Scanning cysteine accessibility of EmrE, an H+-coupled multidrug transporter from Escherichia coli, reveals a hydrophobic pathway for solutes

EmrE is a 12-kDa Escherichia coil multidrug transporter that confers resist ance to a wide variety of toxic reagents by actively removing them in excha nge for hydrogen ions, The three native Cys residues in EmrE are inaccessib le to N-ethylmaleimide (NEM) and a series of other sulfhydryls. In addition , each of the three residues can be replaced with Ser without significant l oss of activity, A protein without all the three Cys residues (Cys-less) ha s been generated and shown to be functional. Using this Cys-less protein, w e have now generated a series of 48 single Cys replacements throughout the protein. The majority of them (43) show transport activity as judged from t he ability of the mutant proteins to confer resistance against toxic compou nds and from in vitro analysis of their activity in proteoliposomes. Here w e describe the use of these mutants to study the accessibility to NEM, a me mbrane permeant sulfhydryl reagent. The study has been done systematically so that in one transmembrane segment (TMS2) each single residue was replace d. In each of the other three transmembrane segments, at least four residue s covering one turn of the helix were replaced. The results show that altho ugh the residues in putative hydrophilic loops readily react with NEM, none of the residues in putative transmembrane domains are accessible to the re agent, The results imply very tight packing of the protein without any cont inuous aqueous domain. Based on the findings described in this work, we con clude that in EmrE the substrates are translocated through a hydrophobic pa thway.