Energetics of wild-type and mutant multidrug resistance secondary transporter LmrP of Lactococcus lactis

LmrP, a proton/multidrug antiporter of Lactococcus lactis, transports a variety of cationic substrates. Previously, two membrane-embedded acidic residues, Asp(142) and Glu(327), have been reported to be important for multidrug transport activity of LmrP. Here we show that neither Glu(327) nor Asp(142) is essential for ethidium binding but that Glu(327) is a critical residue for the high affinity binding of Hoechst 33342. Substitution of these two residues, however, negatively influences the transport activity. The energetics of transport was studied of two closely related cationic substrates ethidium and propidium that carry one and two positive charges, respectively. Extrusion of monovalent ethidium is dependent on both the electrical membrane potential (Deltapsi) and transmembrane proton gradient (DeltapH), while extrusion of propidium predominantly depends on the DeltapH only. The LmrP mutants D142C and E327C, however, mediate electroneutral ethidium extrusion, but are unable to mediate DeltapH-dependent extrusion of propidium. These data indicate that Asp(142) and Glu(327) are involved in proton translocation. (C) 2004 Elsevier B.V. All rights reserved.