Yerushalmi H, Lebendiker M, Schuldiner S
Division of Microbial and Molecular Ecology, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Givat Ram, Israel.
J Biol Chem. 1995 Mar 24;270(12):6856-63. doi: 10.1074/jbc.270.12.6856.
The smallest membrane protein shown to catalyze ion-coupled transport is documented in this report. A gene coding for a small 110-amino acid membrane protein (emrE or mvrC) has been previously identified and cloned and shown to render Escherichia coli cells resistant to methyl viologen and to ethidium. In this report, it is shown that the resistance is due to extrusion of the toxic compounds in a process that requires a proton electrochemical gradient rather than ATP. For this purpose, cells in which the unc gene was inactivated were used so that the interconversion between the proton gradient and ATP is not possible, and the effect of agents, which specifically affect either of them, was tested on transport of ethidium in the intact cell. In addition, EmrE has been overexpressed and metabolically labeled with [35S]methionine. Strikingly, the protein can be quantitatively extracted with a mixture of organic solvents such as chloroform:methanol and is practically pure after this extraction. Moreover, after addition of E. coli lipids to the chloroform:methanol extract, EmrE has been reconstituted in proteoliposomes loaded with ammonium chloride. Upon dilution of the proteoliposomes in ammonium-free medium, a pH gradient was formed that drove transport of ethidium and methyl viologen into the proteoliposome. Both substrates compete with each other and exchange with previously transported solute. EmrE is a multidrug transporter of a novel type, and, because of its size and its solubility properties, it provides a unique model to study structure-function aspects of transport reactions in ion-coupled processes.
本报告记录了已证实可催化离子偶联转运的最小膜蛋白。此前已鉴定并克隆了一个编码110个氨基酸的小膜蛋白（EmrE或MvrC）的基因，该基因可使大肠杆菌细胞对甲基紫精和溴化乙锭产生抗性。本报告表明，这种抗性是由于在一个需要质子电化学梯度而非ATP的过程中排出有毒化合物所致。为此，使用unc基因失活的细胞，以使质子梯度和ATP之间无法相互转换，并测试了特异性影响其中任何一个的试剂对完整细胞中溴化乙锭转运的影响。此外，EmrE已被过量表达并用[35S]甲硫氨酸进行代谢标记。引人注目的是，该蛋白可用氯仿：甲醇等有机溶剂混合物进行定量提取，提取后几乎是纯的。此外，将大肠杆菌脂质添加到氯仿：甲醇提取物中后，EmrE已在装载有氯化铵的蛋白脂质体中重新组装。在无铵培养基中稀释蛋白脂质体时，形成了一个pH梯度，该梯度驱动溴化乙锭和甲基紫精转运到蛋白脂质体中。两种底物相互竞争，并与先前转运的溶质进行交换。EmrE是一种新型的多药转运蛋白，由于其大小和溶解性，它为研究离子偶联过程中转运反应的结构-功能方面提供了一个独特的模型。
