DNA Translocation Across the Agrobacterium Envelope

描述（由申请人提供）：大分子易位 原核和真核细胞膜是生物医学感兴趣的主要领域。在 近年来，分泌系统与细菌鞭毛和 结合系统已被证明在发病机制中起重要作用， 转运效应分子到真核细胞胞质溶胶中， 感染的过程。这些系统现在被指定为第三和第四类 分泌途径，分别。本实验室的工作重点是 A.使用的IV型传输系统。tumefaciens传递致癌T-DNA， 易感植物细胞T-DNA转移系统是一个很好的模型， IV型分泌的详细机制研究。最近的研究表明， T-DNA转移系统在底物 选择和靶细胞识别。除了T-DNA分泌 该系统可以输出其他DNA底物以及效应物 蛋白质独立于DNA。此外，该系统还可以 其他细菌和各种真核细胞类型的底物， 包括植物、真菌和人类。这方面工作的总体目标 实验室将：i）定义这种IV型转移的组装路径 系统，ii）描述其架构安排，iii）阐明 基片处理和输送到反应器的反应机制 分泌通道，和iv）限定跨膜转运的途径。 革兰氏阴性菌包膜。现在已经确定， 需要一个菌毛来介导细胞间的接触， 穿过细菌细胞包膜的易位。我们将结合使用 分子、遗传和生物化学方法，具体目标如下。 首先，我们将探索内膜的结构-功能关系 VirB 11 ATP酶在转运蛋白生物合成和底物中的作用 易位;对于这些研究，我们将利用我们的集合， 大量的功能改变的突变体和晶体结构的一个 VirB 11同源物。其次，我们将确定基板交付的步骤 途径，侧重于模型VirE 2底物和VirE 1分泌 监护人最后，我们将描述组装途径中的关键步骤 这些研究将确定具体的联系人， 涉及三种转运蛋白组装和结构蛋白 完整性，多位内膜蛋白VirB 6和两个外膜 蛋白质，VirB 7脂蛋白和VirB 9。

DESCRIPTION (provided by applicant):Macromolecular translocation across prokaryotic and eukaryotic membranes is a major area of biomedical interest. In recent years, secretion systems ancestrally-related to bacterial flagellar and conjugation systems have been shown to play important roles in pathogenesis by translocating effector molecules to the eukaryotic cell cytosol during the course of infection. These systems are now designated as types III and IV secretion pathways, respectively. The focus of work in this laboratory is the type IV transfer system used by A. tumefaciens to deliver oncogenic T-DNA to susceptible plant cells. The T-DNA transfer system is an excellent model for detailed mechanistic studies of type IV secretion. Recent work has shown that the T-DNA transfer system is exceptionally versatile both in terms of substrate selection and target cell recognition. In addition to the T-DNA secretion substrate, this system can export other DNA substrates as well as effector proteins independently of DNA. Furthermore, this system can translocate substrates to other bacteria and to a wide variety of eukaryotic cell types, including those of plants, fungi, and humans. The overall goals of work in this laboratory are to: i) define the assembly pathway for this type IV transfer system, ii) characterize its architectural arrangement, iii) elucidate the reaction mechanisms underlying substrate processing and delivery to the secretion channel, and iv) define the route of translocation across the Gram-negative bacterial envelope. It is now established that substrate transfer requires a pilus for mediating cell-cell contacts and a channel for translocation across the bacterial cell envelope. We will use a combination of molecular, genetic, and biochemical approaches in the following specific aims. First, we will explore structure - function relationships of the inner membrane VirB11 ATPase to define its role in transporter biogenesis and substrate translocation; for these studies we will capitalize on our assemblage of a large collection of altered-function mutants and a crystal structure of a VirB11 homolog. Second, we will identify steps in the substrate delivery pathway, focusing on the model VirE2 substrate and the VirE1 secretion chaperone. Finally, we will characterize critical steps in the assembly pathway of this transfer system; these studies will identify specific contacts involving three proteins essential for transporter assembly and structural integrity, the polytopic inner membrane protein VirB6 and two outer membrane proteins, VirB7 lipoprotein and VirB9.