Guilvout Ingrid, Chami Mohamed, Berrier Catherine, Ghazi Alexandre, Engel Andreas, Pugsley Anthony P, Bayan Nicolas
Molecular Genetics Unit and CNRS URA2172, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris, France.
J Mol Biol. 2008 Sep 26;382(1):13-23. doi: 10.1016/j.jmb.2008.06.055. Epub 2008 Jun 28.
Synthesis of the Klebsiella oxytoca outer membrane secretin PulD, or its membrane-associated core domain, in a liposome-supplemented Escherichia coli in vitro transcription-translation system resulted in the formation of multimers that appeared as typical dodecameric secretin rings when examined by negative-stain electron microscopy. Cryo-electron microscopy of unstained liposomes and differential extraction by urea indicated that the secretin particles were inserted into the liposome membranes. When made in the presence of the detergent Brij-35, PulD and the core domain were synthesized as monomers. Both proteins caused almost immediate growth cessation when synthesized in E. coli without a signal peptide. The small amounts of PulD synthesized before cell death appeared as multimers with characteristics similar to those of the normal outer membrane secretin dodecamers. It was concluded that multimerization and membrane insertion are intrinsic properties of secretin PulD that are independent of a specific membrane environment or membrane-associated factors. The closely related Erwinia chrysanthemi secretin OutD behaved similarly to PulD in all assays, but the more distantly related Neisseria meningitidis secretin PilQ did not form multimers either when made in vitro in the presence of liposomes or when made in E. coli without its signal peptide. This is the first report of the apparently spontaneous in vitro assembly and membrane insertion of a large outer membrane protein complex. Spontaneous multimerization and insertion appear to be restricted to outer membrane proteins closely related to PulD.
在补充脂质体的大肠杆菌体外转录 - 翻译系统中合成产酸克雷伯菌外膜分泌素PulD或其膜相关核心结构域，结果形成了多聚体，通过负染电子显微镜检查时呈现典型的十二聚体分泌素环。对未染色脂质体的冷冻电子显微镜观察和尿素差异提取表明，分泌素颗粒插入到脂质体膜中。当在去污剂Brij - 35存在下合成时，PulD和核心结构域以单体形式合成。在没有信号肽的大肠杆菌中合成时，这两种蛋白质几乎立即导致生长停止。在细胞死亡前合成的少量PulD呈现出与正常外膜分泌素十二聚体相似特征的多聚体。得出的结论是，多聚化和膜插入是分泌素PulD的固有特性，独立于特定的膜环境或膜相关因子。在所有测定中，密切相关的菊欧文氏菌分泌素OutD的行为与PulD相似，但关系较远的脑膜炎奈瑟菌分泌素PilQ在脂质体存在下体外合成时或在没有信号肽的大肠杆菌中合成时都不形成多聚体。这是关于大型外膜蛋白复合物明显自发体外组装和膜插入的首次报道。自发多聚化和插入似乎仅限于与PulD密切相关的外膜蛋白。
