Bouveret E, Rigal A, Lazdunski C, Bénédetti H
Laboratoire d'Ingénierie et de, Dynamique des Systèmes Membranaires, Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France.
Mol Microbiol. 1997 Mar;23(5):909-20. doi: 10.1046/j.1365-2958.1997.2751640.x.
Colicins use two envelope multiprotein systems to reach their cellular target in susceptible cells of Escherichia coli: the Tol system for group A colicins and the TonB system for group B colicins. The N-terminal domain of colicins is involved in the translocation step. To determine whether it interacts in vivo with proteins of the translocation system, constructs were designed to produce and export to the cell periplasm the N-terminal domains of colicin E3 (group A) and colicin B (group B). Producing cells became specifically tolerant to entire extracellular colicins of the same group. The periplasmic N-terminal domains therefore compete with entire colicins for proteins of the translocation system and thus interact in situ with these proteins on the inner side of the outer membrane. In vivo cross-linking and co-immunoprecipitation experiments in cells producing the colicin E3 N-terminal domain demonstrated the existence of a 120 kDa complex containing the colicin domain and TolB. After in vitro cross-linking experiments with these two purified proteins, a 120 kDa complex was also obtained. This suggests that the complex obtained in vivo contains exclusively TolB and the colicin E3 domain. The N-terminal domain of a translocation-defective colicin E3 mutant was found to no longer interact with TolB. Hence, this interaction must play an important role in colicin E3 translocation.
A 组大肠杆菌素利用 Tol 系统，B 组大肠杆菌素利用 TonB 系统。大肠杆菌素的 N 端结构域参与转运步骤。为了确定它在体内是否与转运系统的蛋白质相互作用，构建了一些载体，用于在细胞周质中产生并输出大肠杆菌素 E3（A 组）和大肠杆菌素 B（B 组）的 N 端结构域。产生这些结构域的细胞对同组的完整细胞外大肠杆菌素产生了特异性耐受性。因此，周质中的 N 端结构域与完整的大肠杆菌素竞争转运系统的蛋白质，从而在原位与外膜内侧的这些蛋白质相互作用。在产生大肠杆菌素 E3 N 端结构域的细胞中进行的体内交联和共免疫沉淀实验表明，存在一种包含大肠杆菌素结构域和 TolB 的 120 kDa 复合物。用这两种纯化的蛋白质进行体外交联实验后，也得到了一个 120 kDa 的复合物。这表明在体内获得的复合物仅包含 TolB 和大肠杆菌素 E3 结构域。发现一种转运缺陷型大肠杆菌素 E3 突变体的 N 端结构域不再与 TolB 相互作用。因此，这种相互作用在大肠杆菌素 E3 的转运中必定起着重要作用。
