Visualizing interactions along the Escherichia coli twin-arginine translocation pathway using protein fragment...

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• The twin-arginine translocation (Tat) pathway is crucial for exporting folded proteins from bacterial cytoplasm.
• Understanding protein-protein interactions within the Tat pathway is key to elucidating protein export mechanisms in bacteria.

Purpose of the Study:

• To adapt and apply bimolecular fluorescence complementation (BiFC) for visualizing protein-protein interactions in the Tat pathway of living bacterial cells.
• To investigate interactions between Tat substrates, chaperones, and the TatABC translocase components.

Main Methods:

• Bimolecular fluorescence complementation (BiFC) using yellow fluorescent protein (YFP) chimeras.
• Fusion of YFP fragments to Tat pathway components including substrates (e.g., DmsA), chaperones (e.g., DmsD), and translocase proteins (TatA, TatB, TatC).
• Fluorescence microscopy to analyze protein-protein interactions and complex localization.

Main Results:

• BiFC successfully visualized diverse protein-protein interactions within the Tat pathway, including substrate-chaperone (DmsA-DmsD) and translocase component interactions.
• Homo- and hetero-oligomeric complexes of TatA, TatB, and TatC were identified, supporting models of translocase assembly.
• Evidence was found for TatBC complex co-localization at bacterial cell poles.
• Interactions between the Tat receptor complex (TatBC) and substrate (DmsA) or chaperone (DmsD) were captured.

Conclusions:

• BiFC is a powerful tool for studying dynamic protein-protein interactions in bacterial cytoplasmic and inner membrane transport pathways.
• The study provides novel insights into the assembly and function of the Tat translocase and its interaction with substrates and chaperones.
• Visualizing these interactions advances our understanding of bacterial protein secretion mechanisms.