Two electrical potential–dependent steps are required for transport by the Escherichia coli Tat machinery

The twin-arginine translocation (Tat) pathway in Escherichia coli transports fully folded and assembled proteins across the energy-transducing periplasmic membrane. In chloroplasts, Tat transport requires energy input only from the proton motive force. To elucidate the mechanism and energetics of bacterial Tat protein transport, we developed an efficient in vitro transport assay using TatABC-enriched inverted membrane vesicles and the physiological precursor pre-SufI. We report transport efficiencies of 60–80% for nanomolar pre-SufI concentrations. Dissipation of the pH gradient does not reduce pre-SufI transport efficiency. Instead, pre-SufI transport requires at least two electrical potential (Δψ)–dependent steps that differ in both the duration and minimum magnitude of the required Δψ. The data are consistent with a model in which a substantial Δψ of short duration is required for an early transport step, and in which a small Δψ of long duration is necessary to drive a later transport step.

[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]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

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

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

[5]  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.

[6]  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.

[7]  S. Kawasaki,et al.  Membrane vesicles containing overproduced SecY and SecE exhibit high translocation ATPase activity and countermovement of protons in a SecA- and presecretory protein-dependent manner. , 1993, The Journal of biological chemistry.

[8]  B. Berks.  A common export pathway for proteins binding complex redox cofactors? , 1996, Molecular microbiology.

[9]  B. Dobberstein,et al.  Common Principles of Protein Translocation Across Membranes , 1996, Science.

[10]  D. Bush,et al.  Sec-independent protein translocation by the maize Hcf106 protein. , 1997, Science.

[11]  A. Ghelli,et al.  Measurement of the membrane potential generated by complex I in submitochondrial particles. , 1997, Journal of biochemistry.

[12]  G. Heijne,et al.  Genome‐wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms , 1998, Protein science : a publication of the Protein Society.

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

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

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

[16]  E. Harlow,et al.  Using Antibodies: A Laboratory Manual , 1999 .

[17]  A. Bolhuis,et al.  Subunit interactions in the twin‐arginine translocase complex of Escherichia coli , 2000, FEBS letters.

[18]  S M Musser,et al.  Characterization of the early steps of OE17 precursor transport by the thylakoid DeltapH/Tat machinery. , 2000, European journal of biochemistry.

[19]  M. Brunori,et al.  The ratio between the fast and slow forms of bovine cytochrome c oxidase is changed by cholate or nucleotides bound to the cholate-binding site close to the cytochrome a3/CuB binuclear centre , 2000, Cellular and Molecular Life Sciences CMLS.

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

[21]  B. Berks,et al.  The Twin Arginine Consensus Motif of Tat Signal Peptides Is Involved in Sec-independent Protein Targeting in Escherichia coli * , 2000, The Journal of Biological Chemistry.

[22]  H. Saibil,et al.  Purified components of the Escherichia coli Tat protein transport system form a double-layered ring structure. , 2001, European journal of biochemistry.

[23]  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.

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

[25]  D. Kramer,et al.  Contribution of electric field (Δψ) to steady-state transthylakoid proton motive force (pmf) in vitro and in vivo. Control of pmf parsing into Δψ and ΔpH by ionic strength , 2001 .

[26]  C. Santini,et al.  Translocation of Jellyfish Green Fluorescent Protein via the Tat System of Escherichia coli and Change of Its Periplasmic Localization in Response to Osmotic Up-shock* , 2001, The Journal of Biological Chemistry.

[27]  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.

[28]  Matthias Müller,et al.  Separate Analysis of Twin-arginine Translocation (Tat)-specific Membrane Binding and Translocation in Escherichia coli * , 2002, The Journal of Biological Chemistry.

[29]  B. Wallace,et al.  Characterization and membrane assembly of the TatA component of the Escherichia coli twin-arginine protein transport system. , 2002, Biochemistry.

[30]  G. Finazzi,et al.  Thylakoid targeting of Tat passenger proteins shows no ΔpH dependence in vivo , 2003 .

[31]  A. Driessen,et al.  The bacterial translocase: a dynamic protein channel complex , 2003, Cellular and Molecular Life Sciences CMLS.

[32]  N. Alder,et al.  Energetics of Protein Transport across Biological Membranes A Study of the Thylakoid ΔpH-Dependent/cpTat Pathway , 2003, Cell.

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

[34]  G. Finazzi,et al.  Thylakoid targeting of Tat passenger proteins shows no delta pH dependence in vivo. , 2003, The EMBO journal.

[35]  W. Kühlbrandt,et al.  Consensus structural features of purified bacterial TatABC complexes. , 2003, Journal of molecular biology.

[36]  Acid denaturation and refolding of green fluorescent protein. , 2004 .

[37]  Topological studies on the twin-arginine translocase component TatC. , 2004, FEMS microbiology letters.

[38]  George Georgiou,et al.  A periplasmic fluorescent reporter protein and its application in high-throughput membrane protein topology analysis. , 2004, Journal of molecular biology.

[39]  C. Santini,et al.  Dual Topology of the Escherichia coli TatA Protein* , 2004, Journal of Biological Chemistry.

[40]  G. Georgiou,et al.  Phage Shock Protein PspA of Escherichia coli Relieves Saturation of Protein Export via the Tat Pathway , 2004, Journal of bacteriology.

[41]  Ehud Y. Isacoff,et al.  How Far Will You Go to Sense Voltage? , 2005, Neuron.

[42]  G. Finazzi,et al.  The energetics of the chloroplast Tat protein transport pathway revisited. , 2005, Trends in plant science.

[43]  C. Robinson,et al.  The thylakoid delta pH/delta psi are not required for the initial stages of Tat-dependent protein transport in tobacco protoplasts. , 2005, The Journal of biological chemistry.

[44]  A. Bolhuis,et al.  The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC complex, spectrum of modular TatA complexes and minor TatAB complex. , 2005, Journal of molecular biology.

[45]  Frank Sargent,et al.  Export of complex cofactor-containing proteins by the bacterial Tat pathway. , 2005, Trends in microbiology.

[46]  T. Palmer,et al.  Common principles in the biosynthesis of diverse enzymes. , 2005, Biochemical Society transactions.

[47]  C. Robinson,et al.  The Thylakoid ΔpH/ΔΨ Are Not Required for the Initial Stages of Tat-dependent Protein Transport in Tobacco Protoplasts* , 2005, Journal of Biological Chemistry.

[48]  Frank Sargent,et al.  Protein targeting by the bacterial twin-arginine translocation (Tat) pathway. , 2005, Current opinion in microbiology.

[49]  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.

[50]  George Georgiou,et al.  The bacterial twin-arginine translocation pathway. , 2006, Annual review of microbiology.

[51]  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.

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

[53]  B. Berks,et al.  Subunit composition and in vivo substrate‐binding characteristics of Escherichia coli Tat protein complexes expressed at native levels , 2006, The FEBS journal.

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

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

[56]  D. Tieleman,et al.  The TatA subunit of Escherichia coli twin-arginine translocase has an N-in topology. , 2007, Biochemistry.