Department of Biomedical Engineering, Cornell University, Ithaca, New York, United States of America.
PLoS One. 2010 Feb 16;5(2):e9225. doi: 10.1371/journal.pone.0009225.
The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of gram-negative and gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways.
双精氨酸转运（Tat）途径以其能够将完全折叠的底物蛋白从革兰氏阴性和革兰氏阳性细菌的细胞质中输出而闻名。对该机制在大肠杆菌中的研究已经确定了许多短暂的蛋白-蛋白相互作用，这些相互作用指导具有出口能力的蛋白通过 Tat 途径。为了可视化这些相互作用，我们已经适应了双分子荧光互补（BiFC）来检测活细胞中 Tat 途径中的蛋白-蛋白相互作用。黄色荧光蛋白（YFP）的片段与参与转运过程的可溶性和跨膜因子融合，包括 Tat 底物、Tat 特异性校对伴侣和形成转运体的整合膜蛋白 TatABC。这些 YFP 嵌合体的荧光分析揭示了广泛的相互作用，例如 Tat 底物二甲亚砜还原酶（DmsA）与其专用校对伴侣 DmsD 之间的相互作用。此外，BiFC 分析阐明了 TatA、TatB 和 TatC 整合膜蛋白的同源和异源寡聚复合物，这与转运体组装的当前模型一致。在 TatBC 组装的情况下，我们提供了第一个证据表明这些复合物在细胞极共定位。最后，我们使用这种 BiFC 方法来捕获 TatBC 形成的假定 Tat 受体复合物与 DmsA 底物或其专用伴侣 DmsD 之间的相互作用。我们的结果表明，BiFC 是研究细菌分泌途径中细胞质和内膜相互作用的有力方法。
