Roles of K+, H+, H2O, and ΔΨ in Solute Transport Mediated by Major Facilitator Superfamily Members ProP and LacY

Abstract

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 (+), H 2O, and DeltaPsi 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 (Deltamu H (+) ), efflux of lactose from MVs was much more sensitive to increasing osmolality than lactose exchange. Thus, H 2O may be directly involved in proton transport via LacY. In the absence of Deltamu H (+) , proline efflux and exchange from MVs were osmolality-independent. In PRLs with a DeltapH of 1 (lumen alkaline), ProP-His 6 was inactive when the membrane potential (DeltaPsi) was zero, was active but insensitive to osmolality when DeltaPsi was -100 mV, and became osmolality-sensitive as DeltaPsi increased further to -137 mV. ProP-His 6 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 Deltamu H (+) represent a ProP population that is predominantly off.