Activities of virE1 and the VirE1 secretion chaperone in export of the multifunctional VirE2 effector via an Agrobacterium type IV secretion pathway.
Activities of virE1 and the VirE1 secretion chaperone in export of the multifunctional VirE2 effector via an Agrobacterium type IV secretion pathway.
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virE1 和 VirE1 分泌伴侣通过农杆菌 IV 型分泌途径输出多功能 VirE2 效应子的活性。
DOI：
10.1128/jb.183.13.3855-3865.2001
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发表时间：
2001
影响因子：
3.2
通讯作者：
Christie,PJ
中科院分区：
文献类型：
--
作者：
Zhao,Z;Sagulenko,E;Ding,Z;Christie,PJ
根癌农杆菌使用IV型分泌系统将致癌核蛋白颗粒和效应蛋白（如多功能VirE 2蛋白）递送至植物细胞。在这项研究中，我们研究了virE 1及其产物，VirE 1分泌伴侣，在介导VirE 2输出的功能。非极性virE 1无效突变体积累低水平的VirE 2，并且在该突变体中virE 1的反式表达仅部分恢复VirE 2丰度。通过对flacZ转录和翻译融合体的分析表明，virE 1的缺失不影响virE 2的转录，但降低了virE 2的翻译。VirE 2是稳定的一个延长的时间，超过6小时，当它与virE 1切割表达，它表现出半衰期约为2小时，当它在transwithvirE 1和不到10分钟，当它在virE 1的情况下表达，如脉冲追踪实验所示。VirE 1通过与VirE 2的N末端附近的结构域的相互作用来稳定VirE 2，如A中合成的VirE 2截短和插入突变体的分析所示。根瘤菌通过噬菌体λ cI阻遏物融合和pull-down试验证明了VirE 1的自结合，并通过非变性聚丙烯酰胺凝胶电泳和凝胶过滤色谱获得了VirE 1体内同源多聚化的证据。一个假定的VirE 1-VirE 2复合物的分子量约为70至80 kDa的检测通过凝胶过滤色谱的提取物从野生型细胞，而高阶VirE 2复合物或聚集体中检测到的提取物从avirE 1突变体。综上所述，我们的研究结果表明，virE 1有助于在几种方式VirE 2的出口：（i）virE 1调节efficientvirE 2翻译的表达从nativePvirE启动子的上下文中;（ii）VirE 1分泌伴侣稳定VirE 2，最有可能通过与N-末端结构域的相互作用;和（iii）VirE 1形成一个VirE 1-VirE 2复合物与预测的2：1的化学计量，抑制组装高阶VirE 2复合物或聚集体。
Agrobacterium tumefaciensuses a type IV secretion system to deliver oncogenic nucleoprotein particles and effector proteins, such as the multifunctional VirE2 protein, to plant cells. In this study, we examined the function ofvirE1and its product, the VirE1 secretion chaperone, in mediating VirE2 export. A nonpolarvirE1null mutant accumulated low levels of VirE2, andtransexpression ofvirE1in this mutant only partially restored VirE2 abundance. Deletion ofvirE1did not affect transcription but decreased translation ofvirE2, as shown by analysis oflacZtranscriptional and translational fusions. VirE2 was stable for a prolonged period, more than 6 h, when it was expressed inciswithvirE1, and it exhibited half-lives of about 2 h when it was expressed intranswithvirE1and less than 10 min when it was expressed in the absence ofvirE1, as shown by pulse-chase experiments. VirE1 stabilized VirE2 via an interaction with a domain near the N terminus of VirE2, as shown by analyses of VirE2 truncation and insertion mutants synthesized inA. tumefaciens. VirE1 self-association was demonstrated by using bacteriophage λ cI repressor fusion and pull-down assays, and evidence of VirE1 homomultimerization in vivo was obtained by native polyacrylamide gel electrophoresis and gel filtration chromatography. A putative VirE1-VirE2 complex with a molecular mass of about 70 to 80 kDa was detected by gel filtration chromatography of extracts from wild-type cells, whereas higher-order VirE2 complexes or aggregates were detected in extracts from avirE1mutant. Taken together, our findings show thatvirE1contributes in several ways to VirE2 export:(i)virE1regulates efficientvirE2translation in the context of expression from the nativePvirEpromoter; (ii) the VirE1 secretion chaperone stabilizes VirE2, most probably via an interaction with an N-terminal domain; and (iii) VirE1 forms a VirE1-VirE2 complex with a predicted 2:1 stoichiometry that inhibits assembly of higher-order VirE2 complexes or aggregates.