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 ΔΨ 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-His6. 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 (ΔμH+), 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 ΔμH+, 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 absence of ΔμH+ represent a ProP population that is predominantly off.