EmrE, an Escherichia coli 12-kDa multidrug transporter, exchanges toxic cations and H+ and is soluble in organic solvents.
The smallest membrane protein shown to catalyze ion-coupled transport is documented in this report. A gene coding for a small 110-amino acid membrane protein (emrE or mvrC) has been previously identified and cloned and shown to render Escherichia coli cells resistant to methyl viologen and to ethidium. In this report, it is shown that the resistance is due to extrusion of the toxic compounds in a process that requires a proton electrochemical gradient rather than ATP. For this purpose, cells in which the unc gene was inactivated were used so that the interconversion between the proton gradient and ATP is not possible, and the effect of agents, which specifically affect either of them, was tested on transport of ethidium in the intact cell. In addition, EmrE has been overexpressed and metabolically labeled with [35S]methionine. Strikingly, the protein can be quantitatively extracted with a mixture of organic solvents such as chloroform:methanol and is practically pure after this extraction. Moreover, after addition of E. coli lipids to the chloroform:methanol extract, EmrE has been reconstituted in proteoliposomes loaded with ammonium chloride. Upon dilution of the proteoliposomes in ammonium-free medium, a pH gradient was formed that drove transport of ethidium and methyl viologen into the proteoliposome. Both substrates compete with each other and exchange with previously transported solute. EmrE is a multidrug transporter of a novel type, and, because of its size and its solubility properties, it provides a unique model to study structure-function aspects of transport reactions in ion-coupled processes.
Yerushalmi
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Division of Microbial and Molecular Ecology, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Givat Ram, Israel.
Lebendiker
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Schuldiner
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D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D001483:Base Sequence; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002352:Carrier Proteins / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D018432:Drug Resistance, Multiple; D004926:Escherichia coli / Q000187:drug effects*; D004996:Ethidium / Q000378:metabolism; D008081:Liposomes; D008969:Molecular Sequence Data; D010269:Paraquat / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D002352:Carrier Proteins; D008081:Liposomes; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D004247:DNA; D004996:Ethidium; D010269:Paraquat