Kinetics of Precursor Interactions with the Bacterial Tat Translocase Detected by Real-time FRET*

Background: The Tat machinery transports folded proteins from the bacterial cytoplasm to the periplasm. Results: A Δψ is required for a Tat cargo to move away from the TatBC receptor complex. Conclusion: Cargo migration away from the TatBC complex requires a Δψ-dependent assembly step or conformational change. Significance: Cargo migration from the TatBC receptor is a major rate-limiting step of Tat transport. The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. Traditionally, in vitro protein translocation studies have been performed using gel-based transport assays. This technique suffers from low time resolution, and often, an inability to distinguish between different steps in a continuously occurring translocation process. To address these limitations, we have developed an in vitro FRET-based assay that reports on an early step in the Tat translocation process in real-time. The natural Tat substrate pre-SufI was labeled with Alexa532 (donor), and the fluorescent protein mCherry (acceptor) was fused to the C terminus of TatB or TatC. The colored Tat proteins were easily visible during purification, enabling identification of a highly active inverted membrane vesicle (IMV) fraction yielding transport rates with NADH almost an order of magnitude faster than previously reported. When pre-SufI was bound to the translocon, FRET was observed for both Tat proteins. FRET was diminished upon addition of nonfluorescent pre-SufI, indicating that the initial binding step is reversible. When the membranes were energized with NADH, the FRET signal was lost after a short delay. These data suggest a model in which a Tat cargo initially associates with the TatBC complex, and an electric field gradient is required for the cargo to proceed to the next stage of transport. This cargo migration away from the TatBC complex requires a significant fraction of the total transport time.

[1]  S. Cohen,et al.  Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[2]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[3]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[4]  R. Mould,et al.  A proton gradient is required for the transport of two lumenal oxygen-evolving proteins across the thylakoid membrane. , 1991, The Journal of biological chemistry.

[5]  K. Cline,et al.  Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP. , 1992, The Journal of biological chemistry.

[6]  J. Weiner,et al.  A Novel and Ubiquitous System for Membrane Targeting and Secretion of Cofactor-Containing Proteins , 1998, Cell.

[7]  B. Berks,et al.  Overlapping functions of components of a bacterial Sec‐independent protein export pathway , 1998, The EMBO journal.

[8]  Jack Benner,et al.  Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. , 1998, Nucleic acids research.

[9]  G. Giordano,et al.  A novel Sec‐independent periplasmic protein translocation pathway in Escherichia coli , 1998, The EMBO journal.

[10]  B. Berks,et al.  Sec-independent Protein Translocation in Escherichia coli , 1999, The Journal of Biological Chemistry.

[11]  B. Berks,et al.  TatD Is a Cytoplasmic Protein with DNase Activity , 2000, The Journal of Biological Chemistry.

[12]  R. Daniel,et al.  Export of active green fluorescent protein to the periplasm by the twin‐arginine translocase (Tat) pathway in Escherichia coli , 2001, Molecular microbiology.

[13]  W. Wickner,et al.  Functional reconstitution of bacterial Tat translocation in vitro , 2001, The EMBO journal.

[14]  G. Sawers,et al.  Constitutive Expression of Escherichia coli tat Genes Indicates an Important Role for the Twin-Arginine Translocase during Aerobic and Anaerobic Growth , 2001, Journal of bacteriology.

[15]  A. Bolhuis,et al.  TatB and TatC Form a Functional and Structural Unit of the Twin-arginine Translocase from Escherichia coli * , 2001, The Journal of Biological Chemistry.

[16]  B. Berks,et al.  Membrane interactions and self‐association of the TatA and TatB components of the twin‐arginine translocation pathway , 2001, FEBS letters.

[17]  B. Berks,et al.  Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes. , 2002, Journal of molecular biology.

[18]  K. Cline,et al.  A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase , 2002, The Journal of cell biology.

[19]  Matthias Müller,et al.  Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli. , 2003, Molecular cell.

[20]  Frank Sargent,et al.  The Tat protein translocation pathway and its role in microbial physiology. , 2003, Advances in microbial physiology.

[21]  R. Tsien,et al.  Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein , 2004, Nature Biotechnology.

[22]  Søren Brunak,et al.  Prediction of twin-arginine signal peptides , 2005, BMC Bioinformatics.

[23]  Helen R Saibil,et al.  The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[24]  K. Cline,et al.  Efficient Twin Arginine Translocation (Tat) Pathway Transport of a Precursor Protein Covalently Anchored to Its Initial cpTatC Binding Site* , 2006, Journal of Biological Chemistry.

[25]  Y. Bollen,et al.  Membrane binding of twin arginine preproteins as an early step in translocation. , 2006, Biochemistry.

[26]  S. Frielingsdorf,et al.  Unassisted membrane insertion as the initial step in DeltapH/Tat-dependent protein transport. , 2006, Journal of molecular biology.

[27]  S. M. Musser,et al.  Two electrical potential–dependent steps are required for transport by the Escherichia coli Tat machinery , 2007, The Journal of cell biology.

[28]  Roland Freudl,et al.  Escherichia coli Twin Arginine (Tat) Mutant Translocases Possessing Relaxed Signal Peptide Recognition Specificities* , 2007, Journal of Biological Chemistry.

[29]  Escherichia coli tatC mutations that suppress defective twin-arginine transporter signal peptides. , 2007, Journal of molecular biology.

[30]  Nikolai A Braun,et al.  The chloroplast Tat pathway utilizes the transmembrane electric potential as an energy source. , 2007, Biophysical journal.

[31]  S. Frielingsdorf,et al.  Prerequisites for Terminal Processing of Thylakoidal Tat Substrates* , 2007, Journal of Biological Chemistry.

[32]  Matthias Müller,et al.  The entire N-terminal half of TatC is involved in twin-arginine precursor binding. , 2007, Biochemistry.

[33]  K. Cline,et al.  The Thylakoid Proton Gradient Promotes an Advanced Stage of Signal Peptide Binding Deep within the Tat Pathway Receptor Complex* , 2007, Journal of Biological Chemistry.

[34]  F. Sargent,et al.  The twin-arginine transport system: moving folded proteins across membranes. , 2007, Biochemical Society transactions.

[35]  A. Driessen,et al.  Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane--distinct translocases and mechanisms. , 2008, Biochimica et biophysica acta.

[36]  Matthias Müller,et al.  Following the Path of a Twin-arginine Precursor along the TatABC Translocase of Escherichia coli* , 2008, Journal of Biological Chemistry.

[37]  T. Palmer,et al.  Proteolytic processing of Escherichia coli twin-arginine signal peptides by LepB , 2009, Archives of Microbiology.

[38]  Helen R Saibil,et al.  Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system , 2009, Proceedings of the National Academy of Sciences.

[39]  Neal Whitaker,et al.  Interconvertibility of lipid‐ and translocon‐bound forms of the bacterial Tat precursor pre‐SufI , 2009, Molecular microbiology.

[40]  René Schlesier,et al.  Twin arginine translocation (Tat)-dependent protein transport: the passenger protein participates in the initial membrane binding step , 2010, Biological chemistry.

[41]  Anna-Carina Jungkamp,et al.  TatB Functions as an Oligomeric Binding Site for Folded Tat Precursor Proteins , 2010, Molecular biology of the cell.

[42]  Matthias Müller,et al.  Early Contacts between Substrate Proteins and TatA Translocase Component in Twin-arginine Translocation* , 2011, The Journal of Biological Chemistry.

[43]  Genetic Evidence for a TatC Dimer at the Core of the Escherichia coli Twin Arginine (Tat) Protein Translocase , 2011, Journal of Molecular Microbiology and Biotechnology.