Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL.
Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL.
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菊欧文氏菌 II 型分泌机制的组装：OutE、假定的 ATP 结合成分和膜蛋白 OutL 之间的直接相互作用和相关构象变化。
DOI：
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发表时间：
1999
影响因子：
5.6
通讯作者：
F. Barras
中科院分区：
文献类型：
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作者：
Béatrice Py;L. Loiseau;F. Barras
菊花欧文氏菌通过II型分泌途径分泌大量的酶，包括纤维素酶和果胶酶。这个过程需要外源基因的产物，这些基因在革兰氏阴性菌中广泛保守。Out蛋白被认为形成一种膜相关的多蛋白复合物。在这里，我们研究了假定的ATP结合成分OutE和细胞膜蛋白OutL之间的相互作用。我们发现，通过有限的蛋白水解、遗传抑制和酵母双杂交系统，OutE和OutL直接相互作用。对截断形式的OutE的分析表明，OutE的N端（残基1-97）对OutE/OutL相互作用很重要。此外，酵母双杂交系统的结果表明，OutE和OutL都能形成同聚体。OutE同源化所需的区域位于其C端。有限的蛋白水解实验表明，OutE诱导了OutL的构象变化，在其细胞质和质周结构域。此外，分泌过程需要OutE的构象改变，这取决于与OutL的相互作用以及OutE中完整的Walker a基序的存在。我们的结果支持细胞质侧发生的相互作用影响外膜发生的事件的观点。我们讨论了一个模型，其中OutE使用ATP来控制II型分泌机制的组装。
Erwinia chrysanthemi secretes, by the type II secretory pathway, a large number of enzymes, including cellulases and pectinases. This process requires the products of the out genes, which are widely conserved in Gram-negative bacteria. The Out proteins are thought to form a membrane-associated multiprotein complex. Here, we investigated interaction between OutE, the putative ATP binding component, and OutL, an inner membrane protein. We showed, by limited proteolysis, genetic suppression and the yeast two-hybrid system, that OutE and OutL interact directly. Analysis of truncated forms of OutE demonstrated that the N terminus of OutE (residues 1-97) is important for the OutE/OutL interaction. Moreover, results from the yeast two-hybrid system suggested that OutE and OutL are each able to form homomultimers. The region required for homomultimerisation of OutE is located in its C terminus. Limited proteolysis assay indicated that OutE induces a conformational change in OutL, in both its cytoplasmic and periplasmic domains. Moreover, the secretion process requires a conformational change in OutE which depends on both the interaction with OutL and on the presence of an intact Walker A motif in OutE. Our results support the view that interaction occurring on the cytoplasmic side influences the events occurring in the outer membrane. We discuss a model in which OutE uses ATP to control the assembly of the type II secretion machinery.