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Awareness of the problem of antimicrobial resistance (AMR) has escalated and drug-resistant infections are named among the most urgent problems facing clinicians today. Our experiments here identify a transporter interactome and portray its essential function in acquisition of antimicrobial resistance. By exposing E. coli cells to consecutive increasing concentrations of the fluoroquinolone norfloxacin we generated in the laboratory highly resistant strains that carry multiple mutations, most of them identical to those identified in clinical isolates. With this experimental paradigm, we show that the MDTs function in a coordinated mode to provide an essential first-line defense mechanism, preventing the drug reaching lethal concentrations, until a number of stable efficient alterations occur that allow survival. Single-component efflux transporters remove the toxic compounds from the cytoplasm to the periplasmic space where TolC-dependent transporters expel them from the cell. We postulate a close interaction between the two types of transporters to prevent rapid leak of the hydrophobic substrates back into the cell. The findings change the prevalent concept that in Gram-negative bacteria a single multidrug transporter, AcrAB-TolC type, is responsible for the resistance. The concept of a functional interactome, the process of identification of its members, the elucidation of the nature of the interactions and its role in cell physiology will change the existing paradigms in the field. We anticipate that our work will have an impact on the present strategy searching for inhibitors of AcrAB-TolC as adjuvants of existing antibiotics and provide novel targets for this urgent undertaking.

The topology of integral membrane proteins with a weak topological tendency can be influenced when fused to tags, such as these used for topological determination or protein purification. Here, we describe a technique for topology determination of an untagged membrane protein. This technique, optimized for bacterial cells, allows the visualization of the protein in the native environment and incorporates the substituted-cysteine accessibility method.

Transporters of the small multidrug resistance (SMR) family are small homo- or heterodimers that confer resistance to multiple toxic compounds by exchanging substrate with protons. Despite the wealth of biochemical information on EmrE, the most studied SMR member, a high-resolution three-dimensional structure is missing. To provide proteins that are more amenable to biophysical and structural studies, we identified and partially characterized SMR transporters from bacteria living under extreme conditions of temperature and radiation. Interestingly, these homologues as well as EmrE confer resistance to streptomycin and tobramycin, two aminoglycoside antibiotics widely used in clinics. These are hydrophilic and clinically important substrates of SMRs, and study of their mode of action should contribute to understanding the mechanism of transport and to combating the phenomenon of multidrug resistance. Furthermore, our study of one of the homologues, a putative heterodimer, supports the suggestion that in the SMR family, heterodimers can also function as homodimers.

Inverted repeats in ion-coupled transporters have evolved independently in many unrelated families. It has been suggested that this inverted symmetry is an essential element of the mechanism that allows for the conformational transitions in transporters. We show here that small multidrug transporters offer a model for the evolution of such repeats. This family includes both homodimers and closely related heterodimers. In the former, the topology determinants, evidently identical in each protomer, are weak, and we show that for EmrE, an homodimer from Escherichia coli, the insertion into the membrane is random, and dimers are functional whether they insert into the cytoplasmic membrane with the N- and C-terminal domains facing the inside or the outside of the cell. Also, mutants designed to insert with biased topology are functional regardless of the topology. In the case of EbrAB, a heterodimer homologue supposed to interact antiparallel, we show that one of the subunits, EbrB, can also function as a homodimer, most likely in a parallel mode. In addition, the EmrE homodimer can be forced to an antiparallel topology by fusion of an additional transmembrane segment. The simplicity of the mechanism of coupling ion and substrate transport and the few requirements for substrate recognition provide the robustness necessary to tolerate such a unique and unprecedented ambiguity in the interaction of the subunits and in the dimer topology relative to the membrane. The results suggest that the small multidrug transporters are at an evolutionary junction and provide a model for the evolution of structure of transport proteins.

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.

Aromatic residues may play several roles in integral membrane proteins, including direct interaction with substrates. In this work, we studied the contribution of tyrosine residues to the activity of EmrE, a small multidrug transporter from Escherichia coli that extrudes various drugs across the plasma membrane in exchange with protons. Each of five tyrosine residues was replaced by site-directed mutagenesis. Two of these residues, Tyr-40 and Tyr-60, can be partially replaced with hydroxyamino acids, but in the case of Tyr-40, replacement with either Ser or Thr generates a protein with modified substrate specificity. Replacement of Tyr-4 with either Trp or Phe generates a functional transporter. A Cys replacement at this position generates an uncoupled protein; it binds substrate and protons and transports the substrate downhill but is impaired in uphill substrate transport in the presence of a proton gradient. The role of these residues is discussed in the context of the published structures of EmrE.

Multidrug transporters are ubiquitous proteins, and, based on amino acid sequence similarities, they have been classified into several families. Here we characterize a cluster of archaeal and bacterial proteins from the major facilitator superfamily (MFS). One member of this family, the vesicular monoamine transporter (VMAT) was previously shown to remove both neurotransmitters and toxic compounds from the cytoplasm, thereby conferring resistance to their effects. A BLAST search of the available microbial genomes against the VMAT sequence yielded sequences of novel putative multidrug transporters. The new sequences along with VMAT form a distinct cluster within the dendrogram of the MFS, drug-proton antiporters. A comparison with other proteins in the family suggests the existence of a potential ion pair in the membrane domain. Three of these genes, from Mycobacterium smegmatis, Corynebacterium glutamicum, and Halobacterium salinarum, were cloned and functionally expressed in Escherichia coli. The proteins conferred resistance to fluoroquinolones and chloramphenicol (at concentrations two to four times greater than that of the control). Measurement of antibiotic accumulation in cells revealed proton motive force-dependent transport of those compounds.

EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu14) from both EmrE monomers. Previous studies implied that other residues in the vicinity of Glu14 are part of the binding domain. Alkylation of Cys replacements in the same transmembrane domain inhibits the activity of the protein and this inhibition is fully prevented by substrates of EmrE. To monitor directly the reaction we tested also the extent of modification using fluorescein-5-maleimide. While most residues are not accessible or only partially accessible, four, Y4C, I5C, L7C, and A10C, were modified at least 80%. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of two of these residues by up to 80%. To study other essential residues we generated functional hetero-oligomers and challenged them with various methane thiosulfonates. Taken together the findings imply the existence of a binding cavity accessible to alkylating reagents where at least three residues from TM1, Tyr40 from TM2, and Trp63 in TM3 are involved in substrate binding.

Tryptophan residues may play several roles in integral membrane proteins including direct interaction with substrates. In this work we studied the contribution of tryptophan residues to substrate binding in EmrE, a small multidrug transporter of Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons. Each of the four tryptophan residues was replaced by site-directed mutagenesis. The only single substitutions that affected the protein's activity were those in position 63. While cysteine and tyrosine replacements yielded a completely inactive protein, the replacement of Trp63 with phenylalanine brought about a protein that, although it could not confer any resistance against the toxicants tested, could bind substrate with an affinity 2 orders of magnitude lower than that of the wild-type protein. Double or multiple cysteine replacements at the other positions generate proteins that are inactive in vivo but regain their activity upon solubilization and reconstitution. The findings suggest a possible role of the tryptophan residues in folding and/or insertion. Substrate binding to the wild-type protein and to a mutant with a single tryptophan residue in position 63 induced a very substantial fluorescence quenching that is not observed in inactive mutants or chemically modified protein. The reaction is dependent on the concentration of the substrate and saturates at a concentration of 2.57 microM with the protein concentration of 5 microM supporting the contention that the functional unit is a dimer. These findings strongly suggest the existence of an interaction between Trp63 and substrate, and the nature of this interaction can now be studied in more detail with the tools developed in this work.

EmrE is a small multidrug transporter from Escherichia coli that provides a unique model for the study of polytopic membrane proteins. Here, we show its synthesis in a cell-free system in a fully functional form. The detergent-solubilized protein binds substrates with high affinity and, when reconstituted into proteoliposomes, transports substrate in a Deltamicro(H)(+)-dependent fashion. Here, we used the cell-free system to study the oligomeric properties of EmrE. EmrE functions as an oligomer, but the size of the functional oligomer has not been established unequivocally. Coexpression of two plasmids in the cell-free system allowed demonstration of functional complementation and pull-down experiments confirmed that the basic functional unit is the dimer. An additional interaction between dimers has been detected by using crosslinking between unique Cys residues. This finding implies the existence of a dimer of dimers.

EmrE is a small multidrug transporter (110 amino acids long) from Escherichia coli that extrudes various drugs in exchange with protons, thereby rendering bacteria resistant to these compounds. Glu-14 is the only charged membrane-embedded residue in EmrE and is evolutionarily highly conserved. This residue has an unusually high pK and is an essential part of the binding domain, shared by substrates and protons. The occupancy of the binding domain is mutually exclusive, and, as such, this provides the molecular basis for the coupling between substrate and proton fluxes. Systematic cysteine-scanning mutagenesis of the residues in the transmembrane segment (TM1), where Glu-14 is located, reveals an amino acid cluster on the same face of TM1 as Glu-14 that is part of the substrate- and proton-binding domain. Substitutions at most of these positions yielded either inactive mutants or mutants with modified affinity to substrates. Substitutions at the Ala-10 position, one helix turn away from Glu-14, yielded mutants with modified affinity to protons and thereby impaired in the coupling of substrate and proton fluxes. Taken as a whole, the results strongly support the concept of a common binding site for substrate and protons and stress the importance of one face of TM1 in substrate recognition, binding, and H(+)-coupled transport.

Esters of amino acids are known to penetrate into cells by simple diffusion. Subsequently, they are hydrolyzed by hydrolases to release the parent amino acid. Due to the abundance of hydrolases in phagolysosomes, amino acids accumulate, there because the rate of influx and hydrolysis exceed the rate of amino acid efflux through specific carriers. The osmotic effect of this accumulation results in the disruption of the organelles. This mechanism has been demonstrated to be responsible for the killing of Leishmania amastigotes by amino acid esters. In this investigation, it is shown that all esters tested, including alcohol esters, N-acetyl esters and the esters of some dipeptides, inhibit the growth of Plasmodium falciparum in culture. Inhibition is time-dependent and, in some cases, ring-stage parasites are more sensitive than trophozoites. Similar to the findings with Leishmania, alcohol esters of Glu, Leu, Met, Phe and Trp are more toxic to Plasmodium whereas Ala, Gly, His and Ile are much less noxious. Esters caused the release of acridine orange that selectively accumulates in the phagolysosome-like food vacuole of the parasite, attesting the ostensible destruction of this organelle by osmotic lysis. The toxicity of the N-acetyl esters is probably associated in part to their ability to inhibit cytosolic proteases. Since excess of amino acids can also inhibit proteolysis, the effect of free amino acids on parasite growth was also tested. Of the 19 odd amino acids tested, only three, namely Cys, His and Trp, were found to be toxic to the parasites at millimolar concentrations and the reasons for their possible specific toxicity are discussed.

EmrE is a small multidrug transporter that extrudes various drugs in exchange with protons, thereby rendering Escherichia coli cells resistant to these compounds. In this study, relative helix packing in the EmrE oligomer solubilized in detergent was probed by intermonomer crosslinking analysis. Unique cysteine replacements in transmembrane domains were shown to react with organic mercurials but not with sulfhydryl reagents, such as maleimides and methanethiosulfonates. A new protocol was developed based on the use of HgCl(2), a compound known to react rapidly and selectively with sulfhydryl groups. The reaction can bridge vicinal pairs of cysteines and form an intermolecular mercury-linked dimer. To circumvent problems inherent to mercury chemistry, a second crosslinker, hexamethylene diisocyanate, was used. After the HgCl(2) treatment, excess reagent was removed and the oligomers were dissociated with a strong denaturant. Only those previously crosslinked reacted with hexamethylene diisocyanate. Thus, vicinal cysteine-substituted residues in the EmrE oligomer were identified. It was shown that transmembrane domain (TM)-1 and TM4 in one subunit are in contact with the corresponding TM1 and TM4, respectively, in the other subunit. In addition, TM1 is also in close proximity to TM4 of the neighboring subunit, suggesting possible arrangements in the binding and translocation domain of the EmrE oligomer. This method should be useful for other proteins with cysteine residues in a low-dielectric environment.

The role of N-glycosylation in the expression, ligand recognition, activity, and intracellular localization of a rat vesicular monoamine transporter (rVMAT1) was investigated. The glycosylation inhibitor tunicamycin induced a dose-dependent decrease in the rVMAT1-mediated uptake of [3H]serotonin. Part of this effect was due to a general toxic effect of the drug. Therefore, to assess the contribution of each of the glycosylation sites to the transporter activity, the three putative N-glycosylation sites were mutated individually, in combination, and in toto ("triple" mutant). Mutation of each glycosylation site caused a minor and additive decrease in activity, up to the triple mutant, which retained at least 50% of the wild-type activity. No significant differences were found either in the time dependence of uptake or the apparent affinity for ligands of the triple mutant compared with the wild-type protein. It is interesting that in contrast to plasma-membrane neurotransmitter transporters, the unglycosylated form of rVMAT1 distributed in the cell as the wild-type protein. Pro43 is a highly conserved residue located at the beginning of the large loop in which all the potential glycosylation sites are found. A Pro43Leu mutant transporter was inactive. It is remarkable that despite the presence of glycosylation sites, the mutant transporter was not glycosylated. Moreover, the distribution pattern of the Pro43Leu mutant clearly differed from that of the wild type. In contrast, a Pro43Gly mutant displayed an activity practically identical to the wild-type protein. As this replacement generated a protein with wild-type characteristics, we suggest that the conformation conferred by the amino acid at this position is essential for activity.

Vesicular monoamine transporters (VMAT) catalyze transport of serotonin, dopamine, epinephrine and norepinephrine into subcellular storage organelles in a variety of cells. Accumulation of the neurotransmitter depends on the proton electrochemical gradient across the organelle membrane and involves VMAT-mediated exchange of two lumenal protons with one cytoplasmic amine. It has been suggested in the past that His residues play a role in H+ movement or in its coupling to active transport in H(+)-symporters and antiporters. Indeed VMAT-mediated transport is inhibited by reagents specific for His residues. We have identified one His residue in VMAT1 from rat which is conserved in other vesicular neurotransmitter transporters. Mutagenesis of this His (H419) to either Arg or Cys completely inhibits [3H]serotonin and [3H]dopamine accumulation. Mutagenesis also inhibits other H(+)-dependent partial reactions of VMAT such as the acceleration of binding of the high affinity ligand reserpine, but does not inhibit the [3H]reserpine binding which is not dependent on H+ translocation. It is concluded that His-419 plays a role in energy coupling in r-VMAT1.

The energetics of reserpine binding to the bovine adrenal biogenic amine transporter suggest that H+ ion translocation converts the transporter to a form which binds reserpine essentially irreversibly. Reserpine binding to bovine adrenal chromaffin granule membrane vesicles is accelerated by generation of a transmembrane pH difference (delta pH) (interior acid) or electrical potential (delta psi) (interior positive). Both components of the electrochemical H+ potential (delta mu H+) must be dissipated to block reserpine binding, and generation of either one stimulates the binding rate. Reserpine binding is less dependent than amine transport on the delta pH, suggesting that translocation of fewer H+ ions is required to expose the high-affinity site than are required for net transport. Bound reserpine dissociates very slowly, if at all, from the transporter. Binding is stable to 1% cholate, 1.5% Triton X-100, 1 M SCN-, and 8 M urea, but sodium dodecyl sulfate (0.035%) and high temperatures (100 degrees C) released bound reserpine, indicating that binding is noncovalent. The results raise the possibility that the transporter, by translocating one H+ ion outward down its concentration gradient, is converted to a form that can either transport a neutral substrate molecule inward or occlude reserpine in a dead-end complex.

Free fatty acids (FFA) uncouple oxidative phosphorylation and reverse electron transport and inhibit ATP-Pi exchange in beef heart submitochondrial particles. In this, they resemble classical uncouplers and ionophores. However, in contrast to the latter agents, FFA do not collapse the substrate generated proton electrochemical potential and do not inhibit ATP synthesis when the latter is driven by artificially imposed delta microH. These results lend further support to the suggestion that oxidative phosphorylation depends, in part, on direct intramembranal proton transfer - a process which is specifically uncoupled by FFA and other membrane perturbing agents (e.g. general anesthetics).

The role of lipids in the interaction of the beta-adrenergic receptor (R) with the regulatory protein (Gs) was investigated. Solubilized preparations of R and of Gs from turkey erythrocytes were delipidated by gel filtration. They were subsequently combined and reconstituted by the addition of various lipids. When reconstitution was carried out in the presence of soybean lipids, Gs could be fully activated via R by addition of hormone plus GTP gamma S. In contrast, purified phospholipids or a phospholipid fraction from soybean failed to produce an active system. Fractionation of soybean lipids revealed that acetone-soluble neutral lipids are essential for the reconstitution of a hormone responsive system. The acetone fraction could be replaced by specific neutral lipids such as alpha-tocopherol or cholesteryl arachidonate while a mixture of phosphatidylethanolamine, -choline and -serine satisfied the phospholipid requirement of the system.

Detergent-solubilized preparations of the beta-adrenergic receptor (R) and of the guanyl nucleotide binding proteins (Gs) were extensively treated to remove phospholipids and cholesterol. Reconstitution of an R-Gs system was subsequently performed in the presence of a mixture of natural phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine or the synthetic dioleoyl derivatives of the same phospholipids. In both cases, an additional lipid was required for the agonist-dependent activation of Gs. The requirement could be fulfilled by alpha-tocopherol, or by unsaturated fatty acids such as oleic acid. Inclusion of this non-phosphorylated lipid in the reconstituted system enhanced the isoproterenol-dependent activation of Gs by guanosine 5'-O-[gamma-thio]triphosphate 16-33-fold. The rate of activation was largely dependent on the addition of the agonist. Efficient functional reconstitution of R-Gs was thus achieved in a totally defined lipid system. Additional studies of the reconstituted system and of the native membrane led to the notion that the non-phosphorylated lipid plays a role in the function of the hormone-R complex.