Abstract
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.
Highlights
The bulk of protein transport across the inner membrane of Gram-negative bacteria occurs via the well-characterized Sec export pathway [1,2,3,4]
To visualize protein interactions between soluble and transmembrane factors that participate in various steps of the Tat export process, we employed bimolecular fluorescence complementation (BiFC) based on split fragments of enhanced yellow fluorescent protein (YFP)
Since the dimethyl sulfoxide reductase (DmsA) signal peptide alone is sufficient to interact with DmsD [26], we first tested whether ssDmsA fused to the N-terminal YFP fragment interacted with DmsD fused to the C-terminal YFP fragment (DmsD-Y2)
Summary
The bulk of protein transport across the inner membrane of Gram-negative bacteria occurs via the well-characterized Sec export pathway [1,2,3,4]. The hallmark of the Tat pathway that distinguishes it from the Sec mechanism is the ability to transport proteins of varying dimension that have acquired a largely, if not completely, folded conformation [7,8,9,10]. Studies on the Tat mechanism have demonstrated that the integral membrane proteins TatA, TatB, and TatC form the minimal components necessary for exporting folded proteins in E. coli. The TatA and TatB components are single-span integral membrane proteins while TatC has been shown to contain six transmembrane spans [11]. These membrane proteins have been observed to form two distinct complexes: one that is comprised of multiple subunits of TatA and a second that contains predominantly TatB and TatC [12,13,14]. The TatB and TatC proteins form a complex to which substrates initially bind [17], suggesting that TatBC serves as the twin-arginine signal peptide binding site
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