Yerushalmi H, Lebendiker M, Schuldiner S
Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
J Biol Chem. 1996 Dec 6;271(49):31044-8. doi: 10.1074/jbc.271.49.31044.
EmrE, the smallest known ion-coupled transporter, is an Escherichia coli 12-kDa protein 80% helical and soluble in organic solvents. EmrE is a polyspecific antiporter that exchanges hydrogen ions with aromatic toxic cations such as methyl viologen. Since it is many times smaller than the classical consensus 12 transmembrane segments transporters, it was particularly interesting to determine its oligomeric state. For this purpose, a series of nonfunctional mutants has been generated and characterized to test their effect on the activity of the wild-type protein upon mixing. As opposed to the wild type, these mutants do not confer resistance to methyl viologen, ethidium bromide, or a series of other toxicants. Co-expression of each of the nonfunctional mutants with the wild-type protein results in a reduction in the ability of the functional transporter to confer resistance to several toxicants. To perform mixing experiments in vitro, all the mutants have been purified by extraction with organic solvents, reconstituted in proteoliposomes, and found to be inactive. When co-reconstituted with wild-type protein, they inhibit the activity of the latter in a dose-dependent form up to full inhibition. We assume that this inhibition is due to the formation of mixed oligomers in which the presence of one nonfunctional subunit causes full inactivation. A binomial analysis of the results based on the latter assumptions do not provide statistically significant answers but suggests that the oligomer is composed of three subunits. The results described provide the first in vitro demonstration of the functional oligomeric structure of an ion-coupled transporter.
EmrE是已知最小的离子偶联转运蛋白，是一种大肠杆菌12千道尔顿的蛋白质，80%为螺旋结构，可溶于有机溶剂。EmrE是一种多特异性反向转运蛋白，可与甲基紫精等芳香族有毒阳离子交换氢离子。由于它比经典的12个跨膜片段的转运蛋白小很多倍，因此确定其寡聚状态特别有趣。为此，已构建并表征了一系列无功能突变体，以测试它们在与野生型蛋白混合时对其活性的影响。与野生型不同，这些突变体对甲基紫精、溴化乙锭或一系列其他毒物不具有抗性。将每个无功能突变体与野生型蛋白共表达会导致功能性转运蛋白赋予对几种毒物抗性的能力降低。为了在体外进行混合实验，所有突变体都通过有机溶剂萃取进行了纯化，重组成蛋白脂质体，结果发现它们无活性。当与野生型蛋白共重组时，它们以剂量依赖的形式抑制后者的活性，直至完全抑制。我们假设这种抑制是由于形成了混合寡聚体，其中一个无功能亚基的存在导致完全失活。基于后一种假设对结果进行的二项式分析没有提供具有统计学意义的答案，但表明寡聚体由三个亚基组成。所述结果首次在体外证明了离子偶联转运蛋白的功能性寡聚结构。
