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

  • Neal Whitaker
    Affiliations
    Department of Molecular and Cellular Medicine, College of Medicine, The Texas A&M Health Science Center, 1114 TAMU, College Station, Texas 77843
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  • Umesh K. Bageshwar
    Affiliations
    Department of Molecular and Cellular Medicine, College of Medicine, The Texas A&M Health Science Center, 1114 TAMU, College Station, Texas 77843
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  • Siegfried M. Musser
    Correspondence
    To whom correspondence should be addressed: Department of Molecular and Cellular Medicine, College of Medicine, The Texas A&M Health Science Center, 1114 TAMU, College Station, TX 77843. Tel.: 979-862-4128; Fax: 979-847-9481
    Affiliations
    Department of Molecular and Cellular Medicine, College of Medicine, The Texas A&M Health Science Center, 1114 TAMU, College Station, Texas 77843
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant R01 GM065534 (to S. M. M.).
    This article contains supplemental Figs. S1–S11.
      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.

      Introduction

      The bacterial twin-arginine translocation (Tat)
      The abbreviations used are: Tat
      twin-arginine translocation
      FRET
      fluorescence resonance energy transfer
      IMV
      inverted membrane vesicle.
      machinery transports protein cargos from the cytoplasm to the periplasm (
      • Santini C.L.
      • Ize B.
      • Chanal A.
      • Müller M.
      • Giordano G.
      • Wu L.F.
      A novel sec-independent periplasmic protein translocation pathway in Escherichia coli.
      ,
      • Thomas J.D.
      • Daniel R.A.
      • Errington J.
      • Robinson C.
      Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli.
      ). A functional Escherichia coli Tat machinery minimally consists of three membrane proteins, TatA (or TatE), TatB, and TatC (
      • Sargent F.
      The twin-arginine transport system: moving folded proteins across membranes.
      ,
      • Natale P.
      • Brüser T.
      • Driessen A.J.
      Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-distinct translocases and mechanisms.
      ,
      • Sargent F.
      • Bogsch E.G.
      • Stanley N.R.
      • Wexler M.
      • Robinson C.
      • Berks B.C.
      • Palmer T.
      Overlapping functions of components of a bacterial Sec-independent protein export pathway.
      ,
      • Sargent F.
      • Stanley N.R.
      • Berks B.C.
      • Palmer T.
      Sec-independent protein translocation in Escherichia coli. A distinct and pivotal role for the TatB protein.
      ,
      • Weiner J.H.
      • Bilous P.T.
      • Shaw G.M.
      • Lubitz S.P.
      • Frost L.
      • Thomas G.H.
      • Cole J.A.
      • Turner R.J.
      A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins.
      ). TatB and TatC are expressed at approximately equal levels (
      • Bolhuis A.
      • Mathers J.E.
      • Thomas J.D.
      • Barrett C.M.
      • Robinson C.
      TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli.
      ), and together act as the receptor for precursor proteins (
      • Alami M.
      • Lüke I.
      • Deitermann S.
      • Eisner G.
      • Koch H.G.
      • Brunner J.
      • Müller M.
      Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli.
      ,
      • Jack R.L.
      • Sargent F.
      • Berks B.C.
      • Sawers G.
      • Palmer T.
      Constitutive expression of Escherichia coli tat genes indicates an important role for the twin-arginine translocase during aerobic and anaerobic growth.
      ). TatA is expressed at higher levels than TatB and TatC (
      • Bolhuis A.
      • Mathers J.E.
      • Thomas J.D.
      • Barrett C.M.
      • Robinson C.
      TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli.
      ), and can form ring-like structures in vitro (
      • Gohlke U.
      • Pullan L.
      • McDevitt C.A.
      • Porcelli I.
      • de Leeuw E.
      • Palmer T.
      • Saibil H.R.
      • Berks B.C.
      The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter.
      ). A popular model is that cargos are first recruited by TatBC complexes, and are then conveyed across the membrane by a protein conducting channel comprised of TatA oligomers (
      • Gohlke U.
      • Pullan L.
      • McDevitt C.A.
      • Porcelli I.
      • de Leeuw E.
      • Palmer T.
      • Saibil H.R.
      • Berks B.C.
      The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter.
      ,
      • De Leeuw E.
      • Porcelli I.
      • Sargent F.
      • Palmer T.
      • Berks B.C.
      Membrane interactions and self-association of the TatA and TatB components of the twin-arginine translocation pathway.
      ). After transport, Tat signal peptides are cleaved from precursor proteins by the LepB peptidase (
      • Yahr T.L.
      • Wickner W.T.
      Functional reconstitution of bacterial Tat translocation in vitro.
      ,
      • Lüke I.
      • Handford J.I.
      • Palmer T.
      • Sargent F.
      Proteolytic processing of Escherichia coli twin-arginine signal peptides by LepB.
      ). Recent studies have suggested that precursor binding to the cytoplasmic face of the inner membrane is a functional intermediate in the transport process (
      • Bageshwar U.K.
      • Whitaker N.
      • Liang F.C.
      • Musser S.M.
      Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.
      ,
      • Hou B.
      • Frielingsdorf S.
      • Klösgen R.B.
      Unassisted membrane insertion as the initial step in ΔpH/Tat-dependent protein transport.
      ). A proton motive force (PMF) is essential for translocon assembly and cargo transport (
      • Mould R.M.
      • Robinson C.
      A proton gradient is required for the transport of two lumenal oxygen-evolving proteins across the thylakoid membrane.
      ,
      • Cline K.
      • Ettinger W.F.
      • Theg S.M.
      Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP.
      ,
      • Braun N.A.
      • Davis A.W.
      • Theg S.M.
      The chloroplast Tat pathway utilizes the transmembrane electric potential as an energy source.
      ). In E. coli, only the membrane potential (Δψ) component of the PMF is required for transport (
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ).
      Both TatB and TatC participate in binding and recognition of signal peptides. Bacterial Tat signal peptides contain an (S/T)RRXFLK consensus motif, a hydrophobic domain, and a short polar domain which precedes the signal sequence cleavage site (
      • Berks B.C.
      • Palmer T.
      • Sargent F.
      The Tat protein translocation pathway and its role in microbial physiology.
      ,
      • Bendtsen J.D.
      • Nielsen H.
      • Widdick D.
      • Palmer T.
      • Brunak S.
      Prediction of twin-arginine signal peptides.
      ,
      • Shanmugham A.
      • Wong Fong Sang H.W.
      • Bollen Y.J.
      • Lill H.
      Membrane binding of twin arginine preproteins as an early step in translocation.
      ). Based on mutant suppression, alanine substitutions and cross-linking experiments, the N-terminal half of TatC interacts with the twin-arginine portion of the signal peptide (
      • Holzapfel E.
      • Eisner G.
      • Alami M.
      • Barrett C.M.
      • Buchanan G.
      • Lüke I.
      • Betton J.M.
      • Robinson C.
      • Palmer T.
      • Moser M.
      • Müller M.
      The entire N-terminal half of TatC is involved in twin-arginine precursor binding.
      ,
      • Kreutzenbeck P.
      • Kröger C.
      • Lausberg F.
      • Blaudeck N.
      • Sprenger G.A.
      • Freudl R.
      Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities.
      ,
      • Strauch E.M.
      • Georgiou G.
      Escherichia coli tatC mutations that suppress defective twin-arginine transporter signal peptides.
      ,
      • Gérard F.
      • Cline K.
      Efficient twin arginine translocation (Tat) pathway transport of a precursor protein covalently anchored to its initial cpTatC binding site.
      ). Cross-linking indicates that TatB interacts with the C-terminal end of the signal peptide (
      • Holzapfel E.
      • Eisner G.
      • Alami M.
      • Barrett C.M.
      • Buchanan G.
      • Lüke I.
      • Betton J.M.
      • Robinson C.
      • Palmer T.
      • Moser M.
      • Müller M.
      The entire N-terminal half of TatC is involved in twin-arginine precursor binding.
      ,
      • Gérard F.
      • Cline K.
      Efficient twin arginine translocation (Tat) pathway transport of a precursor protein covalently anchored to its initial cpTatC binding site.
      ). TatB also makes extensive contacts with the cargo mature domain, presumably through its cytoplasmic domain (
      • Maurer C.
      • Panahandeh S.
      • Jungkamp A.C.
      • Moser M.
      • Müller M.
      TatB functions as an oligomeric binding site for folded Tat precursor proteins.
      ).
      It is unclear how the cargo proceeds across the membrane after recognition by the TatBC complex. A severe constraint on possible models is the finding that the mature domain can be efficiently translocated when the signal sequence is cross-linked to TatC near the twin-arginine motif (
      • Gérard F.
      • Cline K.
      Efficient twin arginine translocation (Tat) pathway transport of a precursor protein covalently anchored to its initial cpTatC binding site.
      ). One possibility is that TatA somehow assists with flipping the mature domain from one side of the membrane to the other while the signal sequence remains tethered (
      • Gérard F.
      • Cline K.
      Efficient twin arginine translocation (Tat) pathway transport of a precursor protein covalently anchored to its initial cpTatC binding site.
      ). In this picture, the signal peptide may initially only partially occupy a transmembrane binding pocket before transport, and full binding only occurs during or after transport, consistent with the identification of deep insertion of the signal peptide and distinct translocation intermediates (
      • Gérard F.
      • Cline K.
      The thylakoid proton gradient promotes an advanced stage of signal peptide binding deep within the Tat pathway receptor complex.
      ,
      • Frielingsdorf S.
      • Klösgen R.B.
      Prerequisites for terminal processing of thylakoidal Tat substrates.
      ,
      • Schlesier R.
      • Klösgen R.B.
      Twin arginine translocation (Tat)-dependent protein transport: the passenger protein participates in the initial membrane binding step.
      ). Electron microscopic images of purified TatBC complexes revealed cargo bound within the membrane plane, possibly reflecting cargo in transit (
      • Tarry M.J.
      • Schäfer E.
      • Chen S.
      • Buchanan G.
      • Greene N.P.
      • Lea S.M.
      • Palmer T.
      • Saibil H.R.
      • Berks B.C.
      Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system.
      ).
      Fluorescence resonance energy transfer (FRET) can provide time-dependent distance information at low protein concentrations. Here we report the use of FRET to probe the binding of a cargo to the TatBC complex, and its migration away from this complex, either by dissociation or by movement along the transport pathway. We found that the TatBC complex has a nanomolar affinity for pre-SufI, and that an electric field gradient is required for migration beyond the initial binding step.

      EXPERIMENTAL PROCEDURES

       Bacterial Strains, Plasmids, and Growth Conditions

      E. coli strains MC4100ΔTatABCDE, JM109, and BL21(λDE3) were described previously (
      • Wexler M.
      • Sargent F.
      • Jack R.L.
      • Stanley N.R.
      • Bogsch E.G.
      • Robinson C.
      • Berks B.C.
      • Palmer T.
      TatD is a cytoplasmic protein with DNase activity. No requirement for TatD family proteins in sec-independent protein export.
      ,
      • Casadaban M.J.
      • Cohen S.N.
      Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences.
      ,
      • Studier F.W.
      • Rosenberg A.H.
      • Dunn J.J.
      • Dubendorff J.W.
      Use of T7 RNA polymerase to direct expression of cloned genes.
      ,
      • Yanisch-Perron C.
      • Vieira J.
      • Messing J.
      Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.
      ). Cultures for IMV preparations were grown at 37 °C as described (
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ). Overexpression cultures for pre-SufI and spTorA-mCherry-MA were grown in Luria-Bertani (LB) medium at 37 °C, and shifted to 15 °C upon induction with 1 mm IPTG or 0.6% arabinose, respectively. All cultures were supplemented with ampicillin (50 μg/ml). Tat proteins were induced for 4 h with 0.7% arabinose, as previously described (
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ).
      The plasmid pIntein-preSufI-MA was generated by inserting a cysteine-free form of pre-SufI with a 6×His tag into the vector pTYB11 (New England Biochem). Plasmid pIntein-preSufI-MA was used as the template for all pre-SufI mutants, as described in supplemental Fig. S1. Plasmids pTatABcherryC and pTatABCcherry were generated by insertion of the mCherry sequence (plasmid pmCherry, Clontech) into pTatABC (
      • Yahr T.L.
      • Wickner W.T.
      Functional reconstitution of bacterial Tat translocation in vitro.
      ) immediately after the coding regions of TatB and TatC, respectively (supplemental Figs. S2 and S3). pTatBC and pTatBCcherry were generated by excising the TatA sequence from pTatABC and pTatABCcherry, respectively (supplemental Fig. S4). All mutations were generated via the Quikchange protocol (Stratagene), and all plasmid coding sequences were verified by DNA sequencing. Plasmid pTorA-mCherry-H6 was generated by inserting mCherry along with the TorA signal peptide and 4 amino acids (AQAA) of the TorA mature domain into the pBAD24 vector (supplemental Fig. S5).

       Protein Purification and Labeling

      All pre-SufI proteins were purified from expression cultures via chitin chromatography as described previously (
      • Chong S.
      • Montello G.E.
      • Zhang A.
      • Cantor E.J.
      • Liao W.
      • Xu M.Q.
      • Benner J.
      Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step.
      ) with an on-column cleavage (50 mm dithiothreitol) at 4 °C for 20 h (supplemental Fig. S6). Excess dithiothreitol was removed from the eluate by dialysis (10,000 MW cutoff) against phosphate-buffered saline (1× PBS: 137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, and 2 mm KH2PO4, pH 7.5) for 4 h. Ni-NTA chromatography was used for further purification (
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ). Alexa532 labeling of pre-SufI proteins was performed as described previously (
      • Bageshwar U.K.
      • Whitaker N.
      • Liang F.C.
      • Musser S.M.
      Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.
      ). After a 4 h dialysis against 1× PBS buffer, excess dye was removed by an additional Ni-NTA purification step (
      • Bageshwar U.K.
      • Whitaker N.
      • Liang F.C.
      • Musser S.M.
      Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.
      ). The final storage buffer was 20 mm Tris-HCl, 50 mm NaCl, 250 mm imidazole, 50% glycerol, pH 8.0. Purification of spTorA-mCherry-H6 was performed by Ni-NTA chromatography, by the procedure used for pre-SufI (
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ). The mCherry protein was obtained by purifying the mature protein from a spTorA-mCherry-H6 preparation via Resource Q (GE Healthcare) chromatography.
      The protein concentrations of all pre-SufI proteins were quantified by SDS-PAGE using bovine serum albumin as the standard. Spot intensities after Coomassie Blue staining were determined with a phosphorimager (model FX PhosphoImager, Bio-Rad). Alexa532 concentrations were determined by absorbance spectroscopy at 532 nm (ϵ = 81,000 cm−1 m−1). Typical dye-to-protein ratios after labeling indicated that 80–90% of the cysteines were successfully tagged. mCherry concentrations were estimated in 2% SDS based on the mCherry absorbance at 587 nm and its extinction coefficient at this wavelength (
      • Shaner N.C.
      • Campbell R.E.
      • Steinbach P.A.
      • Giepmans B.N.
      • Palmer A.E.
      • Tsien R.Y.
      Improved monomeric red, orange, and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.
      ).

       Isolation of IMVs

      IMVs were isolated from MC4100ΔTatABCDE as described (
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ), with the following modifications. Cell lysis by French press was performed at ∼16,000 psi instead of ∼6,000 psi. In addition, the 2.3 m sucrose cushion was replaced with a 3-step (0.5, 1.5, and 2.3 m) sucrose gradient, which enabled enrichment of a highly active inner membrane fraction. A band in the 0.5 m region was faintly pink when mCherry Tat fusions were expressed (supplemental Fig. S7), and translucent dark brown when wild type TatABC was expressed. IMV concentrations were determined as the A280 in 2% SDS (
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ).

       Transport and Membrane Binding Assays

      In vitro transport assays and precursor-membrane binding assays were performed as described (
      • Bageshwar U.K.
      • Whitaker N.
      • Liang F.C.
      • Musser S.M.
      Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.
      ,
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ), unless otherwise indicated. Gel-based transport kinetics were performed in a heated cuvette to mimic conditions in which the real-time FRET assays were performed. Aliquots (35 μl) of the reaction mixture were removed from the cuvette at the indicated time points and quenched with a mixture of 5 μm nigericin and 5 μm valinomycin on ice. Visualization of protein bands was performed by direct in-gel fluorescence imaging (model FX PhosphorImager, Bio-Rad).

       Real-time FRET Assay

      Translocation buffer (5 mm MgCl2, 50 mm KCl, 200 mm sucrose, 57 μg ml−1 BSA, 25 mm MOPS, 25 mm MES, pH 8.0) (
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ) and IMVs (A280 = 2) were added to a heated cuvette (final volume: 800 μl) in an SLM-Aminco fluorometer and allowed to equilibrate to 37 °C for 5 min. Precursor protein was then added, and equilibration was continued for at least 200 s. Reactions were initiated by addition of competitor precursor or NADH. Excitation and emission wavelengths were 500 nm (4 nm slits) and 550 nm (8 nm slits), respectively.

       Analysis

      Kinetic data were fit with a single exponential plus a linear baseline drift,
      y=ax+b(1-e-kx)
      (Eq. 1)


      where a, b, and k (= 1/τ) are fit parameters. The data in Fig. 2D were fit to the equation for single site binding when the KD and receptor concentration are of similar magnitude (derived in the supplemental materials),
      y=C*(x+T0+KD)-(x+T0+KD)2+4(x)T02T0
      (Eq. 2)


      where C, T0, and KD are fit parameters.
      Figure thumbnail gr2
      FIGURE 2FRET between the pre-SufI cargo and the Tat translocon. A, emission scan (EX = 500 nm) of pre-SufI(T96C)Alexa532 (20 nm) and TatABCcherry IMVs (A280 = 2) before (red) and after (blue) addition of 200 nm unlabeled pre-SufI(T96C). The increase in signal at 550 nm indicates loss of FRET. The overall signal change is not a good measure of FRET efficiency since a high fraction of the cargo is not bound to Tat translocons (i.e. free in solution or bound to the lipids). B, time trace of the fluorescence emission at 550 nm (donor) for the experiment in A. Unlabeled pre-SufI(T96C) was added at t = 0 s. The competitor-induced loss of FRET occurred with a time constant of 24 ± 2 s (n = 3). C, total FRET signal observed for various pre-SufI mutants, determined as in B. IMVs contained TatABCcherry (red) or TatABcherryC (blue) (n = 3). The absence of blue bars for some mutants indicates no FRET to TatBcherry. D, precursor binding affinity and receptor concentration estimated from the concentration dependence of the FRET signal. TatABCcherry IMVs were titrated with pre-SufI(T96C)Alexa532 in the presence and absence of 300 nm unlabeled pre-SufI(T96C). Shown here is the average difference (n = 5) between two titration curves, such as those shown in . Three fits are shown in which the receptor concentration (T0) was fixed and the KD and maximum signal were fitting parameters: (red) T0 = 0.1 nm, KD = 23 nm; (blue) T0 = 20 nm, KD = 7.5 nm; (green) T0 = 30 nm, KD = 3.6 nm. These fits indicate that T0 ≈ 0–20 nm and KD ≈ 7–23 nm. Details of the analysis are described in the .

      RESULTS

       Experimental Design

      Our goal was to examine the interaction between the TatBC receptor complex and a cargo under real-time transport conditions. To this end, we used FRET, with a donor fluorophore (Alexa532) on the cargo and an acceptor fluorophore (mCherry) on one of the Tat proteins. We assumed that binding would result in a decrease in donor fluorescence due to FRET. The fluorescent cargo selected was the natural Tat substrate pre-SufI. Eight different single cysteine mutants of pre-SufI were generated. The different cysteine mutations were spread over the surface of the protein (Fig. 1A). These mutants were labeled with Alexa532 maleimide. The C-terminal cysteine mutant, pre-SufI(479C), serves as our wild type (wt) reference because previous experiments indicated that a C-terminal dye had no effect on transport efficiency (
      • Bageshwar U.K.
      • Whitaker N.
      • Liang F.C.
      • Musser S.M.
      Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.
      ). The T96C and 479C mutants yielded the highest transport efficiencies (Fig. 1B).
      Figure thumbnail gr1
      FIGURE 1Pre-SufI cysteine mutants and influence of TatBcherry and TatCcherry on Tat transport. A, location of single cysteine mutations. Residues mutated to cysteine are indicated in red (PDB accession number: 2UXT). Cysteines were labeled with Alexa532 maleimide for transport and FRET experiments. B, transport efficiencies of Alexa532 labeled pre-SufI mutants. Transport reactions (30 min) were performed with TatABC IMVs (A280 = 5), 4 mm NADH and 90 nm pre-SufI. Transport efficiencies were normalized to the 479C mutant (n = 3). C, transport of pre-SufI(479C)Alexa532 into IMVs containing TatABcherryC, TatABCcherry or wt TatABC. Transport requires NADH to generate the necessary Δψ. The mCherry domain has no apparent effect on transport efficiency. Lanes 1–3 are pre-SufI concentration standards. The location of precursor (p) and mature (m) molecular weight bands are identified. Conditions are the same as in B. D, kinetics of pre-SufI(479C)Alexa532 (20 nm) transport into IMVs (A280 = 2) containing wt TatABC (blue), TatABcherryC (green), and TatABCcherry (red). Transport efficiencies were normalized based on the 20 min time point. The black dashed curve is a single exponential plus linear baseline fit to the TatABCcherry data (τ = 81 s).
      To exclude any endogenous (and therefore unlabeled) Tat proteins from IMV preparations, TatBcherry and TatCcherry were expressed in the Tat deletion strain MC4100ΔTatABCDE. The mCherry domain did not significantly affect transport efficiency (Fig. 1C) or transport kinetics (Fig. 1D). The fluorescent TatB and TatC proteins allowed us to visually monitor the IMV purification process. Consequently, we identified a minor membrane-containing band within the 0.5 m sucrose region of the sucrose gradient that had the majority of the mCherry protein (supplemental Fig. S7). The MC4100 membranes recovered from this band catalyzed pre-SufI transport about an order of magnitude faster (Fig. 1D) than reported previously for JM109 IMVs (
      • Bageshwar U.K.
      • Whitaker N.
      • Liang F.C.
      • Musser S.M.
      Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.
      ,
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ). Further, IMVs prepared with our current protocol were more consistently active. Previously, we were not able to consistently obtain active membranes from MC4100 (
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ).

       The FRET Signal

      IMVs isolated from E. coli expressing TatABcherryC or TatABCcherry exhibited two fluorescence peaks at ∼610 nm and ∼641 nm (supplemental Fig. S8, A and B). These peaks both arise from mCherry (supplemental Fig. S8C). Addition of pre-SufIAlexa532 resulted in a third peak at ∼550 nm (Fig. 2A). The decrease in donor fluorescence signal due to FRET was most easily verified by addition of a competitor protein (non-fluorescent cargo), which resulted in an increase in the 550 nm peak, presumably due to replacement of the bound fluorescent protein with the non-fluorescent competitor (Fig. 2A). No increase in 550 nm emission was observed upon competitor addition if mCherry was not attached to the Tat proteins (supplemental Fig. S8D), indicating that the decrease in 550 nm emission when the fluorescent cargo was bound to the Tat translocon (Fig. 2A) was indeed due to FRET. The strong mCherry fluorescence emission far from the excitation wavelength of 500 nm indicates a high concentration relative to the cargo. The mCherry concentration was estimated as 322 ± 88 nm (n = 5) under typical assay conditions, over an order of magnitude higher than the pre-SufI concentration of 20 nm (Fig. 2A). These values are consistent with the amounts of pre-SufIAlexa532 and mCherry needed to approximately reproduce the emission spectra in Fig. 2A in an IMV-free mixture (supplemental Fig. S8D). Any increase in mCherry emission due to FRET was weak and not reliably detected. This is expected if the mCherry acceptor molecules self-quench due to their proximity in a TatBC oligomer (
      • Tarry M.J.
      • Schäfer E.
      • Chen S.
      • Buchanan G.
      • Greene N.P.
      • Lea S.M.
      • Palmer T.
      • Saibil H.R.
      • Berks B.C.
      Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system.
      ).

       Maximizing the FRET Efficiency

      Having determined that FRET indeed occurred upon binding of the cargo to the Tat translocon, we next sought to maximize the FRET signal. This was accomplished by individually attaching the donor dye to the 8 locations identified in Fig. 1A, which cover the surface of pre-SufI. In addition, the mCherry acceptor was attached to either TatB or TatC. FRET signal intensity was determined from kinetic experiments in which non-fluorescent competitor cargo was added to IMVs with prebound fluorescent cargo (Fig. 2B). For pre-SufI(T96C), the competitor released the bound cargo with τ = 24 ± 2 s (Fig. 2B). Control kinetic experiments in the absence of a donor or acceptor fluorophore confirmed the transient FRET signal (supplemental Fig. S9A). We found that TatCcherry yielded stronger FRET signals than TatBcherry (Fig. 2C). Two pre-SufI mutants, G29C and T96C, yielded similarly strong FRET signals when the mCherry domain was attached to TatC (Fig. 2C). However, labeling at G29C reduced transport efficiency (Fig. 1B). To convert the relative FRET signals into reliable distance and/or orientation information, the data in Fig. 2C need to be corrected based on binding affinity. We attempted to do this using our previous binding assay (
      • Bageshwar U.K.
      • Whitaker N.
      • Liang F.C.
      • Musser S.M.
      Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.
      ), but the errors of these binding measurements and the errors of the FRET measurements were too high to provide meaningful information. Instead, we settled on using TatCcherry and the T96C mutant of pre-SufI for all subsequent experiments since these proteins yielded the strongest FRET signal while retaining transport efficiency (Figs. 1B and 2C).

       Binding Affinity

      The FRET signal was used to estimate the cargo-TatBC binding affinity. Two titrations were done. The first titration involved adding increasing amounts of fluorescent pre-SufI(T96C) to IMVs. The second titration was identical to the first, except that a large excess of non-fluorescent cargo was preincubated with the IMVs (supplemental Fig. S10). The difference in donor fluorescence intensity between the two titrations (FRET signal) reflects the amount of fluorescent cargo bound to the translocon (Fig. 2D). Based on the mCherry concentration, the Tat receptor concentration is high relative to the apparent KD. Thus, the data in Fig. 2D were fit with Equation 2 (“Experimental Procedures” and supplemental materials), which explicitly includes the receptor concentration as a fit parameter. Unfortunately, the receptor concentration and KD are not uniquely determined from the data. However, the data indicate that the functional receptor concentration is ≤ ∼20 nm and the KD ≈ 7–23 nm (see Fig. 2D and supplemental materials for a detailed explanation).

       FRET Decreases upon Membrane Energization

      When fluorescent pre-SufI was prebound to IMVs containing Tat-mCherry fusions and the membranes were subsequently energized by the addition of NADH, the donor fluorescence signal increased (a decrease in FRET). This was observed for membranes containing either TatBcherry or TatCcherry (Fig. 3A). Control experiments in the absence of donor or acceptor fluorophore confirmed the NADH-dependent loss of FRET (supplemental Fig. S9B). Since NADH addition initiates transport across the membrane, these data are consistent with migration of the cargo away from its initial binding site on the TatBC complex as part of the transport process. It is unclear if the observed kinetics reflect migration elsewhere within the translocon (e.g. to the TatA pore) or immediate movement across the membrane. NADH generates a PMF, which is necessary for Tat transport. It was shown earlier that the Δψ and not the ΔpH component of the PMF is essential for Tat transport (
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ). We therefore tested whether the observed FRET signal is sensitive to these two PMF components. We found that the decrease in FRET upon NADH addition requires a Δψ and not a ΔpH (Fig. 3B), consistent with the hypothesis that the observed changes in the FRET signal report a transport substep.
      Figure thumbnail gr3
      FIGURE 3Effect of PMF components on the FRET signal. A, time trace of the fluorescence emission at 550 nm (donor) upon membrane energization. Reactions contained pre-SufI(T96C)Alexa532 (20 nm) and IMVs (A280 = 2) with TatABcherryC (blue) or TatABCcherry (red). NADH (4 mm) was added at t = 0 s. B, sensitivity of NADH-dependent fluorescence changes to components of the PMF. The ΔpH and Δψ were reduced with nigericin (5 μm, blue) and valinomycin (5 μm, green), respectively. The control trace (red) contains no ionophores. Conditions as in A with TatABCcherry IMVs (n = 2). C, gradients across membranes of TatABCcherry IMVs (A280 = 2). The presence of Δψ (blue) and ΔpH (red) gradients were determined using 100 nm oxonol VI (EX = 610 nm, EM = 645 nm) and 2.5 μm quinacrine (EX = 420 nm, EM = 510 nm), respectively, as described previously (
      • Bageshwar U.K.
      • Whitaker N.
      • Liang F.C.
      • Musser S.M.
      Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.
      ). NADH (4 mm) was added at t = 50 s.

       A Δψ-dependent Step Precedes Cargo Migration Away from the TatBC Complex

      Exponential NADH-dependent FRET changes (τ = ∼30 s) were preceded by a lag phase (Fig. 3A). The duration of the delay was dependent on the batch of IMVs (e.g. compare Fig. 3, A and B), and ranged from ∼20 to ∼45 s (n = 9). This delay did not arise from slow formation of the Δψ, as the Δψ forms within seconds (Fig. 3C). While it takes ∼25–30 s for the ΔpH to become fully established (Fig. 3C), it was shown earlier (
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ) and here (Fig. 3B) that the ΔpH is not necessary for transport. Therefore, the slow formation of the ΔpH does not explain the lag phase. Consequently, we reasoned that the lag phase could be explained by a Δψ-dependent conformational change or oligomerization process that is required for the cargo to migrate away from the TatBC binding site. We tested this hypothesis by preincubating the IMVs with NADH and then adding the cargo after a short delay (Fig. 4). The lag phase largely disappeared when the cargo was added 200 s after NADH (Fig. 4C). Under these conditions, slower kinetics (τ = ∼70–90 s) were observed.
      Figure thumbnail gr4
      FIGURE 4Cargo migration kinetics depend on the interval between energization and cargo addition times. The time-dependent donor fluorescence emission (550 nm) was measured for transport reactions containing TatABCcherry IMVs (A280 = 2) and pre-SufI(T96C)Alexa532 (20 nm). Membranes were energized with NADH (4 mm) and the cargo was added 0 s (A), 10 s (B), or 200 s (C) later. Cargo was added at t = 0 s. The kinetics after the lag phase in A and B were fit with a single exponential plus a linear baseline drift (“Experimental Procedures”), yielding τ = 33 s and 46 s, respectively. The entire trace in C was fit with the same equation, yielding τ = 93 s. The linear baseline drift accounts for ∼11% of the total fit fluorescence change for all three panels. Each trace represents an average of three individual runs.

       Pre-SufI Is Released from TatBC Complexes when Membranes Are Energized in the Absence of TatA

      As a control experiment for the NADH-dependent loss of FRET (Fig. 3A), we repeated these experiments in the absence of TatA. Similar levels of TatB and TatCcherry were recovered in IMVs in the presence and absence of TatA (supplemental Fig. S11). Since TatA is required for transport, our expectation was that the cargo would remain bound to the TatBC complex, and hence, the FRET signal would remain constant. Experiments similar to those in Fig. 3A were performed, where pre-SufI was allowed to bind to IMVs, and NADH was added at time zero. FRET immediately decreased (τ = 71 s) when the membranes were energized in the absence of TatA (Fig. 5A). This differed from results obtained with IMVs containing TatA (Figs. 3A and 4A), which exhibited a lag-phase and rapid loss of FRET after the lag phase (τ = ∼30 s). Since the Tat machinery is incapable of transport without TatA (or its paralog, TatE) (
      • Sargent F.
      • Bogsch E.G.
      • Stanley N.R.
      • Wexler M.
      • Robinson C.
      • Berks B.C.
      • Palmer T.
      Overlapping functions of components of a bacterial Sec-independent protein export pathway.
      ), the observed loss of FRET is not due to cargo translocation across the membrane, as confirmed in Fig. 5B. Rather, likely possibilities are that the loss of FRET arises either from a conformational change that results in the mature domain of the bound precursor moving away from the mCherry fluorophore, or from the precursor dissociating from the TatBC complex entirely. The latter appears to be the case since TatBC IMVs pelleted after energization retain ∼53% less bound pre-SufI than unenergized IMVs (Fig. 5B). Multiple groups have suggested the possibility that the signal peptide can penetrate fairly deeply into the membrane (
      • Strauch E.M.
      • Georgiou G.
      Escherichia coli tatC mutations that suppress defective twin-arginine transporter signal peptides.
      ,
      • Gérard F.
      • Cline K.
      The thylakoid proton gradient promotes an advanced stage of signal peptide binding deep within the Tat pathway receptor complex.
      ,
      • Schlesier R.
      • Klösgen R.B.
      Twin arginine translocation (Tat)-dependent protein transport: the passenger protein participates in the initial membrane binding step.
      ,
      • Panahandeh S.
      • Maurer C.
      • Moser M.
      • DeLisa M.P.
      • Müller M.
      Following the path of a twin-arginine precursor along the TatABC translocase of Escherichia coli.
      ). The energy-dependent dissociation of pre-SufI from the receptor complex in the absence of TatA suggests the possibility that the signal sequence may not have penetrated as deeply under these conditions.
      Figure thumbnail gr5
      FIGURE 5Transport and binding to energized membranes in the absence of TatA. A, time traces of donor emission (at 550 nm) in the presence or absence of mCherry after energization with 4 mm NADH at t = 0. Reactions contained pre-SufI(T96C)Alexa532 (20 nm) and IMVs with TatBCcherry (red) or TatBC (blue) (A280 = 2). The TatBCcherry data were fit with a single exponential plus a linear baseline drift, yielding τ = 71 s (the baseline drift accounts for 13% of the total fluorescent change). B, membrane binding and cargo transport in the presence and absence of TatA. Pre-SufI(T96C)Alexa532 (20 nm) was allowed to bind to membranes (TatBC or TatABC IMVs, A280 = 2) for 10 min at 37 °C, respectively. After a 10 min binding period, control samples were then centrifuged (16,000 × g, 45 min, room temperature), and the pellet fractions were analyzed by SDS-PAGE (lanes 1–2). Resuspended membranes were energized with 4 mm NADH for 20 min at 37 °C (lanes 3–6). Proteinase K (PK, 0.7 mg/ml) was added to half the reactions and all reactions were immediately centrifuged (16,000 × g, 45 min, room temperature). The resultant membrane pellets were resuspended in translocation buffer containing 68 mm PMSF and resolved via SDS-PAGE. The amounts of recovered precursor (p, blue) and mature (m, red) proteins are shown above the sample gel, as indicated (n = 3). Less pre-SufI was recovered with membranes lacking TatA (compare lanes 3 and 4), indicating that the TatBC affinity for pre-SufI is lower without TatA in the presence of a PMF. Matured protein was protease protected (compare lanes 4 and 6), indicating transport. The average amount of protein initially recovered with TatABC membranes (lane 2) was set to 100%.

      DISCUSSION

      Tat transport requires both binding of a pre-protein to the Tat proteins as well as movement of the cargo across the membrane. Presumably, these two seemingly conflicting processes are coupled, i.e. the binding reaction gates the transport event. Critical to understanding this process is the development and analysis of assays that can identify and discern the conversion between substeps of transport. To this end, we developed a FRET assay that reports the initial cargo binding step. Our major conclusions are: 1) transport occurs on the minute timescale with a highly purified membrane fraction (Fig. 1D); 2) the cargo binding interaction occurs with an apparent KD ≈ 7–23 nm (Fig. 2D) and a competitor-induced koff ≈ 0.042 s−1 (=1/24 s) (Fig. 2B); 3) the cargo mature domain appears to be nearer to the C terminus of TatC than the C terminus of TatB (Fig. 2C); 4) migration of the cargo from its initial binding site requires a Δψ (Fig. 3B); 5) a delay in cargo migration from the initial binding site occurs after membrane energization (FIGURE 3, FIGURE 4), indicating that the Δψ does not directly promote cargo migration; and 6) TatA increases the affinity of the TatBC receptor complex for the precursor in the presence of a membrane potential (Fig. 5). The implications of these results are now discussed.
      As far as we are aware, the binding experiments reported here provide the first estimate of the binding affinity of a signal peptide for functional E. coli Tat translocons. The estimated KD of 7–23 nm indicates a fairly strong, highly specific interaction, as is reasonably expected for a selective process. From this measured KD and the competitor-induced koff ≈ 0.042 s−1 (Fig. 2B), the apparent kon is 106-107 m−1 s−1. This second order rate constant is of the magnitude expected for enzymatic interactions, where orientation of the reactants is clearly important, and is complicated by the fact that it likely reflects both binding to the membrane lipids (
      • Bageshwar U.K.
      • Whitaker N.
      • Liang F.C.
      • Musser S.M.
      Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.
      ) as well as lateral diffusion to the Tat translocon. Whereas the precursor can apparently exchange between lipid- and translocon-bound forms on the tens of seconds timescale, release from the membrane surface is significantly slower (
      • Bageshwar U.K.
      • Whitaker N.
      • Liang F.C.
      • Musser S.M.
      Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.
      ).
      It is unclear whether the signal peptide binding interaction was broken at the point at which the FRET interaction was lost under transport conditions, or whether there is sufficient flexibility between the mature domain and signal peptide such that large movements of the mature domain away from the C termini of TatB and TatC can occur without dissociation of the signal peptide. The latter is consistent with the finding that the thylakoid Tat machinery can transport cargos covalently linked to TatC via their signal peptide (
      • Gérard F.
      • Cline K.
      Efficient twin arginine translocation (Tat) pathway transport of a precursor protein covalently anchored to its initial cpTatC binding site.
      ). In contrast, the NADH-dependent release of the cargo from membranes containing TatBC complexes but no TatA (Fig. 5) indicates a weaker membrane binding affinity in the presence of a Δψ under these conditions, and thus clear dissociation of the signal peptide from the TatBC receptor complex under these conditions.
      The analysis of the data in Fig. 2D indicates that the functional TatBC receptor concentration is ≤∼20 nm (supplemental materials). Thus, the functional binding site concentration is significantly lower than the number of TatC molecules (322 ± 88 nm) calculated based on the mCherry concentration. Note that TatABCcherry IMVs contained only full-length TatCcherry (supplemental Fig. S11), indicating that the mCherry fluorescence signal was not contaminated by degradation products of the fusion protein. One possibility is that most TatC molecules are inaccessible or non-functional, possibly a consequence of overexpression. Tarry et al. (
      • Tarry M.J.
      • Schäfer E.
      • Chen S.
      • Buchanan G.
      • Greene N.P.
      • Lea S.M.
      • Palmer T.
      • Saibil H.R.
      • Berks B.C.
      Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system.
      ) found that cargos bind to only a few available binding sites in TatBC oligomers, which they estimated to be heptamers. According to our data, active heptamers could account for ∼140 nm (=7 × 20 nm) of the TatC molecules present. The remaining ∼180 nm of the TatC molecules present may then be inactive or inaccessible. However, a recent report with covalent TatC dimers suggests that TatBC complexes contain an even number of TatC molecules (
      • Maldonado B.
      • Buchanan G.
      • Müller M.
      • Berks B.C.
      • Palmer T.
      Genetic evidence for a TatC dimer at the core of the Escherichia coli twin arginine (Tat) protein translocase.
      ). Thus, the oligomerization state of active TatBC complexes remains uncertain.
      For all the pre-SufI single cysteine mutants tested, FRET signals were higher with TatCcherry than with TatBcherry. This is seemingly at odds with a recent crosslinking study, which suggested that pre-SufI binds more closely to TatB than to TatC (
      • Maurer C.
      • Panahandeh S.
      • Jungkamp A.C.
      • Moser M.
      • Müller M.
      TatB functions as an oligomeric binding site for folded Tat precursor proteins.
      ). However, the observed FRET signal arises from the proximity of the pre-SufI mature domain to the mCherry protein, which was at the C termini of the Tat proteins. Our data do not exclude the possibility that the dye molecules on the pre-SufI mutants are closer to the amphipathic region of TatB than to the TatB C terminus. Consequently, the models of Maurer et al. (
      • Maurer C.
      • Panahandeh S.
      • Jungkamp A.C.
      • Moser M.
      • Müller M.
      TatB functions as an oligomeric binding site for folded Tat precursor proteins.
      ) are entirely consistent with our FRET data.
      A model that has emerged for the E. coli Tat system and the pre-SufI cargo is that the cargo first binds to the membrane lipids. From there, the cargo laterally diffuses to the TatBC complex. Translocation across the membrane, presumably with the assistance of TatA oligomers, requires a Δψ (
      • Bageshwar U.K.
      • Whitaker N.
      • Liang F.C.
      • Musser S.M.
      Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.
      ). From studies on the thylakoid Tat system, the oligomerization of a TatBC complex with a TatA complex requires a PMF (
      • Mori H.
      • Cline K.
      A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase.
      ). It seems safe to deduce, then, that such an oligomerization process is responsible for the lag phase observed in Fig. 3A. Loss of FRET is fairly rapid after this lag phase, possibly indicating that migration of the cargo mature domain from TatBC is fairly quick after TatABC oligomerization is complete. Fig. 4C indicates that migration after binding occurs essentially immediately if the cargo is added ∼200 s after Δψ generation has been initiated, possibly indicating that TatABC oligomerization occurred before the cargo bound to TatBC. The cargo migration away from TatBC is ∼2.6-fold slower under these conditions, likely due to the fact that the detectable Δψ had already collapsed (Fig. 3C; see also Ref.
      • Bageshwar U.K.
      • Whitaker N.
      • Liang F.C.
      • Musser S.M.
      Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.
      ) when the cargo was added. Receptor binding and migration of the mature domain away from the TatBC complex occurs concurrently in Fig. 4C. The absence of a detectable increase in FRET at the beginning part of the kinetics in Fig. 4C indicates a rapid binding step (τ ≤ ∼3 s), which includes both lipid binding and diffusion to the Tat translocon. Our data are therefore consistent with the hypothesis that TatBC and TatA oligomerization occurs in the presence of a Δψ, with or without the cargo. Further, the Δψ makes the translocation system receptive to cargo movement from the receptor binding site (e.g. by a conformational change, or the oligomerization itself), but does not appear to directly drive it energetically. We expect that under in vivo conditions where a large Δψ is consistently maintained, all three Tat components can and do oligomerize and cargo transport occurs on the sub-minute timescale.
      According to the oligomerization model discussed in the previous paragraph, TatA and TatBC form separate oligomers in the absence of a cargo and a PMF (
      • Bolhuis A.
      • Mathers J.E.
      • Thomas J.D.
      • Barrett C.M.
      • Robinson C.
      TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli.
      ,
      • Gohlke U.
      • Pullan L.
      • McDevitt C.A.
      • Porcelli I.
      • de Leeuw E.
      • Palmer T.
      • Saibil H.R.
      • Berks B.C.
      The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter.
      ,
      • Maldonado B.
      • Buchanan G.
      • Müller M.
      • Berks B.C.
      • Palmer T.
      Genetic evidence for a TatC dimer at the core of the Escherichia coli twin arginine (Tat) protein translocase.
      ,
      • de Leeuw E.
      • Granjon T.
      • Porcelli I.
      • Alami M.
      • Carr S.B.
      • Müller M.
      • Sargent F.
      • Palmer T.
      • Berks B.C.
      Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes.
      ). Thus, our expectation was that the stability of the TatBC-precursor complex should be unaffected by the absence of TatA in the presence or absence of a Δψ. We were therefore surprised that pre-SufI was released from IMVs upon addition of NADH in the absence of TatA (Fig. 5B). We interpret these data to imply that the receptor complex has a weaker affinity for pre-SufI in the absence of TatA when a Δψ is present. This appears inconsistent with an oligomerization model in which TatA is not part of the receptor complex. However, an alternative, revised model is that at least some TatA is part of the receptor complex, consistent with earlier results (
      • Bolhuis A.
      • Mathers J.E.
      • Thomas J.D.
      • Barrett C.M.
      • Robinson C.
      TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli.
      ,
      • de Leeuw E.
      • Granjon T.
      • Porcelli I.
      • Alami M.
      • Carr S.B.
      • Müller M.
      • Sargent F.
      • Palmer T.
      • Berks B.C.
      Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes.
      ). This picture is also consistent with a recent study, which reports some cross-linking of pre-SufI to TatA in the absence of a PMF (
      • Fröbel J.
      • Rose P.
      • Müller M.
      Early contacts between substrate proteins and TatA translocase component in twin-arginine translocation.
      ). In light of these data suggesting that TatA is part of the TatBC receptor complex, it is not at all surprising that the absence of TatA affects the interaction between TatBC and pre-SufI. According to our FRET assay, a weaker receptor complex binding affinity in the absence of TatA is only apparent in the presence of a PMF. TatA is necessary to keep pre-SufI bound to the TatBC complex in the presence of a PMF, eventually leading to transport (compare Figs. 4A and 5A).
      In summary, we have isolated membranes exhibiting rapid Tat translocation activity. For TatABCcherry IMVs, the overall translocation process is characterized by τ = ∼80 s (Fig. 1D). When a Δψ is generated across these IMV membranes with bound precursor protein, migration away from the TatBC binding site occurs with τ = ∼30 s after a significant (20–45 s) lag phase (Fig. 4A). Migration away from the TatBC binding site occurs more slowly, but with no lag phase, if the precursor is added to previously energized membranes (τ = ∼90 s, Fig. 4C), suggesting that the effect of the Δψ slowly wears off or that transport in the presence of a weak, undetectable Δψ (
      • Bageshwar U.K.
      • Whitaker N.
      • Liang F.C.
      • Musser S.M.
      Interconvertibility of lipid- and translocon-bound forms of the bacterial Tat precursor pre-SufI.
      ) occurs more slowly. Binding to the membrane surface and diffusion to the TatBC complex occurs rapidly (τ ≤ ∼3 s). Therefore, the migration process in which the cargo moves away from the TatBC complex requires a significant fraction (∼40%) of the total transport time. It is unclear whether the migration step itself is rate-limiting or whether an upstream kinetic step limits the migration rate. The gel-based transport assay has poor time resolution, and, since it is not a real-time assay, it is not clear whether any transport substeps can occur after quenching the reaction. Consequently, it remains unclear whether the cargo proceeds directly across the membrane bilayer upon leaving the TatBC complex or whether there is an additional kinetically significant intermediate, e.g. cargo within the TatA pore. Further experiments are needed to clarify these downstream events.

      Acknowledgments

      We thank T. L. Yahr for pET-SufI and pTatABC, T. Palmer for MC4100 and MC4100ΔTatABCDE, and Anita Pokharel for technical assistance.

      Supplementary Material

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