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Anti-parallel membrane topology of two components of EbrAB, a multidrug transporter
Authors:Kikukawa Takashi  Miyauchi Seiji  Araiso Tsunehisa  Kamo Naoki  Nara Toshifumi
Affiliation:Laboratory of Biomolecular Systems, Creative Research Initiative Sosei (CRIS), Hokkaido University, Sapporo 001-0021, Japan. kikukawa@cast.hokudai.ac.jp
Abstract:EbrAB is a multidrug-resistance transporter in Bacillus subtilis that belongs to the small multidrug resistance, and requires two polypeptides of both EbrA and EbrB, implying that it functions in the hetero-dimeric state. In this study, we investigated the transmembrane topologies of EbrA and EbrB. Various single-cysteine mutants were expressed in Escherichia coli cells, and the efflux activity was measured. Only mutants having a high activity were used for the topology experiments. The reactivity of a membrane impermeable NEM-fluorescein against the single cysteine of these fully functional mutants was examined when this reactive fluorophore was applied either from the outside or both sides of the cell membrane or in the denatured state. The results clearly showed that EbrA and EbrB have the opposite orientation within the membrane or an anti-parallel configuration.
Keywords:Efflux transporter   EmrE   Small multidrug resistance (SMR)   Dual topology configuration   Fluorescein-5-maleimide (NEM-fluorescein)   Ethidium efflux
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