Jin Jie, Guffanti Arthur A, Bechhofer David H, Krulwich Terry A
Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA.
J Bacteriol. 2002 Sep;184(17):4722-32. doi: 10.1128/JB.184.17.4722-4732.2002.
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.
枯草芽孢杆菌染色体中编码的Tet(L)蛋白和来自金黄色葡萄球菌质粒的密切相关的Tet(K)蛋白是多功能反向转运蛋白，它们有三种细胞质外排底物：带有净单正电荷的四环素-二价金属（TC-Me(2+)）复合物、Na+和K+。Tet(L)和Tet(K)已被证明将这些底物中的每一种的外排与作为偶联离子的H+的内流相偶联。在本研究中，证明了K+与其他细胞质外排底物之间的竞争性交叉抑制。Tet(L)和Tet(K)也已被证明使用K+作为替代偶联离子来支持Na+或K+的外排。在此研究中，它们还被证明将TC-Me(2+)的外排与K+的摄取相偶联，随着外部pH值的增加，对K+作为偶联离子的利用增加。这两种Tet蛋白的底物和偶联离子偏好不同，特别是Tet(K)比Tet(L)对K+有更高的偏好，无论是作为细胞质外排底物还是作为外部偶联离子。采用定点诱变来检验这样的假设，即Tet蛋白假定的“反向转运基序”（基序C）的某些特征会参与这些特征性偏好。将Tet(L)中的A157突变为羟基氨基酸会导致作为偶联离子和外排底物时更像Tet(K)的K+偏好。Tet(K)的反向S157A突变体表现出降低的K+偏好。底物之间的竞争性抑制以及单个突变对K+偏好的平行影响，无论是作为外排底物还是偶联离子，都与一个模型相符，在该模型中，通过Tet(L)和Tet(K)转运体的单个转运途径以交替方式用于细胞质外排底物和偶联离子。然而，Tet(L)的A157和其他突变的影响表明，即使存在共享的结合位点和转运途径，该途径的一些元件被所有底物使用，而其他元件仅对特定底物重要。
