Sandkvist M, Bagdasarian M, Howard S P, DiRita V J
University of Michigan Medical School, Department of Microbiology and Immunology, Ann Arbor, USA.
EMBO J. 1995 Apr 18;14(8):1664-73. doi: 10.1002/j.1460-2075.1995.tb07155.x.
Vibrio cholerae secretes a number of proteins important for virulence, including cholera toxin. This process requires the products of the eps genes which have homologues in genera such as Aeromonas, Klebsiella and Pseudomonas and are thought to form a membrane-associated multiprotein complex. Here we show that the putative nucleotide-binding protein EpsE is associated with and stabilized by the cytoplasmic membrane via interaction with EpsL. Analysis of fusion proteins between EpsE and the homologous ExeE from Aeromonas hydrophila demonstrates that the N-terminus of EpsE contains the EpsL binding domain and determines species specificity. An intact Walker A box, commonly found in ATP-binding proteins, is required for activity of EpsE in vivo and for autophosphorylation of purified EpsE in vitro. These results indicate that both the kinase activity of EpsE as well as its ability to interact with the putative cytoplasmic membrane protein EpsL are required for translocation of toxin across the outer membrane in Vibrio cholerae.
霍乱弧菌分泌多种对毒力至关重要的蛋白质，包括霍乱毒素。这一过程需要eps基因的产物，这些基因在气单胞菌属、克雷伯菌属和假单胞菌属等菌属中有同源物，并且被认为形成一种膜相关的多蛋白复合物。在此我们表明，假定的核苷酸结合蛋白EpsE通过与EpsL相互作用而与细胞质膜相关联并得到稳定。对EpsE与嗜水气单胞菌同源的ExeE之间的融合蛋白的分析表明，EpsE的N端包含EpsL结合结构域并决定物种特异性。完整的沃克A框常见于ATP结合蛋白中，它是EpsE在体内发挥活性以及纯化的EpsE在体外进行自磷酸化所必需的。这些结果表明，EpsE的激酶活性及其与假定的细胞质膜蛋白EpsL相互作用的能力都是霍乱弧菌毒素跨外膜转运所必需的。
