Restrictive use of detergents in the functional reconstitution of the secondary multidrug transporter LmrP

The histidine-tagged secondary multidrug transporter LmrP was overexpressed in Lactococcus lactis, using a novel protein expression system for cytotox ic proteins based on the tightly regulated, nisin-inducible nisA promoter. LmrP-mediated H+/drug antiport activity in inside-out membrane vesicles was inhibited by detergents, such as Triton X-100, Triton X-114, and Tween 80, at low concentrations that did not affect the magnitude or composition of the proton motive force. The inhibition of the activity of LmrP by detergen ts restricted the range of compounds that could be used for the solubilizat ion and reconstitution of the protein because low concentrations of deterge nt are retained in proteoliposomes. Surprisingly, dodecyl maltoside did not modulate the activity of LmrP. Therefore, LmrP was solubilized with dodecy l maltoside, purified by nickel-chelate affinity chromatography, and recons tituted in dodecyl maltoside-destabilized, preformed liposomes prepared fro m Escherichia coli phospholipids and egg phosphatidylcholine. Reconstituted LmrP mediated the transport of multiple drugs in response to an artificial ly imposed pH gradient, demonstrating that the protein functions as a proto n motive force-dependent multidrug transporter, independent of accessory pr oteins. These observations are relevant for the effective solubilization an d reconstitution of multidrug transporters belonging to the major facilitat or superfamily, which, in view of their broad drug specificity, may strongl y interact with detergents.