In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state
In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state
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DOI：
10.1073/pnas.0306533101
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发表时间：
2004-02-10
影响因子：
11.1
通讯作者：
Schuldiner, S
中科院分区：
文献类型：
--
作者：
Elbaz, Y;Steiner-Mordoch, S;Schuldiner, S
EmRE是一种来自大肠杆菌的小型多药转运蛋白，为研究多表位膜蛋白提供了一个独特的模型。在这里，我们展示了它在无细胞系统中以全功能形式合成。洗涤剂溶解的蛋白质以高亲和力结合底物，当重组为蛋白脂质体时，以DelTamu(H)(+)依赖的方式运输底物。在这里，我们使用无细胞体系来研究EmRE的寡聚性质。EmRE是一种低聚物，但功能性低聚物的大小尚未明确确定。在无细胞系统中，两个质粒的共表达证实了功能互补，下拉实验证实了基本功能单位是二聚体。通过使用独特的半胱氨酸残基之间的交联，检测到了二聚体之间的额外相互作用。这一发现暗示了二聚体的存在。
EmrE is a small multidrug transporter from Escherichia coli that provides a unique model for the study of polytopic membrane proteins. Here, we show its synthesis in a cell-free system in a fully functional form. The detergent-solubilized protein binds substrates with high affinity and, when reconstituted into proteoliposomes, transports substrate in a Deltamu(H)(+)-dependent fashion. Here, we used the cell-free system to study the oligomeric properties of EmrE. EmrE functions as an oligomer, but the size of the functional oligomer has not been established unequivocally. Coexpression of two plasmids in the cell-free system allowed demonstration of functional complementation and pull-down experiments confirmed that the basic functional unit is the dimer. An additional interaction between dimers has been detected by using crosslinking between unique Cys residues. This finding implies the existence of a dimer of dimers.