Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University SE-106 91 Stockholm, Sweden.
J Biol Chem. 2012 Jul 27;287(31):26052-9. doi: 10.1074/jbc.M112.357590. Epub 2012 Jun 14.
The bacterial multidrug transporter EmrE is a dual-topology membrane protein and as such is able to insert into the membrane in two opposite orientations. The functional form of EmrE is a homodimer; however, the relative orientation of the subunits in the dimer is under debate. Using EmrE variants with fixed, opposite orientations in the membrane, we now show that, although the proteins are able to form parallel dimers, an antiparallel organization of the subunits in the dimer is preferred. Blue-native PAGE analyses of intact oligomers and disulfide cross-linking demonstrate that in membranes, the proteins form parallel dimers only if no oppositely orientated partner is present. Co-expression of oppositely orientated proteins almost exclusively yields antiparallel dimers. Finally, parallel dimers can be disrupted and converted into antiparallel dimers by heating of detergent-solubilized protein. Importantly, in vivo function is correlated clearly to the presence of antiparallel dimers. Our results suggest that an antiparallel arrangement of the subunits in the dimer is more stable than a parallel organization and likely corresponds to the functional form of the protein.
细菌多药转运蛋白 EmrE 是一种具有双重拓扑结构的膜蛋白，因此能够以两种相反的方向插入膜中。EmrE 的功能形式是同源二聚体；然而，二聚体中亚基的相对取向仍存在争议。使用在膜中具有固定的相反取向的 EmrE 变体，我们现在表明，尽管这些蛋白质能够形成平行二聚体，但二聚体中亚基的反平行组织更为优选。完整寡聚体的蓝色非变性 PAGE 分析和二硫键交联表明，在膜中，只有不存在相反取向的伴侣时，蛋白质才会形成平行二聚体。相反取向的蛋白质的共表达几乎只产生反平行二聚体。最后，通过去污剂溶解蛋白的加热可以破坏平行二聚体并将其转化为反平行二聚体。重要的是，体内功能与反平行二聚体的存在明显相关。我们的结果表明，二聚体中亚基的反平行排列比平行组织更稳定，并且可能对应于蛋白质的功能形式。
