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 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 (Cys-less) 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.
Mordoch
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Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Granot
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Lebendiker
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Schuldiner
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D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000167:Acriflavine / Q000494:pharmacology; D000595:Amino Acid Sequence; D017920:Antiporters; D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D004996:Ethidium / Q000494:pharmacology; D005033:Ethylmaleimide / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010641:Phenotype; D017433:Protein Structure, Secondary; D012996:Solutions
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017920:Antiporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D012996:Solutions; C075436:EmrE protein, E coli; D000167:Acriflavine; D004996:Ethidium; D003545:Cysteine; D005033:Ethylmaleimide