Roles of K⁺, H⁺, H₂O, and ΔΨ in solute transport mediated by major facilitator superfamily members ProP and LacY

H⁺-solute symporters ProP and LacY are members of the major facilitator superfamily. ProP mediates osmoprotectant (e.g., proline) accumulation, whereas LacY transports the nutrient lactose. The roles of K ⁺, H⁺, H₂O, and ΓΨ in H ⁺-proline and H⁺-lactose symport were compared using right-side-out cytoplasmic membrane vesicles (MVs) from bacteria expressing both transporters and proteoliposomes (PRLs) reconstituted with pure ProP-His ₆. ProP activity increased as LacY activity decreased when osmotic stress (increasing osmolality) was imposed on MVs. The activities of both transporters decreased to similar extents when Na⁺ replaced K ⁺ in MV preparations. Thus, K⁺ did not specifically control ProP activity. As with LacY, an increasing extravesicular pH stimulated ProP-mediated proline efflux much more than ProP-mediated proline exchange from de-energized MVs. In contrast to that of LacY, ProP-mediated exchange was only 2-fold faster than ProP-mediated efflux and was inhibited by respiration. In the absence of the protonmotive force (Δμ_H⁺), efflux of lactose from MVs was much more sensitive to increasing osmolality than lactose exchange. Thus, H₂O may be directly involved in proton transport via LacY. In the absence of αμ_H+, proline efflux and exchange from MVs were osmolality-independent. In PRLs with a ΔpH of 1 (lumen alkaline), ProP-His₆ was inactive when the membrane potential (ΔΨ) was zero, was active but insensitive to osmolality when ΔΨ was -100 mV, and became osmolality-sensitive as ΔΨ increased further to -137 mV. ProP-His6 had the same membrane orientation in PRLs as in cells and MVs. ProP switches among "off", "on", and "osmolality-sensitive" states as the membrane potential increases. Kinetic parameters determined in the zbsence of Δμ_H+ represent a ProP population that is predominantly off.