Affiliation:
1. Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029
Abstract
ABSTRACT
The Tet(L) protein encoded in the
Bacillus subtilis
chromosome and the closely related Tet(K) protein from
Staphylococcus aureus
plasmids are multifunctional antiporters that have three cytoplasmic efflux substrates: a tetracycline-divalent metal (TC-Me
2+
) complex that bears a net single positive charge, Na
+
, and K
+
. Tet(L) and Tet(K) had been shown to couple efflux of each of these substrates to influx of H
+
as the coupling ion. In this study, competitive cross-inhibition between K
+
and other cytoplasmic efflux substrates was demonstrated. Tet(L) and Tet(K) had also been shown to use K
+
as an alternate coupling ion in support of Na
+
or K
+
efflux. Here they were shown to couple TC-Me
2+
efflux to K
+
uptake as well, exhibiting greater use of K
+
as a coupling ion as the external pH increased. The substrate and coupling ion preferences of the two Tet proteins differed, especially in the higher preference of Tet(K) than Tet(L) for K
+
, both as a cytoplasmic efflux substrate and as an external coupling ion. Site-directed mutagenesis was employed to test the hypothesis that some feature of the putative “antiporter motif,” motif C, of Tet proteins would be involved in these characteristic preferences. Mutation of the A157 in Tet(L) to a hydroxyamino acid resulted in a more Tet(K)-like K
+
preference both as coupling ion and efflux substrate. A reciprocal S157A mutant of Tet(K) exhibited reduced K
+
preference. Competitive inhibition among substrates and the parallel effects of the single mutation upon K
+
preference, as both an efflux substrate and coupling ion, are compatible with a model in which a single translocation pathway through the Tet(L) and Tet(K) transporters is used both for the cytoplasmic efflux substrates and for the coupling ions, in an alternating fashion. However, the effects of the A157 and other mutations of Tet(L) indicate that even if there are a shared binding site and translocation pathway, some elements of that pathway are used by all substrates and others are important only for particular substrates.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Reference52 articles.
1. Blostein, R., A. Wilczynska, S. J. D. Karlish, J. M. Arguello, and J. B. Lingrel. 1999. Evidence that Ser775 in the alpha subunit of the Na,K-ATPase is a residue in the cation binding pocket. J. Biol. Chem.272:24987-24993.
2. VmrA, a Member of a Novel Class of Na
+
-Coupled Multidrug Efflux Pumps from
Vibrio parahaemolyticus
3. Cheng, J., A. A. Guffanti, and T. A. Krulwich. 1994. The chromosomal tetracycline resistance locus of Bacillus subtilis encodes a Na+/H+ antiporter that is physiologically important at elevated pH. J. Biol. Chem.269:27365-27371.
4. Chromosomal tetA(L) gene of Bacillus subtilis: regulation of expression and physiology of a tetA(L) deletion strain
5. Cheng, J., D. B. Hicks, and T. A. Krulwich. 1996. The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline-cobalt/H+ and Na+/H+ exchange. Proc. Natl. Acad. Sci. USA93:14446-14451.
Cited by
41 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献