In vitro monomer swapping in EmrE, a multidrug transporter from Escherichia coli, reveals that the oligomer is the functional unit.
EmrE is a small multidrug transporter, 110 amino acids long that extrudes various drugs in exchange with protons, thereby rendering Escherichia coli cells resistant to these compounds. Negative dominance studies and radiolabeled substrate-binding studies suggested that EmrE functions as an oligomer. Projection structure of two-dimensional crystals of the protein revealed an asymmetric dimer. To identify the functional unit of EmrE, a novel approach was developed. In this method, quantitative monomer swapping is induced in detergent-solubilized EmrE by exposure to 80 degrees C, a treatment that does not impair transport activity. Oligomer formation is highly specific as judged by several criteria, among them the fact that (35)S-EmrE can be "pulled out" from a mixture prepared from generally labeled cells. Using this technique, we show that inactive mutant subunits are functionally complemented when mixed with wild type subunits. The hetero-oligomers thus formed display a decreased affinity to substrates. In addition, sulfhydryl reagents inhibit the above hetero-oligomer even though Cys residues are present only in the inactive monomer. It is concluded that, in EmrE, the oligomer is the functional unit.
Rotem
D
D
Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Sal-man
N
N
Schuldiner
S
S
D017920:Antiporters / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism*; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000378:metabolism*
D017920:Antiporters; D001426:Bacterial Proteins; D001704:Biopolymers; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D004364:Pharmaceutical Preparations; C075436:EmrE protein, E coli