Koch H G, Hengelage T, Neumann-Haefelin C, MacFarlane J, Hoffschulte H K, Schimz K L, Mechler B, Müller M
Institut für Biochemie und Molekularbiologie, Universität Freiburg, D-79104 Freiburg, Germany.
Mol Biol Cell. 1999 Jul;10(7):2163-73. doi: 10.1091/mbc.10.7.2163.
The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coli which, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4. 5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and DeltamicroH+. In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.
比较了分泌蛋白跨大肠杆菌质膜转运以及膜蛋白整合到大肠杆菌质膜中的分子要求。这是在一种来自大肠杆菌的新型无细胞系统中实现的，该系统通过广泛的亚分级分离，同时使SecA/SecB和信号识别颗粒（SRP）组分Ffh（P48）、4.5S RNA和FtsY缺失。两种膜蛋白整合到大肠杆菌的内翻质膜囊泡中需要所有三种SRP组分，且不能由SecA、SecB和DeltamicroH +驱动。相比之下，这些是分泌蛋白转运到膜囊泡中所需的唯一组分，在这个过程中SRP组分完全无活性。我们的结果在证实先前体内研究的同时，首次提供了体外证据，证明大肠杆菌中多聚体内膜蛋白的整合依赖于SRP。此外，它们表明SRP和SecA/SecB具有不同的底物特异性，导致大肠杆菌中膜蛋白和分泌蛋白有两种不同的靶向机制。两种靶向途径在转运孔处相交，因为它们同样受到受阻转运通道的影响。
