THE PURIFIED BACILLUS-SUBTILIS TETRACYCLINE EFFLUX PROTEIN TETA(L) RECONSTITUTES BOTH TETRACYCLINE-COBALT H+ AND NA+(K+)/H+ EXCHANGE/

Recent work has suggested that the chromosomally encoded TetA(L) trans porter of Bacillus subtilis, for which no physiological function had b een shown earlier, not only confers resistance to low concentrations o f tetracycline but is also a multifunctional antiporter protein that h as dominant roles in bath Na+- and K+-dependent pH humeostasis and in Na+ resistance during growth at alkaline pH. To rigorously test this h ypothesis, TetA(L) has been purified with a hexahistidine tag at its C terminus and reconstituted into proteoliposomes. The TetA(L)-hexahist idine proteoliposomes exhibit high activities of tetracycline-cobalt/H +, Na+/H+, and K+/H+ antiport in an assay in which an outwardly direct ed proton gradient is artificially imposed and solute uptake is monito red. Tetracycline uptake depends on the presence of cobalt and vice ve rsa, with the cosubstrates being transported in a 1:1 ratio. Evidence for the electrogenicity of both tetracycline-cobalt/H+ and Na+/H+ anti ports is presented. K+ and Li+ inhibit Na+ uptake, but there is little cross-inhibition between Na+ and tetracycline-cobalt uptake activitie s. The results strongly support the conclusion that TetA(L) is a multi functional antiporter. They expand the roster of such porters to encom pass one with a complex organic substrate and monovalent cation substr ates that may have distinct binding domains, and provide the first fun ctional reconstitution of a member of the 14-transmembrane segment tra nsporter family.