In Vitro Monomer Swapping in EmrE, a Multidrug Transporter from Escherichia coli, Reveals That the Oligomer Is the Functional Unit

Dvir Rotem, Neta Sal-man, Shimon Schuldiner

Research output: Contribution to journalArticlepeer-review

58 Scopus citations

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 °C, a treatment that does not impair transport activity. Oligomer formation is highly specific as judged by several criteria, among them the fact that 35S-EmrE can be "pulled out" from a mixture prepared from generally labeled cells. Using this technique, we show that inactive mutant sub-units 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.

Original languageEnglish
Pages (from-to)48243-48249
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number51
DOIs
StatePublished - 21 Dec 2001
Externally publishedYes

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