tandems (KD=59 nM). Figure 6.Negative dominance by design: a tandem with the single Glu 14. (A) Ethidium efflux activity in whole cells expressing Q6EMH and E6EMH was measured as described in Figure 2. (B) [3H]MPP+ uptake by EmrE, EmrE E14Q and Q6EMH was tested with ammonium-loaded proteoliposomes (2 μl, containing ∼0.5 μg of protein). They were diluted into an ammonium-free medium with pH 8.5 containing 1 μM [3H]MPP+, 140 mM K2SO4 with or without addition of 100 nM valinomycin. Hollow triangles display MPP+ incorporated into proteoliposomes containing E14Q in the presence of 100 nM valinomycin. (C) Inhibition of [3H]MPP+ uptake by MV2+. Uptake was measured as above in the presence of 100 nM valinomycin and the indicated amounts of MV2+. (D) [14C]MV2+ efflux activity of EmrE, EmrE E14Q and Q6EMH. K2SO4-containing proteoliposomes were assayed as described under Materials and methods. (E) Substrate induced proton release from EmrE and Q6EMH. The pH of the unbuffered solution containing 105 μg Q6EMH or EmrE was monitored by absorbance of Phenol Red as described under Materials and methods. Addition of 5 μM TPP+ is indicated by the arrow. Addition of 4 nmol of NaOH was to estimate the amount of protons released from the protein upon substrate binding. Download figure Download PowerPoint The reconstituted proteoliposomes were assayed for transport of two different substrates: the monovalent 1-methyl-4-phenylpyridinium (MPP+) (Figure 6B) and a divalent one, MV2+ (Figure 3B). Wild-type EmrE and E6EMH accumulate both the divalent (Figure 3B) and the monovalent substrates (Figures 3B and 6B). As expected from an electrogenic exchange of 2H+/MPP+, transport of the latter is stimulated by a membrane potential generated by valinomycin in the presence of external potassium (Figure 6B). Strikingly, Q6EMH transports MPP+ but does not catalyze accumulation of MV2+ (Figures 3B and 6B, respectively). To distinguish between the effect of a modified stoichiometry and loss of recognition of the divalent substrate by a protein with a single Glu14, we assessed its ability to inhibit transport of MPP+ (Figure 6C). While 10 mM MV2+ significantly inhibits MPP+ accumulation in EmrE and in E6EMH (IC50 <1 mM), it only partially inhibits transport catalyzed by Q6EMH and inhibition plateaus above 10 mM to a level of about 40%. To further test the finding that the tandems with only one Glu14 are impaired in the recognition of divalent substrates, we assayed downhill efflux of MV2+ from proteoliposomes preloaded with the substrate, and diluted to a medium without it. Wild type (Figure 6D) and E6EMH (not
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