Electrogenic antiport activities of the Gram-positive Tet proteins include a Na+(K+)/K+ mode that mediates net K+ uptake.

ABSTRACT

Two Gram-positive Tet proteins, TetA(L) from Bacillus subtilis and TetK from a Staphylococcus aureus plasmid, have previously been suggested to have multiple catalytic modes and roles. These include tetracycline (Tc)-metal/H+ antiport for both proteins (Yamaguchi, A., Shiina, Y., Fujihira, E., Sawai, T., Noguchi, N., and Sasatsu, M. (1995) FEBS Lett. 365, 193-197; Cheng, J. Guffanti, A. A., Wang, W., Krulwich, T. A., and Bechhofer, D. H. (1996) J. Bacteriol. 178, 2853-2860); Na+(K+)/H+ antiport for both proteins (Cheng et al. (1996)); and an electrical potential-dependent K+ leak mode for TetK and highly truncated segments thereof that can facilitate net K+ uptake (Guay, G. G., Tuckman, M., McNicholas, P., and Rothstein, D. M. (1993) J. Bacteriol. 175, 4927-4929). Studies of membrane vesicles from Escherichia coli expressing low levels of complete and 3'-truncated versions of tetA(L) or tetK, now show that the full-length versions of both transporters catalyze electrogenic antiport and that demonstration of electrogenicity depends upon use of a low chloride buffer for the assay. The K+ uptake mode, assayed via 86Rb+ uptake, was also catalyzed by both full-length TetA(L) and TetK. This mode does not represent a potential-dependent leak. Such a leak was not demonstrable in energized membrane vesicles. Rather, Rb+ uptake occurred in right-side-out vesicles when the intravesicular space contained either Na+ or K+ but not choline. If an outwardly directed gradient of Na+ or K+ was present, Rb+ uptake occurred without energization in vesicles from cells transformed with a plasmid containing tetA(L) or tetK but not a control plasmid. Experiments in which a comparable exchange was carried out in low chloride buffers to which oxonol was added confirmed that the exchange was electrogenic. Thus, the K+ uptake mode is proposed to be a mode of the electrogenic monovalent cation/H+ antiport activity of TetA(L) and TetK in which K+ takes the place of the external protons. Truncated TetK and TetA(L) failed to catalyze either Tc-metal/H+ or Na+/H+ antiport in energized everted vesicles. Truncated TetK, but not TetA(L), did, however, exhibit modest, electrogenic Na+(K+)/Rb+ exchange as well as a small, potential-dependent leak of Rb+. The C-terminal halves of the TetA(L) and TetK proteins are thus required both for proton-coupled active transport activities of the multifunctional transporter and, perhaps, for minimizing cation leakiness.