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Mechanistic Aspects of Folded Protein Transport by the Twin Arginine Translocase (Tat)*

Open AccessPublished:May 14, 2015DOI:https://doi.org/10.1074/jbc.R114.626820
      The twin arginine translocase (Tat) transports folded proteins of widely varying size across ionically tight membranes with only 2–3 components of machinery and the proton motive force. Tat operates by a cycle in which the receptor complex combines with the pore-forming component to assemble a new translocase for each substrate. Recent data on component and substrate organization in the receptor complex and on the structure of the pore complex inform models for translocase assembly and translocation. A translocation mechanism involving local transient bilayer rupture is discussed.

      Introduction

      Cells and membranes contain multiple protein translocases, i.e. the enzymes that catalyze protein translocation across or into membranes. This allows for specific targeting to distinct cellular locations and, through varied transport mechanisms, solutions to a range of translocation problems. For example, the prokaryotic and endoplasmic reticulum Sec system and several other translocases transport proteins in a mostly unfolded conformation. This permits a single mechanistic strategy for those substrates that can be unfolded for translocation and folded following transport. By contrast, the twin arginine translocase (Tat)
      The abbreviations used are: Tat
      twin arginine translocase
      PMF
      proton motive force
      APH
      amphipathic helix
      TM
      transmembrane domain
      tOE17
      truncated OE17.
      system transports proteins that are in a folded conformation during translocation. The Tat system is present in most bacteria, in some archaea, in chloroplasts of plants and algae, and in some mitochondria (
      • Cline K.
      • Theg S.
      The Sec and Tat protein translocation pathways in chloroplasts.
      • Palmer T.
      • Berks B.C.
      The twin-arginine translocation (Tat) protein export pathway.
      ,
      • Fröbel J.
      • Rose P.
      • Müller M.
      Twin-arginine-dependent translocation of folded proteins.
      ,
      • Celedon J.M.
      • Cline K.
      Intra-plastid protein trafficking: how plant cells adapted prokaryotic mechanisms to the eukaryotic condition.
      • Berks B.C.
      The twin-arginine protein translocation pathway.
      ). The prevalence of Tat in prokaryotes and prokaryote-derived organelles argues that Tat is an ancient translocation system. The fact that Tat operates with as few as two membrane components of machinery (TatA and TatC) (
      • Goosens V.J.
      • Monteferrante C.G.
      • van Dijl J.M.
      The Tat system of Gram-positive bacteria.
      ) and the proton motive force (PMF) implies a simple mechanism. Although it may be mechanistically simple, Tat behaves in mystifying ways and performs astonishing feats. It exists as oligomeric structures, with a multivalent receptor complex composed of TatC and TatB multimers and a “pore” complex of TatA oligomers that are thought to form the protein-conducting element. Tat can transport folded proteins from ∼20 Å (
      • Henry R.
      • Kapazoglou A.
      • McCaffery M.
      • Cline K.
      Differences between lumen targeting domains of chloroplast transit peptides determine pathway specificity for thylakoid transport.
      ) to ∼70 Å (
      • Berks B.C.
      • Palmer T.
      • Sargent F.
      The Tat protein translocation pathway and its role in microbial physiology.
      ). It can transport heterodimers, where one subunit has the signal peptide and the other hitchhikes the ride (
      • Rodrigue A.
      • Chanal A.
      • Beck K.
      • Müller M.
      • Wu L.F.
      Co-translocation of a periplasmic enzyme complex by a hitchhiker mechanism through the bacterial tat pathway.
      ), and cross-linked tetramers, where each subunit is bound via its own signal peptide (
      • Ma X.
      • Cline K.
      Multiple precursor proteins bind individual Tat receptor complexes and are collectively transported.
      ). Tat will even transport engineered unstructured polypeptides (
      • Cline K.
      • McCaffery M.
      Evidence for a dynamic and transient pathway through the TAT protein transport machinery.
      ,
      • Richter S.
      • Lindenstrauss U.
      • Lücke C.
      • Bayliss R.
      • Brüser T.
      Functional Tat transport of unstructured, small, hydrophilic proteins.
      ). Thus, Tat has solved the daunting mechanistic challenge of transporting both large and small protein structures without opening large holes that would dissipate the PMF.
      The Tat substrate repertoire depends on the organism and ranges from 1 to ∼150 substrates (reviewed in Ref.
      • Palmer T.
      • Berks B.C.
      The twin-arginine translocation (Tat) protein export pathway.
      ). Tat plays critical roles in respiratory and photosynthetic energy production, animal and plant pathogenesis, symbiosis, etc. (reviewed in Refs.
      • Palmer T.
      • Berks B.C.
      The twin-arginine translocation (Tat) protein export pathway.
      ,
      • Berks B.C.
      • Palmer T.
      • Sargent F.
      The Tat protein translocation pathway and its role in microbial physiology.
      , and
      • Meloni S.
      • Rey L.
      • Sidler S.
      • Imperial J.
      • Ruiz-Argüeso T.
      • Palacios J.M.
      The twin-arginine translocation (Tat) system is essential for Rhizobium-legume symbiosis.
      ,
      • Voelker R.
      • Barkan A.
      Two nuclear mutations disrupt distinct pathways for targeting proteins to the chloroplast thylakoid.
      • De Buck E.
      • Lammertyn E.
      • Anné J.
      The importance of the twin-arginine translocation pathway for bacterial virulence.
      ). Among reasons that proteins require transport in a folded conformation include: the absence of folding and cofactor insertion machinery on the trans side of the membrane, the need to control metal cofactor specificity, and the fact that some proteins rapidly fold upon synthesis or import into the organelle (
      • Palmer T.
      • Berks B.C.
      The twin-arginine translocation (Tat) protein export pathway.
      ,
      • Berks B.C.
      The twin-arginine protein translocation pathway.
      ).
      Unraveling the mechanisms of Tat operation has been challenging because of the ephemeral nature of the translocase (
      • Mori H.
      • Cline K.
      A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase.
      ). However, mechanistic knowledge has advanced due to studies with the thylakoid membrane of plant chloroplasts and the cytoplasmic membrane of Escherichia coli. A combination of staged in vitro assays, protein cross-linking, molecular structural analyses, and fluorescence imaging in living cells has increased knowledge sufficiently that informed models are possible. Both systems employ three components, a TatC protein and two members of the TatA family (TatA and TatB). For historical reasons, the nomenclature of the two systems has not been consistent; TatA and TatB in bacteria are called Tha4 and Hcf106 in thylakoids. However, considerable research has shown that Tha4 and Hcf106 function similarly to TatA and TatB. In this review, I will refer to these proteins simply as TatA and TatB and note the system from which results were obtained. In addition, the term cis will designate the compartment of substrate origin, and trans will designate the destination compartment. The scope of this review is limited to recent insights into Tat mechanisms. The reader is referred to several other excellent reviews for a broader view of Tat (
      • Cline K.
      • Theg S.
      The Sec and Tat protein translocation pathways in chloroplasts.
      • Palmer T.
      • Berks B.C.
      The twin-arginine translocation (Tat) protein export pathway.
      ,
      • Fröbel J.
      • Rose P.
      • Müller M.
      Twin-arginine-dependent translocation of folded proteins.
      ,
      • Celedon J.M.
      • Cline K.
      Intra-plastid protein trafficking: how plant cells adapted prokaryotic mechanisms to the eukaryotic condition.
      • Berks B.C.
      The twin-arginine protein translocation pathway.
      ).

      A Twin Arginine Signal Peptide Targets the Substrate

      Tat substrates are targeted to Tat machinery by hydrophobic signal peptides that superficially resemble Sec signal peptides in tripartite organization: an amino proximal N domain, a hydrophobic H domain, and a polar C domain that contains the signal peptidase cleavage site (Fig. 1). The Tat recognition sequence at the N/H junction is the most distinctive feature of Tat signal peptides. It consists of two adjacent Arg residues within a broader consensus (Fig. 1). The RR motif is essential; mutation to KK eliminates interaction with the Tat machinery (
      • Nivière V.
      • Wong S.L.
      • Voordouw G.
      Site-directed mutagenesis of the hydrogenase signal peptide consensus box prevents export of a β-lactamase fusion protein.
      • Chaddock A.M.
      • Mant A.
      • Karnauchov I.
      • Brink S.
      • Herrmann R.G.
      • Klösgen R.B.
      • Robinson C.
      A new type of signal peptide: central role of a twin-arginine motif in transfer signals for the ΔpH-dependent thylakoidal protein translocase.
      ,
      • Henry R.
      • Carrigan M.
      • McCaffrey M.
      • Ma X.
      • Cline K.
      Targeting determinants and proposed evolutionary basis for the Sec and the ΔpH protein transport systems in chloroplast thylakoid membranes.
      ,
      • 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.
      ,
      • Gérard F.
      • Cline K.
      Efficient twin arginine translocation (Tat) pathway transport of a precursor protein covalently anchored to its initial cpTatC binding site.
      • Kwan D.
      • Bolhuis A.
      Analysis of the twin-arginine motif of a haloarchaeal Tat substrate.
      ). Other consensus residues are of lesser importance (
      • Kwan D.
      • Bolhuis A.
      Analysis of the twin-arginine motif of a haloarchaeal Tat substrate.
      ,
      • Stanley N.R.
      • Palmer T.
      • Berks B.C.
      The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli.
      ), with the exception of the Phe at RR+2, which has a critical positive effect on transport efficiency. Most thylakoid Tat signal peptides lack this Phe, but introducing a Phe into the thylakoid OE17 substrate (Fig. 1) increased the binding affinity 10-fold to ∼1 nm Kd (
      • Gérard F.
      • Cline K.
      The thylakoid proton gradient promotes an advanced stage of signal peptide binding deep within the Tat pathway receptor complex.
      ,
      • Celedon J.
      • Cline K.
      Stoichiometry for binding and transport by the twin arginine translocation system.
      ). The resulting tOE17-20F (Fig. 1) was used in many studies cited here. Some Tat signal peptides have an extended N domain of uncertain function. Deleting the extended N domain of several thylakoid Tat substrates improved in vitro transport efficiency (
      • Henry R.
      • Carrigan M.
      • McCaffrey M.
      • Ma X.
      • Cline K.
      Targeting determinants and proposed evolutionary basis for the Sec and the ΔpH protein transport systems in chloroplast thylakoid membranes.
      ,
      • Ma X.
      • Cline K.
      Precursors bind to specific sites on thylakoid membranes prior to transport on the ΔpH protein translocation system.
      ). Deleting the extended N domain of the E. coli hydrogenase-1 small subunit similarly improved transport efficiency of fused reporter proteins but impaired maturation of hydrogenase 1 (
      • Bowman L.
      • Palmer T.
      • Sargent F.
      A regulatory domain controls the transport activity of a twin-arginine signal peptide.
      ). This suggests that the extended N domain serves as a timing device, slowing translocation to allow holoenzyme folding and assembly.
      Figure thumbnail gr1
      FIGURE 1Architecture of Tat signal peptides. Tat signal peptides have three domains, an N-terminal N domain, a hydrophobic helical H domain, and a C-terminal C domain that contains the cleavage site (generally AXA, where X is any residue) for a trans facing signal peptidase. Tat signal peptides differ from Sec signal peptides by the Tat motif, a somewhat lower hydrophobicity H domain, and a C domain often containing basic residue(s). Some Tat signal peptides possess an extended N domain of uncertain function (hatched). Consensus Tat motifs for bacteria (
      • Berks B.C.
      • Sargent F.
      • Palmer T.
      The Tat protein export pathway.
      ), halophilic archaea (
      • Kwan D.
      • Bolhuis A.
      Analysis of the twin-arginine motif of a haloarchaeal Tat substrate.
      ), and thylakoids (
      • Peltier J.B.
      • Emanuelsson O.
      • Kalume D.E.
      • Ytterberg J.
      • Friso G.
      • Rudella A.
      • Liberles D.A.
      • Söderberg L.
      • Roepstorff P.
      • von Heijne G.
      • van Wijk K.J.
      Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.
      ) are shown.

      Cycle of Substrate Binding, Translocase Assembly, and Translocation

      Tat systems operate by a cycle in which the components (Fig. 2A) assemble a translocase on demand (Fig. 2B). This was shown with thylakoid in vitro assays and with fluorescence imaging in living E. coli. In non-transporting thylakoids, TatB and TatC are present as a receptor complex in a 1:1 ratio; TatA is present as a separate pool (
      • Mori H.
      • Cline K.
      A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase.
      ,
      • Cline K.
      • Mori H.
      Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport.
      ). The receptor complex binds the substrate signal peptide, triggering PMF-dependent TatA assembly and oligomerization at the substrate-TatBC interface (
      • Mori H.
      • Cline K.
      A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase.
      ,
      • Dabney-Smith C.
      • Cline K.
      Clustering of C-terminal stromal domains of Tha4 homo-oligomers during translocation by the Tat protein transport system.
      ,
      • Dabney-Smith C.
      • Mori H.
      • Cline K.
      Oligomers of Tha4 organize at the thylakoid Tat translocase during protein transport.
      ). The assembled complex is called the “translocase.” After translocase assembly, the substrate is transported, the signal peptide is cleaved, and the TatA oligomer is disassembled in an uncertain order. The in vitro cycle is sluggish. A time course starting with substrate-bound TatBC and initiated with the PMF exhibited a 1–3-min transport lag (
      • Celedon J.
      • Cline K.
      Stoichiometry for binding and transport by the twin arginine translocation system.
      ) during which TatA could be increasingly cross-linked to TatBC (
      • Mori H.
      • Cline K.
      A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase.
      ). Substrate transport then occurred with first order kinetics and a transport time (τ) of ∼80 s (
      • Celedon J.
      • Cline K.
      Stoichiometry for binding and transport by the twin arginine translocation system.
      ). When all substrate was transported, the ability to cross-link TatA dropped to background. A similar in vitro rate was measured for SufI transport into E. coli vesicles (
      • Whitaker N.
      • Bageshwar U.K.
      • Musser S.M.
      Kinetics of precursor interactions with the bacterial Tat translocase detected by real-time FRET.
      ).
      Figure thumbnail gr2
      FIGURE 2Tat operates by a cyclical mechanism with a signal-assembled translocase. A, three components of Tat machinery in chloroplasts and E. coli. Chloroplast TatC has a very long N terminus of unknown function that is absent from bacterial TatC. APH domains are shown with a striped pattern. The relative molar quantities of components in situ are indicated in parentheses. B, cyclical mechanism for Tat protein transport. The TatBC receptor complex binds the substrate signal peptide in an energy-independent step. The receptor complex is depicted in the figure as a TatBC heterodimer, but it is actually a multimer estimated to contain up to eight TatBC units. Signal peptide binding triggers PMF-dependent assembly and oligomerization of TatA. The resulting complex is the translocase. Changes in the TatA oligomer are thought to facilitate protein transport, after which the translocase dissociates.
      Fluorescence imaging of TatA-YFP (or TatA-GFP) in E. coli cells showed a similar cycle. TatA-XFP exists as small oligomers in non-transporting cells (
      • Leake M.C.
      • Greene N.P.
      • Godun R.M.
      • Granjon T.
      • Buchanan G.
      • Chen S.
      • Berry R.M.
      • Palmer T.
      • Berks B.C.
      Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.
      ,
      • Alcock F.
      • Baker M.A.
      • Greene N.P.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system.
      • Rose P.
      • Fröbel J.
      • Graumann P.L.
      • Müller M.
      Substrate-dependent assembly of the Tat translocase as observed in live Escherichia coli cells.
      ). Substrate expression resulted in coalescence of TatA-XFP into large fluorescent foci termed “TatA assemblies” that co-localized with TatB (
      • Rose P.
      • Fröbel J.
      • Graumann P.L.
      • Müller M.
      Substrate-dependent assembly of the Tat translocase as observed in live Escherichia coli cells.
      ) and presumably TatC. Dissipating the PMF with carbonyl cyanide m-chlorophenylhydrazone prevented the formation of TatA assemblies and resulted in disassembly when added during the protein transport phase. A transport-inactive TatA F39A-YFP mutant formed TatA assemblies but did not disassemble with carbonyl cyanide m-chlorophenylhydrazone (
      • Alcock F.
      • Baker M.A.
      • Greene N.P.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system.
      ). This suggests that, whereas TatA assembly requires the PMF, disassembly requires substrate transport (
      • Alcock F.
      • Baker M.A.
      • Greene N.P.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system.
      ), raising the possibility that the PMF may not be required for the transport step per se. The rate of assembling (20–120 s) and disassembling TatA-YFP (5–15 s) was somewhat faster than the in vitro rate (
      • Alcock F.
      • Baker M.A.
      • Greene N.P.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system.
      ), but overall was still much slower than other protein transport systems (see e.g. Refs.
      • Cline K.
      • Henry R.
      • Li C.
      • Yuan J.
      Multiple pathways for protein transport into or across the thylakoid membrane.
      and
      • De Keyzer J.
      • Van Der Does C.
      • Driessen A.J.
      Kinetic analysis of the translocation of fluorescent precursor proteins into Escherichia coli membrane vesicles.
      ).

      TatC Scaffolds TatB, TatA, and Substrate to Form the Translocase

      Recent NMR and crystal structures provide valuable insights into the individual Tat components, whereas several other approaches yield information on the associations of components and substrate. The accumulated findings support a model in which TatC is the initial specific receptor for the signal peptide RR motif and also the organizing scaffold for the other components. TatB and then TatA, while bound to TatC, sequentially contact the substrate signal peptide and folded domain. TatC spans the membrane six times with N and C termini exposed to the cis compartment (Fig. 2A). The crystal structure of Aquifex aeolicus TatC (
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jäger F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • Sansom M.S.
      • Palmer T.
      • Högbom M.
      • Berks B.C.
      • Lea S.M.
      Structure of the TatC core of the twin-arginine protein transport system.
      ,
      • Ramasamy S.
      • Abrol R.
      • Suloway C.J.
      • Clemons Jr., W.M.
      The glove-like structure of the conserved membrane protein TatC provides insight into signal sequence recognition in twin-arginine translocation.
      ) portrays the TM helices folded into the shape of a cupped hand or glove. This arrangement produces a “palm” with a strikingly broad concave cavity (Fig. 3A) in which the TatA oligomer is thought to form. The six TMs appear to be stabilized by an overhanging cap structure formed by trans loops 1 and 2.
      Figure thumbnail gr3
      FIGURE 3Models for the association of substrate, TatA, and TatB with TatC and for a TatA oligomer. Homology-based models for pea chloroplast TatC (blue), TatB TM (yellow), and TatA TM (red) based on structures of A. aeolicus TatC and E. coli TatB and TatA are shown. A, TatC is labeled for the cis (stroma) 1–4 exposed loops and tails, the TMs 1–6, and the trans (lumen) 1–3 loops. The pivotal C-proximal helix of TM5 is colored cyan. Residues of cis 1 and 2 important for RR binding are colored orange and red to designate loss of binding upon mutation to alanine, with red also designating essential glutamate residues that may coordinate the arginine guanidinium groups, and green designating residues that direct disulfide cross-linking to RR proximal residues of the signal peptide (
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ). B, arrangement of TatB (
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jäger F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • Sansom M.S.
      • Palmer T.
      • Högbom M.
      • Berks B.C.
      • Lea S.M.
      Structure of the TatC core of the twin-arginine protein transport system.
      ,
      • Kneuper H.
      • Maldonado B.
      • Jäger F.
      • Krehenbrink M.
      • Buchanan G.
      • Keller R.
      • Müller M.
      • Berks B.C.
      • Palmer T.
      Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.
      ,

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ) and TatA (
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ) with TatC TM5 under non-transporting conditions (see text). The patterned cylinders represent the APH segments and are approximately placed based on cross-linking. Docking of TatA on the Gln-234 (magenta) of TM4 in the translocase is proposed to initiate TatA oligomerization to form the pore (
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jäger F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • Sansom M.S.
      • Palmer T.
      • Högbom M.
      • Berks B.C.
      • Lea S.M.
      Structure of the TatC core of the twin-arginine protein transport system.
      ,
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ). C, a head-to-tail arrangement of two TatC subunits can explain how the signal peptide RR domain binds to TatC cis 1 and cis 2 regions and the H domain binds the TatB TM. In the panel, the signal peptide is hand-sketched from the RR through the helical H domain and is continuous with the early mature domain that is designated by the dashed line. D, a model of the TatA nonamer in detergent micelles adapted from Ref.
      • Rodriguez F.
      • Rouse S.L.
      • Tait C.E.
      • Harmer J.
      • De Riso A.
      • Timmel C.R.
      • Sansom M.S.
      • Berks B.C.
      • Schnell J.R.
      Structural model for the protein-translocating element of the twin-arginine transport system.
      (Protein Data Bank (PDB) accession 2LZS). The curved APH domains likely reflect the curvature of the micelle surface. The nonamer structure overlays a sketch of a lipid bilayer deformed by hydrophobic mismatch into a lipid half-pore based on ideas presented in Refs.
      • Rodriguez F.
      • Rouse S.L.
      • Tait C.E.
      • Harmer J.
      • De Riso A.
      • Timmel C.R.
      • Sansom M.S.
      • Berks B.C.
      • Schnell J.R.
      Structural model for the protein-translocating element of the twin-arginine transport system.
      and
      • Stoddart D.
      • Ayub M.
      • Höfler L.
      • Raychaudhuri P.
      • Klingelhoefer J.W.
      • Maglia G.
      • Heron A.
      • Bayley H.
      Functional truncated membrane pores.
      .

      TatB and TatA

      TatB and TatA are highly homologous proteins with a short N-terminal TM, a hinge, an amphipathic helix (APH), and a natively unstructured C-tail (Fig. 2A). Solution and solid-state NMR structures of bacterial TatB (
      • Zhang Y.
      • Wang L.
      • Hu Y.
      • Jin C.
      Solution structure of the TatB component of the twin-arginine translocation system.
      ) and TatA (
      • Walther T.H.
      • Grage S.L.
      • Roth N.
      • Ulrich A.S.
      Membrane alignment of the pore-forming component TatAd of the twin-arginine translocase from Bacillus subtilis resolved by solid-state NMR spectroscopy.
      ,
      • Hu Y.
      • Zhao E.
      • Li H.
      • Xia B.
      • Jin C.
      Solution NMR structure of the TatA component of the twin-arginine protein transport system from Gram-positive bacterium Bacillus subtilis.
      • Rodriguez F.
      • Rouse S.L.
      • Tait C.E.
      • Harmer J.
      • De Riso A.
      • Timmel C.R.
      • Sansom M.S.
      • Berks B.C.
      • Schnell J.R.
      Structural model for the protein-translocating element of the twin-arginine transport system.
      ) show that the TM and APH bend at the hinge to form an L-shaped molecule. Both of the E. coli proteins have a kink in their APH domain, with the TatB APH showing a more acute change in direction (
      • Zhang Y.
      • Wang L.
      • Hu Y.
      • Jin C.
      Solution structure of the TatB component of the twin-arginine translocation system.
      ). TatB also has a longer C-tail than the TatA C-tail, both of which can be deleted without eliminating function (
      • Lee P.A.
      • Buchanan G.
      • Stanley N.R.
      • Berks B.C.
      • Palmer T.
      Truncation analysis of TatA and TatB defines the minimal functional units required for protein translocation.
      ,
      • Dabney-Smith C.
      • Mori H.
      • Cline K.
      Requirement of a Tha4-conserved transmembrane glutamate in thylakoid Tat translocase assembly revealed by biochemical complementation.
      ).
      Suppressor analysis (
      • Kneuper H.
      • Maldonado B.
      • Jäger F.
      • Krehenbrink M.
      • Buchanan G.
      • Keller R.
      • Müller M.
      • Berks B.C.
      • Palmer T.
      Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.
      ), site-directed photocross-linking (

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ), and disulfide scanning cross-linking (
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jäger F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • Sansom M.S.
      • Palmer T.
      • Högbom M.
      • Berks B.C.
      • Lea S.M.
      Structure of the TatC core of the twin-arginine protein transport system.
      ,
      • Kneuper H.
      • Maldonado B.
      • Jäger F.
      • Krehenbrink M.
      • Buchanan G.
      • Keller R.
      • Müller M.
      • Berks B.C.
      • Palmer T.
      Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.
      ) in non-transporting conditions indicate that the E. coli TatB TM is associated with the C-proximal helix of TatC TM5 (colored cyan in Fig. 3). Because the TatB TM is matched in length to the TatC TM5 helix, it is thought that they align along their length (
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jäger F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • Sansom M.S.
      • Palmer T.
      • Högbom M.
      • Berks B.C.
      • Lea S.M.
      Structure of the TatC core of the twin-arginine protein transport system.
      ). Photocross-linking also showed that the TatC cis 1 domain and trans 2 loop cross-link to TatB (

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ,
      • Zoufaly S.
      • Fröbel J.
      • Rose P.
      • Flecken T.
      • Maurer C.
      • Moser M.
      • Müller M.
      Mapping precursor-binding site on TatC subunit of twin arginine-specific protein translocase by site-specific photo cross-linking.
      ). Considering TatB topology (
      • 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.
      • Fincher V.
      • Dabney-Smith C.
      • Cline K.
      Functional assembly of thylakoid ΔpH-dependent/Tat protein transport pathway components in vitro.
      ,
      • Koch S.
      • Fritsch M.J.
      • Buchanan G.
      • Palmer T.
      The Escherichia coli TatA and TatB proteins have an N-out C-in topology in intact cells.
      • Aldridge C.
      • Storm A.
      • Cline K.
      • Dabney-Smith C.
      The chloroplast twin arginine transport (Tat) component, Tha4, undergoes conformational changes leading to Tat protein transport.
      ), a tentative arrangement of TatB association with TatC is as shown (Fig. 3B).
      In non-transporting membranes, TatA exists primarily as a large pool of small homo-oligomers (
      • Dabney-Smith C.
      • Cline K.
      Clustering of C-terminal stromal domains of Tha4 homo-oligomers during translocation by the Tat protein transport system.
      ,
      • Leake M.C.
      • Greene N.P.
      • Godun R.M.
      • Granjon T.
      • Buchanan G.
      • Chen S.
      • Berry R.M.
      • Palmer T.
      • Berks B.C.
      Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.
      ). Some TatA is also associated with isolated E. coli TatBC when components are overexpressed (
      • 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.
      ), although TatA is absent from isolated thylakoid TatBC (
      • Mori H.
      • Cline K.
      A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase.
      ,
      • Cline K.
      • Mori H.
      Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport.
      ). Nevertheless, cross-linking studies in E. coli and thylakoids demonstrate TatA contact with TatC in non-transporting conditions in a remarkably similar location as that occupied by TatB. Disulfide scanning (
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ) and photocross-linking (

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ,
      • Zoufaly S.
      • Fröbel J.
      • Rose P.
      • Flecken T.
      • Maurer C.
      • Moser M.
      • Müller M.
      Mapping precursor-binding site on TatC subunit of twin arginine-specific protein translocase by site-specific photo cross-linking.
      ) demonstrated contact of the TatA N terminus, TM, and APH with the corresponding TatC trans loops 2 and 3, TM5, and TatC cis regions, respectively. Photocross-linking from TatB suggests juxtaposition of TatA and TatB in the non-transporting Tat complex (

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ). Together, these results support the arrangement of TatA with TatC shown in Fig. 3B. TatA is placed distal to the TatC cavity with respect to TatB because TatB, but not TatA, contacts the substrate H domain in the absence of the PMF (
      • 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.
      ,
      • Gérard F.
      • Cline K.
      Efficient twin arginine translocation (Tat) pathway transport of a precursor protein covalently anchored to its initial cpTatC binding site.
      ) (see below).
      Upon assembly of the thylakoid Tat translocase, TatA makes a new contact in the center of the concave cavity of TatC such that TatA Glu-10 is aligned with TatC Gln-234 on TM4 (Fig. 3B, Gln-234 colored magenta in Fig. 3, A and B) (
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ). This TatA-TatC contact exhibits the same requirements as translocase assembly: an RR signal peptide, the PMF, and the TatA TM Glu-10. These results support the proposal (
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jäger F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • Sansom M.S.
      • Palmer T.
      • Högbom M.
      • Berks B.C.
      • Lea S.M.
      Structure of the TatC core of the twin-arginine protein transport system.
      ) that TatA initiates assembly of the transport-active oligomer by hydrogen bonding between a glutamine and a glutamate of partner proteins. The thylakoid TatC Gln-234 and TatA Glu-10 are conserved in the same relative positions among virtually all chloroplast and cyanobacterial TatC and TatA proteins. Bacterial TatC and TatA proteins commonly have a glutamate at the Gln-234 position and a polar residue at the Glu-10 position (
      • Berks B.C.
      The twin-arginine protein translocation pathway.
      ).

      Substrate

      Substrate binding triggers transition from receptor complex to the assembled translocase. Substrates exhibit a wide range of affinity for TatBC. Some bind in a highly reversible manner, their association only detected by cross-linking (
      • Mori H.
      • Cline K.
      A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase.
      ,
      • Ma X.
      • Cline K.
      Precursors bind to specific sites on thylakoid membranes prior to transport on the ΔpH protein translocation system.
      ). Others remain bound during detergent solubilization and purification (
      • Celedon J.
      • Cline K.
      Stoichiometry for binding and transport by the twin arginine translocation system.
      ,
      • Ma X.
      • Cline K.
      Precursors bind to specific sites on thylakoid membranes prior to transport on the ΔpH protein translocation system.
      ,
      • Cline K.
      • Mori H.
      Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport.
      ,
      • McDevitt C.A.
      • Buchanan G.
      • Sargent F.
      • Palmer T.
      • Berks B.C.
      Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels.
      ,
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      • 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.
      ). The signal peptides of tightly bound substrates are deeply inserted into the TatBC complex (
      • Gérard F.
      • Cline K.
      The thylakoid proton gradient promotes an advanced stage of signal peptide binding deep within the Tat pathway receptor complex.
      ,

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ), an arrangement considered to be an advanced binding stage. Interestingly, Gerard and Cline (
      • Gérard F.
      • Cline K.
      The thylakoid proton gradient promotes an advanced stage of signal peptide binding deep within the Tat pathway receptor complex.
      ) found that a weakly binding substrate similarly inserted into TatBC when thylakoids were energized with the PMF, suggesting that all substrates achieve the advanced stage in actively transporting membranes. Some studies suggest that substrate can initially bind to the lipid bilayer before accessing TatBC (
      • Shanmugham A.
      • Wong Fong Sang H.W.
      • Bollen Y.J.
      • Lill H.
      Membrane binding of twin arginine preproteins as an early step in translocation.
      ,
      • 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.
      ). By contrast, binding of at least some thylakoid substrates to the lipid bilayer appears to be a dead end (
      • Celedon J.
      • Cline K.
      Stoichiometry for binding and transport by the twin arginine translocation system.
      ).
      Binding of the high affinity tOE17-20F saturated at one substrate per TatC (
      • Celedon J.
      • Cline K.
      Stoichiometry for binding and transport by the twin arginine translocation system.
      ). With sufficient TatA, all bound tOE17-20F was efficiently transported. This initially suggested that each TatBC pair constitutes one binding and transport site as illustrated in Fig. 2A (but see below). A synthetic SufI signal peptide also bound with a 1:1 stoichiometry to purified A. aeolicus TatC, although with dramatically reduced affinity (
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jäger F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • Sansom M.S.
      • Palmer T.
      • Högbom M.
      • Berks B.C.
      • Lea S.M.
      Structure of the TatC core of the twin-arginine protein transport system.
      ).
      Knowledge of the precise location of bound signal peptide is critical for understanding how signal peptide alone can induce PMF-dependent translocase assembly (
      • Mori H.
      • Cline K.
      A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase.
      ,
      • Dabney-Smith C.
      • Cline K.
      Clustering of C-terminal stromal domains of Tha4 homo-oligomers during translocation by the Tat protein transport system.
      ,
      • Dabney-Smith C.
      • Mori H.
      • Cline K.
      Oligomers of Tha4 organize at the thylakoid Tat translocase during protein transport.
      ). Early studies showed that TatC binds to the RR motif and TatB contacts the H domain (
      • 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.
      ,
      • Gérard F.
      • Cline K.
      Efficient twin arginine translocation (Tat) pathway transport of a precursor protein covalently anchored to its initial cpTatC binding site.
      ). Recent extensive and complementary data indicate that residues of the TatC cis 1 domain and cis 2 loop form a specific RR binding site (
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jäger F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • Sansom M.S.
      • Palmer T.
      • Högbom M.
      • Berks B.C.
      • Lea S.M.
      Structure of the TatC core of the twin-arginine protein transport system.
      ,
      • Zoufaly S.
      • Fröbel J.
      • Rose P.
      • Flecken T.
      • Maurer C.
      • Moser M.
      • Müller M.
      Mapping precursor-binding site on TatC subunit of twin arginine-specific protein translocase by site-specific photo cross-linking.
      ,
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ,
      • Buchanan G.
      • de Leeuw E.
      • Stanley N.R.
      • Wexler M.
      • Berks B.C.
      • Sargent F.
      • Palmer T.
      Functional complexity of the twin-arginine translocase TatC component revealed by site-directed mutagenesis.
      • 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.
      • Lausberg F.
      • Fleckenstein S.
      • Kreutzenbeck P.
      • Fröbel J.
      • Rose P.
      • Müller M.
      • Freudl R.
      Genetic evidence for a tight cooperation of TatB and TatC during productive recognition of twin-arginine (Tat) signal peptides in Escherichia coli.
      ). Fig. 3A shows residues of chloroplast TatC (colored orange and red) that, when mutated to alanine, impair signal peptide binding. Residues colored red (glutamate) are thought to coordinate the arginine guanidinium groups; residues colored green direct disulfide cross-linking to RR proximal residues of the signal peptide (
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ). Complementary evidence also suggests that signal peptide H domain uniformly binds to the TatB TM, which is simultaneously associated with TatC TM5. Reciprocal photocross-linking experiments indicate that several regions of the signal peptide H domain contact the TatB TM (
      • 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.
      ,
      • Gérard F.
      • Cline K.
      Efficient twin arginine translocation (Tat) pathway transport of a precursor protein covalently anchored to its initial cpTatC binding site.
      ,

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ). Signal peptides and the early mature domain bind in an inverted hairpin configuration, placing the H domain at the same membrane height as the TatB TM (
      • Fincher V.
      • McCaffery M.
      • Cline K.
      Evidence for a loop mechanism of protein transport by the thylakoid ΔpH pathway.
      ,
      • Hou B.
      • Frielingsdorf S.
      • Klösgen R.B.
      Unassisted membrane insertion as the initial step in ΔpH/Tat-dependent protein transport.
      • Fröbel J.
      • Rose P.
      • Lausberg F.
      • Blümmel A.S.
      • Freudl R.
      • Müller M.
      Transmembrane insertion of twin-arginine signal peptides is driven by TatC and regulated by TatB.
      ). When TatB was absent from E. coli membranes, the signal peptide of some substrates was cleaved (without substrate transport) by the trans facing signal peptidase (
      • Fröbel J.
      • Rose P.
      • Lausberg F.
      • Blümmel A.S.
      • Freudl R.
      • Müller M.
      Transmembrane insertion of twin-arginine signal peptides is driven by TatC and regulated by TatB.
      ), implying that the TatB TM binding to the H domain prevents premature C domain exposure to the trans compartment. This is supported by photo- and disulfide cross-linking analyses that place the C-terminal end of the bound signal peptide in proximity with the trans-proximal end of TatC TM5 (

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ,
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ). Finally, suppressors of defective TorA signal peptides map to the TatB N terminus (
      • 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.
      ,
      • Lausberg F.
      • Fleckenstein S.
      • Kreutzenbeck P.
      • Fröbel J.
      • Rose P.
      • Müller M.
      • Freudl R.
      Genetic evidence for a tight cooperation of TatB and TatC during productive recognition of twin-arginine (Tat) signal peptides in Escherichia coli.
      ). It is apparent that all of these conditions can be met only if the signal peptide H domain binds to the TatB TM attached to a different TatC subunit (
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jäger F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • Sansom M.S.
      • Palmer T.
      • Högbom M.
      • Berks B.C.
      • Lea S.M.
      Structure of the TatC core of the twin-arginine protein transport system.
      ,
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ) as depicted in the head-to-tail TatC arrangement in Fig. 3C. This will be further explored below.
      Contacts between TatC and the folded substrate domain have not been detected. However, the TatB APH makes extensive contact with the folded domain of receptor-bound SufI (
      • Maurer C.
      • Panahandeh S.
      • Jungkamp A.C.
      • Moser M.
      • Müller M.
      TatB functions as an oligomeric binding site for folded Tat precursor proteins.
      ), suggesting that the TatB APH serves as a platform for the folded domain of bound substrates (
      • Maurer C.
      • Panahandeh S.
      • Jungkamp A.C.
      • Moser M.
      • Müller M.
      TatB functions as an oligomeric binding site for folded Tat precursor proteins.
      ). TatA contacts the signal peptide (
      • 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.
      ) and folded domain of the substrate later in the reaction cycle. Photocross-linking showed that TatA TM, APH, and C-tail are in contact with substrate only in the presence of PMF (
      • Fröbel J.
      • Rose P.
      • Müller M.
      Early contacts between substrate proteins and TatA translocase component in twin-arginine translocation.
      ). These contacts precede transport and may represent translocase assembly events. Disulfide exchange cross-linking with thylakoids captured interactions between the substrate folded domain and the TatA APH and the N terminus (
      • Pal D.
      • Fite K.
      • Dabney-Smith C.
      Direct interaction between a precursor mature domain and transport component Tha4 during twin arginine transport of chloroplasts.
      ). The latter contacts occurred during protein transport, implying that the substrate encounters the TatA TM during passage across the membrane.

      Models for TatA-facilitated Translocation

      Several observations suggest that TatA forms the translocation pore. It contacts substrate late in the transport reaction, is required only for the transport step (
      • Cline K.
      • Mori H.
      Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport.
      ), is present in superabundance over TatB and TatC (Fig. 2), forms large oligomeric assemblies (
      • Dabney-Smith C.
      • Cline K.
      Clustering of C-terminal stromal domains of Tha4 homo-oligomers during translocation by the Tat protein transport system.
      ,
      • Dabney-Smith C.
      • Mori H.
      • Cline K.
      Oligomers of Tha4 organize at the thylakoid Tat translocase during protein transport.
      ,
      • Leake M.C.
      • Greene N.P.
      • Godun R.M.
      • Granjon T.
      • Buchanan G.
      • Chen S.
      • Berry R.M.
      • Palmer T.
      • Berks B.C.
      Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.
      ) (Fig. 2A), and was found associated with an apparent transport intermediate (
      • 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.
      ).
      Two general pore models have been proposed. The first, invoking form-fitting channels lined with TatA subunits (
      • 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.
      ,
      • Sargent F.
      • Berks B.C.
      • Palmer T.
      Pathfinders and trailblazers: a prokaryotic targeting system for transport of folded proteins.
      ), was inspired by the appearance of channel-like structures of varying diameter when TatA was detergent-extracted from non-transporting membranes (
      • 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.
      ). Such channels might be formed by concerted bilayer insertion of TatA APHs (
      • Walther T.H.
      • Gottselig C.
      • Grage S.L.
      • Wolf M.
      • Vargiu A.V.
      • Klein M.J.
      • Vollmer S.
      • Prock S.
      • Hartmann M.
      • Afonin S.
      • Stockwald E.
      • Heinzmann H.
      • Nolandt O.V.
      • Wenzel W.
      • Ruggerone P.
      • Ulrich A.S.
      Folding and self-assembly of the TatA translocation pore based on a charge zipper mechanism.
      ) to produce a water-filled passageway, similar to the action of amphipathic antimicrobial peptides (
      • Huang H.W.
      Molecular mechanism of antimicrobial peptides: the origin of cooperativity.
      ). However, this model is less attractive because recent analyses show that TatA exists as small oligomers in non-transporting membranes (
      • Dabney-Smith C.
      • Cline K.
      Clustering of C-terminal stromal domains of Tha4 homo-oligomers during translocation by the Tat protein transport system.
      ,
      • Leake M.C.
      • Greene N.P.
      • Godun R.M.
      • Granjon T.
      • Buchanan G.
      • Chen S.
      • Berry R.M.
      • Palmer T.
      • Berks B.C.
      Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.
      ,
      • Alcock F.
      • Baker M.A.
      • Greene N.P.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system.
      • Rose P.
      • Fröbel J.
      • Graumann P.L.
      • Müller M.
      Substrate-dependent assembly of the Tat translocase as observed in live Escherichia coli cells.
      ). In addition, the expected topology inversion entailed by APH bilayer insertion was not found in two recent studies that analyzed TatA topology before and during translocation (
      • Koch S.
      • Fritsch M.J.
      • Buchanan G.
      • Palmer T.
      The Escherichia coli TatA and TatB proteins have an N-out C-in topology in intact cells.
      ,
      • Aldridge C.
      • Storm A.
      • Cline K.
      • Dabney-Smith C.
      The chloroplast twin arginine transport (Tat) component, Tha4, undergoes conformational changes leading to Tat protein transport.
      ).
      A second model proposes that TatA assemblies “weaken” the bilayer (
      • Brüser T.
      • Sanders C.
      An alternative model of the twin arginine translocation system.
      ). Such weakening might occur by severe bilayer thinning due to hydrophobic mismatch between the very short TatA TM and the lipid bilayer. This model is supported by a structure and modeling analysis of E. coli TatA (
      • Rodriguez F.
      • Rouse S.L.
      • Tait C.E.
      • Harmer J.
      • De Riso A.
      • Timmel C.R.
      • Sansom M.S.
      • Berks B.C.
      • Schnell J.R.
      Structural model for the protein-translocating element of the twin-arginine transport system.
      ) showing that, whereas an oligomer of 25 TatA subunits is stable in detergent, small oligomers would be unstable in a lipid bilayer. An oligomer of between four and nine subunits would produce severe distortions that destabilize intermolecular TM contacts and lead to local bilayer rupture (
      • Rodriguez F.
      • Rouse S.L.
      • Tait C.E.
      • Harmer J.
      • De Riso A.
      • Timmel C.R.
      • Sansom M.S.
      • Berks B.C.
      • Schnell J.R.
      Structural model for the protein-translocating element of the twin-arginine transport system.
      ) (Fig. 3D). The translocase-associated TatA assemblies are estimated to contain ∼25 TatA subunits by fluorescence imaging (
      • Leake M.C.
      • Greene N.P.
      • Godun R.M.
      • Granjon T.
      • Buchanan G.
      • Chen S.
      • Berry R.M.
      • Palmer T.
      • Berks B.C.
      Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.
      ) and kinetic measurements (
      • Celedon J.
      • Cline K.
      Stoichiometry for binding and transport by the twin arginine translocation system.
      ), and disulfide cross-linking places oligomer size at 8–16 TatA subunits (
      • Dabney-Smith C.
      • Cline K.
      Clustering of C-terminal stromal domains of Tha4 homo-oligomers during translocation by the Tat protein transport system.
      ). Thus, the TatA oligomers deemed completely unstable in lipid bilayers have been shown to exist in the translocase. This evokes a model whereby the translocase creates an environment that permits the assembly of bilayer-destabilizing TatA oligomers and harnesses this instability to produce transient pores for substrate passage (
      • Dabney-Smith C.
      • Cline K.
      Clustering of C-terminal stromal domains of Tha4 homo-oligomers during translocation by the Tat protein transport system.
      ).
      The strength of this model is that it agrees with and can explain experimentally determined characteristics of the protein transport reaction. Topology studies with thylakoid membranes (
      • Aldridge C.
      • Storm A.
      • Cline K.
      • Dabney-Smith C.
      The chloroplast twin arginine transport (Tat) component, Tha4, undergoes conformational changes leading to Tat protein transport.
      ) confirm structural studies of TatA in bicelles (
      • Walther T.H.
      • Grage S.L.
      • Roth N.
      • Ulrich A.S.
      Membrane alignment of the pore-forming component TatAd of the twin-arginine translocase from Bacillus subtilis resolved by solid-state NMR spectroscopy.
      ) that the APH is angled in non-transporting conditions, effectively relieving hydrophobic mismatch. The same topology studies suggest that the APH is more evenly partitioned into the bilayer interface in the translocase. Similarly, cross-linking studies indicate that the translocase TatA docking site on TatC places it higher in the membrane than the constitutively docked TatA (Fig. 3B) (
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ). Both observations support the bilayer thinning arrangement of translocase-associated TatA (Fig. 3D). The model also explains the observation that translocase-associated TatA oligomers are the same size whether induced by a synthetic signal peptide or by a complete substrate protein (
      • Dabney-Smith C.
      • Cline K.
      Clustering of C-terminal stromal domains of Tha4 homo-oligomers during translocation by the Tat protein transport system.
      ), and it gives a rationale for the translocase being assembled anew for each substrate (
      • Mori H.
      • Cline K.
      A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase.
      ,
      • Alcock F.
      • Baker M.A.
      • Greene N.P.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system.
      ). Collapse of a metastable TatA oligomer might be subject to stochastic thermal fluctuations, which would be consistent with the slow first order transport kinetics following TatA assembly (
      • Celedon J.
      • Cline K.
      Stoichiometry for binding and transport by the twin arginine translocation system.
      ).

      The Oligomeric TatBC Structure May Enable Gated TatA Assembly to Form the Translocase

      Regardless of the precise translocation mechanism, it is clear that the environment in the translocase allows the TatA oligomer to form. Stability may come from interactions between the folded substrate and the TatA APH, but TatC scaffolding surely provides considerable stability. It is also likely (
      • Rodriguez F.
      • Rouse S.L.
      • Tait C.E.
      • Harmer J.
      • De Riso A.
      • Timmel C.R.
      • Sansom M.S.
      • Berks B.C.
      • Schnell J.R.
      Structural model for the protein-translocating element of the twin-arginine transport system.
      ) that the TatA oligomer would assemble by stepwise addition of TatA monomers or very small oligomers.
      The higher order arrangement of Tat (A)BC probably plays a critical role in assembly of the TatA oligomer. Until now, I have discussed the TatB-TatC heterodimer as a functional unit (Figs. 2A and 3, A and B). However, TatBC actually exists as a multimer and appears to function as one. TatBC is estimated to contain 6–8 copies of each protein based primarily on the electrophoretic and chromatographic migration of the detergent-solubilized complex (
      • Cline K.
      • Mori H.
      Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport.
      ,
      • 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.
      ,
      • 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.
      ). Several studies show that this large complex is present in situ (
      • Alcock F.
      • Baker M.A.
      • Greene N.P.
      • Palmer T.
      • Wallace M.I.
      • Berks B.C.
      Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system.
      ,
      • Rose P.
      • Fröbel J.
      • Graumann P.L.
      • Müller M.
      Substrate-dependent assembly of the Tat translocase as observed in live Escherichia coli cells.
      ,
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ). The functional necessity of multimeric TatBC is indicated by the fact that TatC mutations that impair TatBC assembly also impair substrate binding and transport (
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ,
      • Buchanan G.
      • de Leeuw E.
      • Stanley N.R.
      • Wexler M.
      • Berks B.C.
      • Sargent F.
      • Palmer T.
      Functional complexity of the twin-arginine translocase TatC component revealed by site-directed mutagenesis.
      ), whereas mutations in the RR binding site do not affect assembly (
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ,
      • Buchanan G.
      • de Leeuw E.
      • Stanley N.R.
      • Wexler M.
      • Berks B.C.
      • Sargent F.
      • Palmer T.
      Functional complexity of the twin-arginine translocase TatC component revealed by site-directed mutagenesis.
      ). Multimeric TatBC also appears capable of collectively binding and transporting multiple substrates. Specifically, disulfide cross-linking of individually bound substrates via their signal peptide H/C (
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ,
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ) and early mature domains (
      • Ma X.
      • Cline K.
      Multiple precursor proteins bind individual Tat receptor complexes and are collectively transported.
      ) produced substrate dimers and tetramers, respectively, indicating a common compartment for binding multiple substrates. Efficient transport of the dimers and tetramers without breaking the cross-links suggests that TatBC units can combine to form a common translocation pore. Genetic data suggest that the TatBC complex is built from dimeric TatC units (
      • 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.
      ). In particular, a fused TatC dimer was fully functional when one, not both, RR binding sites was mutated. This is consistent with the positioning of the signal peptide between two TatC subunits as illustrated in Figs. 3C and Fig. 4.
      Figure thumbnail gr4
      FIGURE 4Proposed arrangement of TatABC units and bound substrate in the multimeric receptor complex. Graphic models of TatB (yellow), TatA (red), TatC (blue), and substrate (gray/black) are viewed from the cis compartment. The arrangements are based on cross-linking among Tat components and the substrate signal peptide and early mature domain. TatC TMs are labeled 1–6 on the top of the TMs, and only the TM helices of TatA and TatB are shown, except in C, which shows N-terminal tails of TatB, which are known to collectively cross-link. A and B, the face-to-face arrangement of dimers and tetramers is based on disulfide cross-linking, photocross-linking, and bismaleimide cross-linking (

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ,
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ,
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ) of TatC to TatC and photocross-linking between TatB and TatC (

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ). C and D, the arrangement of the substrate-bound TatABC is based on signal peptide cross-linking to TatC (
      • 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.
      ,
      • Gérard F.
      • Cline K.
      Efficient twin arginine translocation (Tat) pathway transport of a precursor protein covalently anchored to its initial cpTatC binding site.
      ,
      • Zoufaly S.
      • Fröbel J.
      • Rose P.
      • Flecken T.
      • Maurer C.
      • Moser M.
      • Müller M.
      Mapping precursor-binding site on TatC subunit of twin arginine-specific protein translocase by site-specific photo cross-linking.
      ,
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ,
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ) and TatB, and the decrease of certain disulfide cross-links when signal peptide is bound. The signal peptide is depicted as a helical black line, the early mature domain is depicted as a dashed line, and the folded domain of the substrate is depicted as a sphere. Note that substrate binding increases the size of the chamber created by TatBC units where the TatA pore is proposed to assemble. It is also apparent that TatB controls TatA entry into the chamber. See the text for additional detail.
      The next major challenge will be to determine how TatBC subunits are arranged to produce such a functional structure. Photo- and disulfide cross-linking provide some insight. Certain TatC-TatC cross-links support a face-to-face dimer arrangement of TatBC units (Fig. 4A). Disulfide scanning of thylakoid TatC (
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ) produced TatC dimers from three different concave face positions, whereas dimers were not obtained from any of eight different convex side (back) positions (
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ,
      • Ma X.
      • Cline K.
      Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
      ). Disulfide scanning with E. coli TatC produced four physiologically relevant dimers (
      • Punginelli C.
      • Maldonado B.
      • Grahl S.
      • Jack R.
      • Alami M.
      • Schröder J.
      • Berks B.C.
      • Palmer T.
      Cysteine scanning mutagenesis and topological mapping of the Escherichia coli twin-arginine translocase TatC component.
      ): one in trans 1 loop on the concave face (

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ), one in trans 2 loop that may face front or back, and two in TM1 at the edge of the TatC cup. These latter two cross-links are consistent if face-to-face TatC dimers are arranged end-to-end. This configuration might allow a pair of dimers to unfold into a tetramer (Fig. 4). In fact, certain cross-links produced by bismaleimide cross-linking of asymmetric pairs of Cys residues support dynamic arrangements of TatBC dimers, tetramers, and hexamers in which the concave faces of all TatCs point inward (

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ) (Fig. 4). The exact pairing geometry of TatBC units is not entirely clear, but certain TatC-TatB photocross-links suggest a possible arrangement. Blümmel et al. (

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ) observed photocross-linking to TatB directed from TatC TM2 and TM4 as well as from TM5. Their interpretation is that the TM of each TatB subunit in the multimeric TatBC complex bridges TM5 of one TatC subunit and the TM2–TM4 cleft of an adjacent or opposing TatC subunit (Fig. 4, A and B). This arrangement can explain the observed cross-linking of E. coli TatB trimers and tetramers (
      • Maurer C.
      • Panahandeh S.
      • Jungkamp A.C.
      • Moser M.
      • Müller M.
      TatB functions as an oligomeric binding site for folded Tat precursor proteins.
      ,
      • Lee P.A.
      • Orriss G.L.
      • Buchanan G.
      • Greene N.P.
      • Bond P.J.
      • Punginelli C.
      • Jack R.L.
      • Sansom M.S.
      • Berks B.C.
      • Palmer T.
      Cysteine-scanning mutagenesis and disulfide mapping studies of the conserved domain of the twin-arginine translocase TatB component.
      ). It is also an intriguing pairing because it places the TatB TM in the approximate site where TatA appears to initiate translocase oligomer assembly. Considering the high level of homology between the TMs of TatA and TatB, this supports a role for TatB as regulatory surrogate for TatA. In this regard, the simplest Tat systems function with just a TatC and a TatA.
      Substrate binding appears to “subtly” change the arrangement of TatBC subunits. Two of three disulfide cross-links between thylakoid TatC subunits were greatly diminished by prior binding of substrate. Substrate binding also increases the stability of detergent-solubilized thylakoid TatBC complex on blue native PAGE, increases stability of the TatB-GFP-TatC complex in living E. coli cells (
      • Rose P.
      • Fröbel J.
      • Graumann P.L.
      • Müller M.
      Substrate-dependent assembly of the Tat translocase as observed in live Escherichia coli cells.
      ), and changes the appearance of TatBC in single particle EM imaging (
      • 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.
      ). Nevertheless, substrate binding does not change the face-to-face arrangement of TatBC units. As evidence, a study with thylakoids produced a disulfide cross-linking product in which two bound substrates were linked to each other via their signal peptide C domains and were independently linked to separate TatC subunits via the RR and RR binding site domains (
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ). Suggested arrangements for TatABC units and substrate are shown in Fig. 4, C and D. These arrangements preserve the head-to-tail arrangement of TatC subunits for signal peptide binding and also create an enclosed chamber for assembly of the TatA translocation oligomer.

      PMF and Signal Peptide Binding as Assembly Triggers

      Mechanisms for how the PMF and signal peptide trigger TatA assembly are speculative. For thylakoids, the ΔpH component of PMF probably protonates the TatA Glu-10, making it energetically feasible to move up in the membrane to its docking site on TM4 and hydrogen-bond with TatC Gln-234 (Fig. 3B) (
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ). It is unclear how the PMF facilitates assembly of E. coli TatA, where only the electrical component of the PMF is required (
      • Bageshwar U.K.
      • Musser S.M.
      Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.
      ). Signal peptide binding must alter the association of components to provide a TatA pathway to the interior as well as an open assembly site on the concave TatC face. As depicted in Fig. 4, this would involve altering the association of TatB with TatC, which appears to block TatA entry into and assembly within the central compartment (

      46. Blümmel, A., Haag, L. A., Elmer, E., Müller, M., Fröbel, J., (2015) Initial assembly steps of a translocase for folded proteins. Nat. Commun., 10.1038/ncomms8234,

      ,
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ). Perhaps signal peptide H domain binding displaces the TatB TM from the TatC TM2–TM4 meshing site and also counterbalances the attraction of TatB TM for TatC TM5, opening the way for TatA movement to the interior. Opening of the TatA pathway might be infrequent and transient, considering the time scale of 1–3 min for assembling the translocase (
      • Celedon J.
      • Cline K.
      Stoichiometry for binding and transport by the twin arginine translocation system.
      ). Such a transient “breathing” of the Tat assembly might also be facilitated by an increased lateral pressure on chamber walls due to the insertion of signal peptides and early mature domains into the chamber. At present, the nature of the TatBC enclosed compartment is not known and has been speculated to be either aqueous or polar lipid-filled (
      • Rollauer S.E.
      • Tarry M.J.
      • Graham J.E.
      • Jääskeläinen M.
      • Jäger F.
      • Johnson S.
      • Krehenbrink M.
      • Liu S.M.
      • Lukey M.J.
      • Marcoux J.
      • McDowell M.A.
      • Rodriguez F.
      • Roversi P.
      • Stansfeld P.J.
      • Robinson C.V.
      • Sansom M.S.
      • Palmer T.
      • Högbom M.
      • Berks B.C.
      • Lea S.M.
      Structure of the TatC core of the twin-arginine protein transport system.
      ,
      • Ramasamy S.
      • Abrol R.
      • Suloway C.J.
      • Clemons Jr., W.M.
      The glove-like structure of the conserved membrane protein TatC provides insight into signal sequence recognition in twin-arginine translocation.
      ).

      Perspectives

      The mechanistic models presented here are preliminary, with some aspects based on a single study; clearly these await replication. However, they emphasize that a detailed map of the subunit organization of TatABC with and without substrate is crucial to further progress. Especially important are the interactions of components and signal peptide that occur at TatC TM5 because these appear to regulate assembly of the translocase. Molecular structures of relevant complexes would be a major advance toward deciphering Tat mechanism, but even additional cross-linking with functionally substituted components and substrate would refine current maps. Certainly, other approaches to these questions are essential.
      The core issue, i.e. how do TatA assemblies enable passage of the substrate across the membrane, is the most challenging because transmembrane passage may occur quite rapidly. Indirect clues to the mechanism may come by manipulating membrane composition of bilayer-forming and non-bilayer-forming polar lipids. The recently acquired ability to stabilize the translocase (
      • Aldridge C.
      • Ma X.
      • Gerard F.
      • Cline K.
      Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.
      ) may permit freezing the translocase for structural analysis. Nevertheless, even at this relatively early stage, it is apparent to me that Tat transports proteins by the novel strategy of organizing an unstable lipid/protein combination into the nano-environment of the translocase. This creates a restricted permeation pathway, whereas on a cellular level, the same instability would be catastrophic.

      Acknowledgments

      I thank Carole Dabney-Smith and Michael McCaffery for insightful comments and critical review of the manuscript.

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