Bacterial multidrug resistance is due to a single membrane protein which functions as a drug pump.
Multidrug transport system in proteoliposomes was reconstituted using the highly purified membrane transport protein responsible for bacterial multidrug resistance. This protein (named Smr, for staphylococcal multidrug resistance) consists of 107 amino acid residues and displays four putative transmembrane domains. The Smr protein was tagged with a FLAG epitope, and the modified protein was expressed, purified, characterized, and reconstituted into proteoliposomes. With this in vitro experimental system, it has been demonstrated that a highly purified multidrug resistance protein functions as a drug pump, which transports methyltriphenylphosphonium actively against a 10(3)-fold concentration gradient. Delta mu H+ was shown to be a driving force, and an electrogenic drug/proton antiport was suggested as the molecular mechanism of the drug transport. Of the 2 Glu residues in putative extramembrane loops of the Smr polypeptide chain, Glu-24 was shown to be involved in determining the specificity of drug resistance. Replacement of both of these Glu residues with Asp produced active Smr. In contrast, Smr was unable to protect cells from multiple drugs when a Glu-13-->Asp-13 replacement was made. We suggest that Glu-13, a unique acidic residue located in the hydrophobic domain of Smr, is directly involved in the drug/proton antiport.
Grinius
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LL
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Goldberg
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EB
D000595:Amino Acid Sequence; D017920:Antiporters / Q000235:genetics / Q000302:isolation & purification*; D001224:Aspartic Acid / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004352:Drug Resistance, Microbial; D018432:Drug Resistance, Multiple; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010641:Phenotype; D011510:Proteolipids / Q000378:metabolism; D013210:Staphylococcus / Q000187:drug effects* / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics
D017920:Antiporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011510:Proteolipids; C020485:proteoliposomes; C521590:small multidrug-resistance pump, Staphylococcus aureus; C075436:EmrE protein, E coli; D001224:Aspartic Acid; D018698:Glutamic Acid; D014364:Tryptophan