Zhang Wei, Bogdanov Mikhail, Pi Jing, Pittard A James, Dowhan William
Department of Biochemistry and Molecular Biology, University of Texas, Medical School, Houston, Texas 77030, USA.
J Biol Chem. 2003 Dec 12;278(50):50128-35. doi: 10.1074/jbc.M309840200. Epub 2003 Oct 1.
Once inserted, transmembrane segments of polytopic membrane proteins are generally considered stably oriented due to the large free energy barrier to topological reorientation of adjacent extramembrane domains. However, the topology and function of the polytopic membrane protein lactose permease of Escherichia coli are dependent on the membrane phospholipid composition, revealing topological dynamics of transmembrane domains after stable membrane insertion (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116). In this study, we show that the high affinity phenylalanine permease PheP shares many similarities with lactose permease. PheP assembled in a mutant of E. coli lacking phosphatidylethanolamine (PE) exhibited significantly reduced active transport function and a complete inversion in topological orientation of the N terminus and adjoining transmembrane hairpin loop compared with PheP in a PE-containing strain. Introduction of PE following the assembly of PheP triggered a reorientation of the N terminus and adjacent hairpin to their native orientation associated with regain of wild-type transport function. The reversible orientation of these secondary transport proteins in response to a change in phospholipid composition might be a result of inherent conformational flexibility necessary for transport function or during protein assembly.
多跨膜蛋白一旦插入，由于相邻膜外结构域拓扑重排的自由能障碍较大，其跨膜片段通常被认为是稳定取向的。然而，大肠杆菌多跨膜蛋白乳糖通透酶的拓扑结构和功能取决于膜磷脂组成，这揭示了其在稳定插入膜后跨膜结构域的拓扑动力学（博格达诺夫，M.，希科克，P. N.，和多万，W.（2002年）《欧洲分子生物学组织杂志》21，2107 - 2116）。在本研究中，我们表明高亲和力苯丙氨酸通透酶PheP与乳糖通透酶有许多相似之处。与含磷脂酰乙醇胺（PE）菌株中的PheP相比，在缺乏PE的大肠杆菌突变体中组装的PheP表现出显著降低的主动运输功能，并且其N端和相邻跨膜发夹环的拓扑取向完全反转。在PheP组装后引入PE会触发N端和相邻发夹向其天然取向的重新定向，同时恢复野生型运输功能。这些次级转运蛋白响应磷脂组成变化的可逆取向可能是运输功能或蛋白质组装过程中所需固有构象灵活性的结果。
