Open AccessThe purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H + and Na + (K + )/H + exchange
Author(s) -
Jianbo Cheng,
David Hicks,
Terry A. Krulwich
Publication year - 1996
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.93.25.14446
Subject(s) - antiporter , tetracycline , bacillus subtilis , efflux , chemistry , cobalt , membrane transport , membrane transport protein , biochemistry , nuclear chemistry , membrane , inorganic chemistry , biology , bacteria , membrane protein , genetics , antibiotics
Recent work has suggested that the chromosomally encoded TetA(L) transporter ofBacillus subtilis , for which no physiological function had been shown earlier, not only confers resistance to low concentrations of tetracycline but is also a multifunctional antiporter protein that has dominant roles in both Na+ - and K+ -dependent pH homeostasis and in Na+ resistance during growth at alkaline pH. To rigorously test this hypothesis, TetA(L) has been purified with a hexahistidine tag at its C terminus and reconstituted into proteoliposomes. The TetA(L)–hexahistidine proteoliposomes exhibit high activities of tetracycline–cobalt/H+ , Na+ /H+ , and K+ /H+ antiport in an assay in which an outwardly directed proton gradient is artificially imposed and solute uptake is monitored. Tetracycline uptake depends on the presence of cobalt andvice versa , with the cosubstrates being transported in a 1:1 ratio. Evidence for the electrogenicity of both tetracycline–cobalt/H+ and Na+ /H+ antiports is presented. K+ and Li+ inhibit Na+ uptake, but there is little cross-inhibition between Na+ and tetracycline–cobalt uptake activities. The results strongly support the conclusion that TetA(L) is a multifunctional antiporter. They expand the roster of such porters to encompass one with a complex organic substrate and monovalent cation substrates that may have distinct binding domains, and provide the first functional reconstitution of a member of the 14-transmembrane segment transporter family.