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, 49274929). 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.