VirE2, a type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens
VirE2, a type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens
复制标题
DOI：
10.1046/j.1365-2958.2003.03669.x
复制
发表时间：
2003-09-01
影响因子：
3.6
通讯作者：
Christie, PJ
中科院分区：
文献类型：
--
作者：
Atmakuri, K;Ding, ZY;Christie, PJ
根癌农杆菌在感染过程中将致癌DNA和效应蛋白转移到植物细胞中。底物跨细菌细胞包膜的易位由IV型分泌（TFS）系统介导，该系统由VirB蛋白以及VirD 4组成，VirD 4是涉及DNA转移中间体与分泌机器偶联的内膜蛋白大家族的成员。在这项研究中，我们证明了新的细胞学筛选-双杂交（C2 H）测定和双分子荧光互补（BiFC）-和化学交联蛋白复合物的免疫沉淀，VirE 2效应蛋白直接与VirD 4偶联蛋白在细胞极的A。根瘤菌截短衍生物的分析表明，VirE 2通过其C末端与VirD 4相互作用，并且，进一步，VirD 4的NH 2-末端跨膜结构域被破坏以形成复合物。VirE 2与VirD 4相互作用独立于virB编码的转移机器和T菌毛、推定的周质伴侣AcvB和VirJ以及T-DNA转移中间体。最后，VirE 2被募集到极性定位的VirD 4作为与其稳定分泌伴侣VirE 1的复合物，但效应子-偶联蛋白相互作用不依赖于伴侣结合。总之，我们的研究结果首次建立了IV型分泌系统的蛋白质底物被招募到偶联蛋白超家族的成员中。
Agrobacterium tumefaciens transfers oncogenic DNA and effector proteins to plant cells during the course of infection. Substrate translocation across the bacterial cell envelope is mediated by a type IV secretion (TFS) system composed of the VirB proteins, as well as VirD4, a member of a large family of inner membrane proteins implicated in the coupling of DNA transfer intermediates to the secretion machine. In this study, we demonstrate with novel cytological screens - a two-hybrid (C2H) assay and bimolecular fluorescence complementation (BiFC) - and by immunoprecipitation of chemically cross-linked protein complexes that the VirE2 effector protein interacts directly with the VirD4 coupling protein at cell poles of A. tumefaciens. Analyses of truncation derivatives showed that VirE2 interacts via its C terminus with VirD4, and, further, an NH2-terminal membrane-spanning domain of VirD4 is dispensable for complex formation. VirE2 interacts with VirD4 independently of the virB - encoded transfer machine and T pilus, the putative periplasmic chaperones AcvB and VirJ, and the T-DNA transfer intermediate. Finally, VirE2 is recruited to polar-localized VirD4 as a complex with its stabilizing secretion chaperone VirE1, yet the effector - coupling protein interaction is not dependent on chaperone binding. Together, our findings establish for the first time that a protein substrate of a type IV secretion system is recruited to a member of the coupling protein superfamily.