Scanning Cysteine Accessibility of EmrE, an H+-coupled Multidrug Transporter from Escherichia coli, Reveals a Hydrophobic Pathway for Solutes

Abstract

EmrE is a 12-kDa Escherichia coli multidrug transporter that confers resistance to a wide variety of toxic reagents by actively removing them in exchange for hydrogen ions. The three native Cys residues in EmrE are inaccessible to N-ethylmaleimide (NEM) and a series of other sulfhydryls. In addition, each of the three residues can be replaced with Ser without significant loss of activity. A protein without all the three Cys residues (Cysless) has been generated and shown to be functional. Using this Cys-less protein, we have now generated a series of 48 single Cys replacements throughout the protein. The majority of them (43) show transport activity as judged from the ability of the mutant proteins to confer resistance against toxic compounds and from in vitro analysis of their activity in proteoliposomes. Here we describe the use of these mutants to study the accessibility to NEM, a membrane permeant sulfhydryl reagent. The study has been done systematically so that in one transmembrane segment (TMS2) each single residue was replaced. In each of the other three transmembrane segments, at least four residues covering one turn of the helix were replaced. The results show that although the residues in putative hydrophilic loops readily react with NEM, none of the residues in putative transmembrane domains are accessible to the reagent. The results imply very tight packing of the protein without any continuous aqueous domain. Based on the findings described in this work, we conclude that in EmrE the substrates are translocated through a hydrophobic pathway.

Structure, Dynamics, and Substrate-induced Conformational Changes of the Multidrug Transporter EmrE in Liposomes

“…For this purpose, overnight cultures were diluted, spotted on agar plates containing 200 g/ml EtBr, and allowed to grow for 24 h at 37°C (26). Bacterial growth was qualitatively characterized as described under "Experimental Procedures."…”

“…Residues in the fourth transmembrane segment were cloned in pET21a ϩ WT* between NdeI and HindIII. This construct has a spacer and a His 6 tag added at the C terminus as described previously (26). All 110 cysteine mutants were also generated in pET20b ϩ WT* as described previously (27) for use in resistance phenotype activity assays.…”

“…Ethidium Resistance Assay-The ability of mutants to confer resistance to ethidium bromide was tested as described previously (26). Overnight cultures of E. coli expressing mutant EmrE were grown to saturation.…”

Determinants of Substrate Recognition by the Escherichia coli Multidrug Transporter MdfA Identified on Both Sides of the Membrane

“…However, there were clear differences in the amounts of the radiolabeled products, indicating different levels of reactivity with NEM. These differences might be due to changes within the local environment of the various Cys side chains, because water exposed Cys side chains are usually highly reactive, whereas those tightly packed in a hydrophobic environment are usually less reactive or inaccessible to the sulfhydryl reagent (25)(26)(27)(28)(29). The single-Cys mutants V54C, T56C, Y127C, A128C, A147C, A191C, S204C, and V335C exhibited high reactivity with NEM, whereas the mutants G39C, V125C, S133C, M146C, and…”

“…Characterization of these mutants revealed that although none of the Cys replacements affected the expression of MdfA, many influenced the substrate recognition spectrum of MdfA. Previously, systematic Cys-scanning mutagenesis studies of other transporters (24,25,29) demonstrated the structural and/or functional importance of several sites. In the present study the genetic screen enabled us to direct the scanning mutagenesis to 18 sites.…”