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 ⌬ 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.ion-coupled transporter ͉ drug resistance ͉ membrane protein ͉ oligomer ͉ cell-free protein synthesis

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 HgCl2, 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 HgCl2 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.ion-coupled transporter ͉ o-PDM ͉ mercurials ͉ drug resistance ͉ helix packing E mrE, a protein from Escherichia coli, provides a unique model for the study of multidrug transporters (1-3). It is a small transporter, 110 aa in length (Fig. 1), that extrudes various drugs in exchange for protons, thereby rendering bacteria resistant to these drugs (2, 3). The protein has been characterized, purified, and reconstituted in a functional form (4). EmrE has only one membrane-embedded charged residue, Glu-14, which is conserved in more than 60 homologous proteins (5). Glu-14 was shown to be part of a binding site for both protons and substrates (6, 7). The oligomeric structure seen in twodimensional crystals of EmrE is a dimer (8). Substrate binding to purified EmrE and negative dominance experiments support the contention that the functional unit of the protein is a dimer of dimers (1, 9, 10).Extensive use of cysteine-specific reagents has been made in many crosslinking experiments in membrane proteins (see, for example, refs. 11-14). The reagents commonly used react more rapidly with thiolates than with thiols. Therefore, their use is limited to hydrophilic loops or to areas of the protein that are accessible to solvent. The method described here supplies a tool to explore domains with low dielectric constants. It should be suitable to any protein that can be visualized, for example, by radiolabeling or by immunological techniques.In this study, relative helix packing in the EmrE oligomer was probed by intermonomer homo-a...

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 µM with the protein concentration of 5 µM 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.

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.

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 (⌬ H؉ ) across the organelle membrane and involves VMAT-mediated exchange of two lumenal protons with one cytoplasmic amine. Mutagenic analysis of the role of two conserved Asp residues located in transmembrane segments X and XI of rat VMAT type I reveals an important role of these two residues in catalysis. Accumulation of the neurotransmitter depends on the proton electrochemical gradient generated by the vesicular H ϩ -ATPase and involves the VMAT-mediated exchange of two lumenal protons with one cytoplasmic amine (1-5).A model of the mechanism of action of VMAT has been proposed based on a large body of biochemical data. In this model, the first step in the cycle is translocation of a single H ϩ that generates the binding form of the transporter (6). The energy invested in the transporter by H ϩ flux is released by ligand binding and is converted into vectorial movement of a substrate molecule across the membrane or directly into binding energy as measured with the high affinity ligand [ 3 H]reserpine. In the case of a substrate, a second conformational change results in the ligand binding site being exposed to the vesicle interior, where the substrate can dissociate. The second H ϩ in the cycle may be required to facilitate the conformational change or to allow for release of the positively charged substrate from the protein (6). Binding occurs also in the absence of a proton electrochemical gradient (⌬ Hϩ ) but many times slower (7,8).A clue for the molecular basis of some of these processes was obtained using diethyl pyrocarbonate (DEPC), a reagent relatively specific for His residues (9, 10). The inhibition by DEPC was specific for His groups because transport could be restored by hydroxylamine (9). DEPC inhibited transport, although it had no effect on binding of reserpine, indicating that the inhibition of transport was not due to a direct interaction with either of the known binding sites. Interestingly, however, the acceleration of reserpine binding by ⌬ Hϩ was inhibited (9). The results suggested that either proton transport or a conformational change induced by proton transport was inhibited by DEPC. Practically identical results were obtained with phenylglyoxal, a reagent specific for Arg residues.Cloning of VMAT (11,12) made it feasible to try to identify the residue(s) modified by DEPC. Only one His (His-419) is conserved in the VMATs from different species and in the two subtypes (5). Replacement of His-419 with either Cys (H419C) 2 or Arg (H419R) completely abolished transport as measured in permeabilized CV1 cells transiently transformed with plasmids coding for the mutant proteins. Reserpine binding to the mutant proteins in the absence of ⌬ Hϩ was at levels comparable with those detected in the wild type. However, ⌬ Hϩ did not accelera...