LmrA activity enhances cell survival.

<p>A, Viability of energized cells (solid bar, LmrA; grey bar, non-expressing control) adapted for 30 min in buffer containing 0.5 M sucrose without or with 100 mM NaCl, 100 mM KCl or 50 mM Na₂SO₄, followed by 100-fold dilution into ultrapure water, or in buffer containing 0.125 M sucrose and 25 mM NaCl (control referred to as 4-fold dilution). B, Effect of mutations in LmrA on the viability after dilution of cells pre-exposed to 100 mM NaCl plus 0.5 M sucrose under conditions as in (A). (<i>n</i> = 10)</p

Ion transport in intact cells.

<p>A, Cl⁻ efflux in cells preloaded with Na³⁶Cl (100 µM) upon the addition of glucose (▪, LmrA; □, non-expressing control; ⧫, EE LmrA; ○, ΔK388 LmrA). B, Effect of the concentration of Na⁺, NMG⁺ or Cl⁻ on the ATPase activity of purified LmrA (open symbols) or LmrCD (•) measured at 2 mM Mg-ATP. C,D,E, H⁺ efflux in energized cells, loaded with pH probe CFDASE to monitor the intracellular pH (pH_in) in the absence (C) or presence of (D) 0.25 M NaCl or (E) 0.672 M sucrose in the external buffer (each equivalent to 521 mOsm) (▪, LmrA; □, non-expressing control; ⧫, EE LmrA; •, E314A LmrA; ▴, ΔK388 LmrA). Metabolic energy was generated in the cells by the addition of 20 mM glucose (at t = 0 min in the figures), 15 min after the addition of the NaCl or sucrose or solvent control. (<i>n</i> = 8)</p

Ion-coupled transport in proteoliposomes.

<p>A, B, ³⁶Cl⁻ uptake (100 µM) by LmrA-MD (•), E314A LmrA-MD (⋄), EE LmrA-MD (▪) or empty liposomes (▵) in the presence of a Δψ (interior negative) of −120 mV (A) or -ZΔpH (interior alkaline) of −49 mV (B). In the duplicate experiment for LmrA-MD (□) in (B), the addition of uncoupler (valinomycin plus nigericin, 1 µM each) at the arrow resulted in efflux of accumulated ³⁶Cl⁻, indicating concentrative uptake of the ion. C, ΔpH (interior alkaline)-dependent ³⁶Cl⁻ uptake by LmrA (•), EE LmrA (▪) or empty liposomes (▵). D, ΔpH (interior alkaline)-dependent uptake of non-radioactive Cl⁻ (1 mM) by LmrA is observed as a quench in the fluorescence of the SPQ fluorophore trapped in the lumen of the proteoliposomes. Quenching was also observed in empty liposomes (control) in the presence of the Cl⁻/OH⁻ antiporter TBT-Cl (1 µM). E, Kinetic analysis of ΔpH (interior alkaline)-dependent ³⁶Cl⁻ uptake by LmrA. F, ΔpH (interior alkaline)-dependent uptake of ²²Na (25 µM) by LmrA-MD. G, Uptake of unlabelled Na⁺ (10 mM) by LmrA was detected as an increase in the fluorescence of the membrane-impermeable sodium green probe trapped in the lumen. H, Na⁺ (100 µM) stimulates the ΔpH-dependent uptake of ³⁶Cl⁻ (100 µM) by LmrA compared to control containing 99 µM NMG⁺ plus 1 µM Na⁺. I, H⁺ efflux in proteoliposomes loaded with pH probe BCECF in the presence of an outwardly directed NaCl gradient. Control, empty liposomes. (<i>n</i> = 5)</p

Mass spectra of purified LmrA and LmrA-MD showing predominant homodimer formation for both proteins.

<p>Peaks assigned to binding of one cardiolipin molecule are labelled (blue stars), and measured molecular masses are shown.</p