PMID:11572877
| Citation |
Rotem, D, Sal-man, N and Schuldiner, S (2001) In vitro monomer swapping in EmrE, a multidrug transporter from Escherichia coli, reveals that the oligomer is the functional unit. J. Biol. Chem. 276:48243-9 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1074/jbc.M108229200 |
| Keywords |
Antiporters/chemistry; Antiporters/metabolism; Bacterial Proteins/metabolism; Biopolymers/chemistry; Biopolymers/metabolism; Drug Resistance, Microbial; Escherichia coli/metabolism; Escherichia coli Proteins; Membrane Proteins/chemistry; Membrane Proteins/metabolism; Pharmaceutical Preparations/metabolism |
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