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

PLoS One. 2010 Feb 16;5(2):e9225. doi: 10.1371/journal.pone.0009225.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Binding Sites / genetics
  • Blotting, Western
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Flow Cytometry
  • Intracellular Signaling Peptides and Proteins
  • Iron-Sulfur Proteins / genetics
  • Iron-Sulfur Proteins / metabolism
  • Luminescent Measurements / instrumentation
  • Luminescent Measurements / methods*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Membrane Transport Proteins / chemistry
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism*
  • Molecular Chaperones / genetics
  • Molecular Chaperones / metabolism
  • Mutation
  • Oxidoreductases / genetics
  • Oxidoreductases / metabolism
  • Protein Binding
  • Protein Multimerization
  • Protein Transport
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction*
  • Substrate Specificity

Substances

  • Carrier Proteins
  • DmsD protein, E coli
  • Escherichia coli Proteins
  • Intracellular Signaling Peptides and Proteins
  • Iron-Sulfur Proteins
  • Luminescent Proteins
  • Membrane Transport Proteins
  • Molecular Chaperones
  • Recombinant Fusion Proteins
  • TatA protein, E coli
  • TatB protein, E coli
  • TatC protein, E coli
  • Oxidoreductases
  • dimethyl sulfoxide reductase