Identification of residues in the translocation pathway of EmrE, a multidrug antiporter from Escherichia coli.

EmrE is a small, 12-kDa, highly polyspecific antiporter, which exchanges hydrogen ions with aromatic cations such as methyl viologen. EmrE-mediated transport is inhibited by the sulfhydryl-reactive reagent 4-(chloromercuri)benzoic acid (PCMB) but not by a variety of other sulfhydryl reagents. This differential effect is due to the fact that the organic mercurial is a substrate of the transporter and can reach domains otherwise inaccessible to the different reagents. To find out which of the three cysteine residues in EmrE is reacting with PCMB, each was replaced with serine and it was shown that none of them is essential for transport activity. A protein completely devoid of Cys residues (CL) is also capable of substrate accumulation albeit at a slower rate. Mutated proteins in which only one of the native cysteines was left whereas the other changed to serine were also constructed. The use of these proteins demonstrated that two of the three Cys in EmrE, Cys-41 and Cys-95, but not Cys-39, react with PCMB. A related mercurial, 4-(chloromercuri)benzenesulfonic acid (PCMBS), is only a very poor inhibitor, probably because of the negative charge it bears. PCMBS reacts with EmrE in an asymmetric and unique way. It reacts with the mutant bearing a single Cys residue in position 95 (CL-C95) only when the reagent is present in the outside face of the membrane and with the mutant CL-C41 only when allowed to permeate to the cell interior; as expected, it does not react with the mutant protein bearing a single Cys at position 39 (CL-C39). It is concluded that PCMB permeates through the substrate pathway of EmrE and covalently reacts with the two exposed residues, Cys-95 and Cys-41, but not with Cys-39, located on the opposite face of the helix relative to residue 41. In addition, because of the asymmetric reactivity to PCMBS, an inhibitor that does not permeate through the protein, it is concluded that Cys-41 is closer to the cytoplasmic face than Cys-95. The results demonstrate the existence of a domain accessible only to substrates and provide a unique tool for studying the substrate permeation pathway of an ion-coupled transporter.