Membrane topology of a multidrug efflux transporter, AcrB, in Escherichia coli
Membrane topology of a multidrug efflux transporter, AcrB, in Escherichia coli
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DOI：
10.1093/oxfordjournals.jbchem.a003069
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发表时间：
2002-01-01
影响因子：
2.7
通讯作者：
Yamaguchi, A
中科院分区：
文献类型：
--
作者：
Fujihira, E;Tamura, N;Yamaguchi, A
大肠杆菌中的AcrA/B是一个多组分系统，负责对多种有毒化合物的内在抗性，并可能与外膜蛋白TolC合作。本研究从大肠杆菌W3104染色体上克隆了acrAB基因。为了确定内膜成分AcrB的拓扑结构，我们采用化学标记方法分析了引入单个半胱氨酸残基的AcrB突变体。无半胱氨酸的AcrB突变体，其中两个固有的Cys残基被Ala取代，保留了完全的耐药性。我们构建了33个半胱氨酸突变体，其中一个半胱氨酸被引入到每个假定的无半胱氨酸AcrB的亲水性环区。研究了[C-14] n -乙基马来酰亚胺（NEM）与Cys残基的结合，以及NEM与膜外马来酰亚胺（4-乙酰酰胺-4'-马来酰亚胺-2,2'-二磺酸）在完整细胞中的结合竞争。结果表明，N端和c端位于细胞膜的细胞质表面，两个大环位于细胞膜的质周表面。结果支持AcrB的12膜跨越结构。四个短的周质环区域中的三个被两个大的周质环域覆盖，直到两个大的周质环中的一个被移除才暴露于水相。
AcrA/B in Escherichia coli is a multicomponent system responsible for intrinsic resistance to a wide range of toxic compounds, and probably cooperates with the outer membrane protein TolC. In this study, acrAB genes were cloned from the E. coli W3104 chromosome. To determine the topology of the inner membrane component AcrB, we employed a chemical labeling approach to analyse mutants of AcrB in which a single cysteine residue had been introduced. The cysteine-free AcrB mutant, in which the two intrinsic Cys residues were replaced by Ala, retained full drug resistance. We constructed 33 cysteine mutants in which a single cysteine was introduced into each putative hydrophilic loop region of the cysteine-free AcrB. The binding of [C-14]N-ethylmaleimide (NEM) to the Cys residue and the competition of NEM binding with the binding of a membrane-impermeant maleimide, 4-acetamide-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), in intact cells were investigated. The results revealed that the N- and C-terminals are localized on the cytoplasmic surface of the membrane and the two large loops are localized on the periplasmic surface of the membrane. The results supported the 12-membrane-spanning structure of AcrB. Three of the four short periplasmic loop regions were covered by the two large periplasmic loop domains and were not exposed to the water phase until one of the two large periplasmic loops was removed.