Tamura N, Konishi S, Iwaki S, Kimura-Someya T, Nada S, Yamaguchi A
Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
J Biol Chem. 2001 Jun 8;276(23):20330-9. doi: 10.1074/jbc.M007993200. Epub 2001 Feb 6.
Bacterial Tn10-encoded metal-tetracycline/H(+) antiporter was the first found drug exporter and has been studied as a paradigm of antiporter-type major facilitator superfamily transporters. Here the 400 amino acid residues of this protein were individually replaced by cysteine except for the initial methionine. As a result, we could obtain a complete map of the functionally or structurally important residues. In addition, we could determine the precise boundaries of all the transmembrane segments on the basis of the reactivity with N-ethylmaleimide (NEM). The NEM binding results indicated the presence of a transmembrane water-filled channel in the transporter. The twelve transmembrane segments can be divided into three groups; four are totally embedded in the hydrophobic interior, four face a putative water-filled channel along their full length, and the remaining four face the channel for half their length, the other halves being embedded in the hydrophobic interior. These three types of transmembrane segments are mutually arranged with a 4-fold symmetry. The competitive binding of membrane-permeable and -impermeable SH reagents in intact cells indicates that the transmembrane water-filled channel has a thin barrier against hydrophilic molecules in the middle of the transmembrane region. Inhibition and stimulation of NEM binding in the presence of tetracycline reflects the substrate-induced protection or conformational change of the Tn10-encoded metal-tetracycline/H(+) antiporter. The mutations protected from NEM binding by tetracycline were mainly located around the permeability barrier in the N-terminal half, suggesting the location of the substrate binding site.
细菌Tn10编码的金属四环素/H(+)反向转运蛋白是最早发现的药物外排蛋白，已作为反向转运体类型的主要易化子超家族转运蛋白的范例进行研究。在此，除起始甲硫氨酸外，该蛋白的400个氨基酸残基均被半胱氨酸逐个取代。结果，我们能够获得功能或结构重要残基的完整图谱。此外，我们可以根据与N-乙基马来酰亚胺（NEM）的反应性确定所有跨膜区段的精确边界。NEM结合结果表明该转运蛋白中存在一个跨膜的充满水的通道。十二个跨膜区段可分为三组；四个完全嵌入疏水内部，四个全长面对一个假定的充满水的通道，其余四个一半长度面对通道，另一半嵌入疏水内部。这三种类型的跨膜区段以四重对称相互排列。完整细胞中膜可透性和膜不可透性SH试剂的竞争性结合表明，跨膜充满水的通道在跨膜区域中间对亲水分子有一个薄屏障。在四环素存在下对NEM结合的抑制和刺激反映了底物诱导的Tn10编码的金属四环素/H(+)反向转运蛋白的保护或构象变化。被四环素保护而免受NEM结合的突变主要位于N端一半的通透性屏障周围，提示底物结合位点的位置。
