Negative Dominance Studies Demonstrate the Oligomeric Structure of EmrE, a Multidrug Antiporter from Escherichia coli*
Negative Dominance Studies Demonstrate the Oligomeric Structure of EmrE, a Multidrug Antiporter from Escherichia coli*
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负显性研究证明了 EmrE 的寡聚结构，EmrE 是一种来自大肠杆菌的多药逆向转运蛋白*
DOI：
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发表时间：
1996
影响因子：
4.8
通讯作者：
S. Schuldiner
中科院分区：
文献类型：
--
作者：
H. Yerushalmi;M. Lebendiker;S. Schuldiner
EmrE是已知的最小的离子偶联转运蛋白，是大肠杆菌12-kDa蛋白，80%螺旋，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。由于它比经典的共有12个跨膜片段转运蛋白小许多倍，因此确定其寡聚状态特别有趣。为此目的，已经产生了一系列非功能性突变体，并进行了表征，以测试它们在混合后对野生型蛋白质活性的影响。与野生型相反，这些突变体不赋予对甲基紫精、溴化乙锭或一系列其他毒物的抗性。每个非功能性突变体与野生型蛋白质的共表达导致功能性转运蛋白赋予对几种毒物的抗性的能力降低。为了在体外进行混合实验，所有的突变体已通过用有机溶剂提取纯化，在脂蛋白体中重构，并发现无活性。当与野生型蛋白共重构时，它们以剂量依赖性形式抑制后者的活性直至完全抑制。我们认为，这种抑制是由于混合寡聚体的形成，其中一个非功能性亚基的存在导致完全失活。基于后一种假设的结果的二项式分析没有提供统计学上显著的答案，但表明寡聚体由三个亚基组成。所描述的结果提供了第一个在体外演示的功能性低聚体结构的离子耦合转运蛋白。
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.
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DOI：
10.1073/pnas.82.4.1074
发表时间：
1985-01-01
影响因子：
11.1
作者：
TABOR, S;RICHARDSON, CC
通讯作者：
RICHARDSON, CC
葡萄球菌多重耐药基因产物是新蛋白质家族的成员。
影响因子：
2.6
作者：
Grinius,L;Dreguniene,G;Goldberg,EB;Liao,CH;Projan,SJ
通讯作者：
Projan,SJ
影响因子：
3.5
作者：
HO, SN;HUNT, HD;PEASE, LR
通讯作者：
PEASE, LR
影响因子：
10.5
作者：
C. A. Doige;G. Ames
通讯作者：
C. A. Doige;G. Ames
影响因子：
7.5
作者：
GRIFFITH J K;BAKER M E;HENDERSON P J F
通讯作者：
HENDERSON P J F