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J Biol Chem, Vol. 273, Issue 41, 26447-26454, October 9, 1998
From the Department of Biochemistry, Mount Sinai School of
Medicine, New York, New York 10029
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.
Electrogenic Antiport Activities of the Gram-positive Tet
Proteins Include a Na+(K+)/K+ Mode
That Mediates Net K+ Uptake
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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