Higgs P I, Myers P S, Postle K
Departments of Microbiology, Washington State University, Pullman, Washington 99164-4233, USA.
J Bacteriol. 1998 Nov;180(22):6031-8. doi: 10.1128/JB.180.22.6031-6038.1998.
The cytoplasmic membrane proteins ExbB and ExbD support TonB-dependent active transport of iron siderophores and vitamin B12 across the essentially unenergized outer membrane of Escherichia coli. In this study, in vivo formaldehyde cross-linking analysis was used to investigate the interactions of T7 epitope-tagged ExbB or ExbD proteins. ExbB and ExbD each formed two unique cross-linked complexes which were not dependent on the presence of TonB, the outer membrane receptor protein FepA, or the other Exb protein. Cross-linking analysis of ExbB- and ExbD-derived size variants demonstrated instead that these ExbB and ExbD complexes were homodimers and homotrimers and suggested that ExbB also interacted with an unidentified protein(s). Cross-linking analysis of epitope-tagged ExbB and ExbD proteins with TonB antisera afforded detection of a previously unrecognized TonB-ExbD cross-linked complex and confirmed the composition of the TonB-ExbB cross-linked complex. The implications of these findings for the mechanism of TonB-dependent energy transduction are discussed.
细胞质膜蛋白ExbB和ExbD支持铁载体和维生素B12通过大肠杆菌基本无能量的外膜进行TonB依赖性主动运输。在本研究中，体内甲醛交联分析用于研究T7表位标签化的ExbB或ExbD蛋白之间的相互作用。ExbB和ExbD各自形成了两种独特的交联复合物，它们不依赖于TonB、外膜受体蛋白FepA或另一种Exb蛋白的存在。相反，对ExbB和ExbD衍生的大小变体进行交联分析表明，这些ExbB和ExbD复合物是同型二聚体和同型三聚体，并表明ExbB还与一种未鉴定的蛋白质相互作用。用TonB抗血清对表位标签化的ExbB和ExbD蛋白进行交联分析，检测到一种以前未识别的TonB-ExbD交联复合物，并证实了TonB-ExbB交联复合物的组成。讨论了这些发现对TonB依赖性能量转导机制的影响。
