Electrogenic antiport activities of the gram-positive tet proteins include a Na+(K+)/K+ mode that mediates net K+ uptake
Electrogenic antiport activities of the gram-positive tet proteins include a Na+(K+)/K+ mode that mediates net K+ uptake
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DOI：
10.1074/jbc.273.41.26447
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发表时间：
1998-10-09
影响因子：
4.8
通讯作者：
Krulwich, TA
中科院分区：
文献类型：
--
作者：
Guffanti, AA;Cheng, JB;Krulwich, TA
两种革兰氏阳性Tet蛋白，枯草芽孢杆菌的TetA(L)和金黄色葡萄球菌的TetK，先前已被认为具有多种催化模式和作用。其中包括：四环素(TC)-金属/H+两种蛋白质的对位蛋白(Yamaguchi，A.，Shiina，Y，，Fujihira，E.，Sawai，T.，Noguchi，N.和Sasatsu，M.(1995)费用。365,193-197；Cheng，J.Guffanti，A.，Wang，W.，Krulwich，T.A.和Bechhofer，D.H.(1996)J.细菌学。178,2853-2860)；两种蛋白的Na+(K+)/H+反向转运(程ct.(1996))；以及TetK及其高度截断的片段的电势依赖的K+泄漏模式，其可以促进净K+吸收(Guay，G.，Tuckman，M.，McNicholas，P.和Rothstein，D.M.(1993)J.细菌素。175,4927-4929)。对表达低水平完整和3‘-截短型TETA(L)或TETK的大肠杆菌膜小泡的研究表明，这两种转运蛋白的全长版本都能催化电生抗，而且电生活性的显示依赖于低氯缓冲液的使用。通过摄取Rb-86(+)检测的K+摄取模式也被全长TETA(L)和TetK所催化。这种模式并不代表潜在的泄漏。这种渗漏在通电的膜小泡中不明显。相反，当囊泡内间隙含有Na+或K+而不含胆碱时，Rb+摄取发生在右侧向外的囊泡中。如果存在向外定向的Na+或K+梯度，则转化含有TETA(L)或TETK而不含对照质粒的细胞的囊泡中发生RBI摄取，而不是能量消耗。在添加了羟苯酚的低氯缓冲液中进行可比交换的实验证实，该交换是电生的。因此，K+摄取模式被认为是TetA(L)和TetK的一价阳离子/H+反转运活性的一种模式，其中K+取代外加质子。截短型TetK和TetA(L)在外翻小泡中不能催化TC-金属/H+或Na+/H+逆向转运蛋白。然而，截短的TetK，而不是Teta(L)，确实表现出适度的、电生的Na+(K+)/Rb+交换以及小的、电位依赖的Rb+泄漏。因此，TetA(L)和TetK蛋白的C末端的一半既是多功能转运蛋白的质子耦合主动转运活动所必需的，也可能是为了最小化阳离子泄漏而需要的。
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) FEES 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 ct at. (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 Rb-86(+) 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, Rbi 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.