Escherichia coli TatA and TatB proteins have N-out, C-in topology in intact cells

The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. A complex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport.     

Structural Basis for TatA Oligomerization: An NMR Study of Escherichia coli TatA Dimeric Structure

Many proteins are transported across lipid membranes by protein translocation systems in living cells. The twin-arginine transport (Tat) system identified in bacteria and plant chloroplasts is a unique system that transports proteins across membranes in their fully-folded states. Up to date, the detailed molecular mechanism of this process remains largely unclear. The Escherichia coli Tat system consists of three essential transmembrane proteins: TatA, TatB and TatC. Among them, TatB and TatC form a tight complex and function in substrate recognition. The major component TatA contains a single transmembrane helix followed by an amphipathic helix, and is suggested to form the translocation pore via self-oligomerization. Since the TatA oligomer has to accommodate substrate proteins of various sizes and shapes, the process of its assembly stands essential for understanding the translocation mechanism. A structure model of TatA oligomer was recently proposed based on NMR and EPR observations, revealing contacts between the transmembrane helices from adjacent subunits. Herein we report the construction and stabilization of a dimeric TatA, as well as the structure determination by solution NMR spectroscopy. In addition to more extensive inter-subunit contacts between the transmembrane helices, we were also able to observe interactions between neighbouring amphipathic helices. The side-by-side packing of the amphipathic helices extends the solvent-exposed hydrophilic surface of the protein, which might be favourable for interactions with substrate proteins. The dimeric TatA structure offers more detailed information of TatA oligomeric interface and provides new insights on Tat translocation mechanism.     

The Tat System for Membrane Translocation of Folded Proteins Recruits the Membrane-stabilizing Psp Machinery in Escherichia coli

Tat systems transport folded proteins across energized membranes of bacteria, archaea, and plant plastids. In Escherichia coli, TatBC complexes recognize the transported proteins, and TatA complexes are recruited to facilitate transport. We achieved an abstraction of TatA from membranes without use of detergents and observed a co-purification of PspA, a membrane-stress response protein. The N-terminal transmembrane domain of TatA was required for the interaction. Electron microscopy displayed TatA complexes in direct contact with PspA. PspB and PspC were important for the TatA-PspA contact. The activator protein PspF was not involved in the PspA-TatA interaction, demonstrating that basal levels of PspA already interact with TatA. Elevated TatA levels caused membrane stress that induced a strictly PspBC- and PspF-dependent up-regulation of PspA. TatA complexes were found to destabilize membranes under these conditions. At native TatA levels, PspA deficiency clearly affected anaerobic TMAO respiratory growth, suggesting that energetic costs for transport of large Tat substrates such as TMAO reductase can become growth limiting in the absence of PspA. The physiological role of PspA recruitment to TatA may therefore be the control of membrane stress at active translocons.     

Early contacts between substrate proteins and TatA translocase component in twin-arginine translocation

Twin-arginine translocation (Tat) is a unique protein transport pathway in bacteria, archaea, and plastids. It mediates the transmembrane transport of fully folded proteins, which harbor a consensus twin-arginine motif in their signal sequences. In Gram-negative bacteria and plant chloroplasts, three membrane proteins, named TatA, TatB, and TatC, are required to enable Tat translocation. Available data suggest that TatA assembles into oligomeric pore-like structures that might function as the protein conduit across the lipid bilayer. Using site-specific photo-cross-linking, we have investigated the molecular environment of TatA under resting and translocating conditions. We find that monomeric TatA is an early interacting partner of functionally targeted Tat substrates. This interaction with TatA likely precedes translocation of Tat substrates and is influenced by the proton-motive force. It strictly depends on the presence of TatB and TatC, the latter of which is shown to make contacts with the transmembrane helix of TatA.     

Salt sensitivity of minimal twin arginine translocases

Bacterial twin arginine translocation (Tat) pathways have evolved to facilitate transport of folded proteins across membranes. Gram-negative bacteria contain a TatABC translocase composed of three subunits named TatA, TatB, and TatC. In contrast, the Tat translocases of most Gram-positive bacteria consist of only TatA and TatC subunits. In these minimal TatAC translocases, a bifunctional TatA subunit fulfils the roles of both TatA and TatB. Here we have probed the importance of conserved residues in the bifunctional TatAy subunit of Bacillus subtilis by site-specific mutagenesis. A set of engineered TatAy proteins with mutations in the cytoplasmic hinge and amphipathic helix regions were found to be inactive in protein translocation under standard growth conditions for B. subtilis or when heterologously expressed in Escherichia coli. Nevertheless, these mutated TatAy proteins did assemble into TatAy and TatAyCy complexes, and they facilitated membrane association of twin arginine precursor proteins in E. coli. Interestingly, most of the mutated TatAyCy translocases were salt-sensitive in B. subtilis. Similarly, the TatAC translocases of Bacillus cereus and Staphylococcus aureus were salt-sensitive when expressed in B. subtilis. Taken together, our present observations imply that salt-sensitive electrostatic interactions have critical roles in the preprotein translocation activity of certain TatAC type translocases from Gram-positive bacteria.     

Cysteine scanning mutagenesis and disulfide mapping studies of the TatA component of the bacterial twin arginine translocase

The Tat (twin arginine translocation) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The integral membrane proteins TatA, TatB, and TatC are essential components of the Tat pathway. TatA forms high order oligomers and is thought to constitute the protein-translocating unit of the Tat system. Cysteine scanning mutagenesis was used to systematically investigate the functional importance of residues in the essential N-terminal transmembrane and amphipathic helices of Escherichia coli TatA. Cysteine substitutions of most residues in the amphipathic helix, including all the residues on the hydrophobic face of the helix, severely compromise Tat function. Glutamine 8 was identified as the only residue in the transmembrane helix that is critical for TatA function. The cysteine variants in the transmembrane helix were used in disulfide mapping experiments to probe the oligomeric arrangement of TatA protomers within the larger TatA complex. Residues in the center of the transmembrane helix (including residues 10-16) show a distinct pattern of cross-linking indicating that this region of the protein forms well defined interactions with other protomers. At least two interacting faces were detected. The results of our TatA studies are compared with analogous data for the homologous, but functionally distinct, TatB protein. This comparison reveals that it is only in TatA that the amphipathic helix is sensitive to amino acid substitutions. The TatA amphipathic helix may play a role in forming and controlling the path of substrate movement across the membrane.     

Evidence for interactions between domains of TatA and TatB from mutagenesis of the TatABC subunits of the twin-arginine translocase

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. Three subunits, TatA, B and C, are known to be involved but their modes of action are poorly understood, as are the inter-subunit interactions occurring within Tat complexes. We have generated mutations in the single transmembrane (TM) spans of TatA and TatB, with the aim of generating structural distortions. We show that substitution in TatB of three residues by glycine, or a single residue by proline, has no detectable effect on translocation, whereas the presence of three glycines in the TatA TM span completely blocks Tat translocation activity. The results show that the integrity of the TatA TM span is vital for Tat activity, whereas that of TatB can accommodate large-scale distortions. Near-complete restoration of activity in TatA mutants is achieved by the simultaneous presence of a V12P mutation in the TatB TM span, strongly implying a direct functional interaction between the TatA/B TM spans. We also analyzed the predicted amphipathic regions in TatA and TatB and again find evidence of direct interaction; benign mutations in either subunit completely blocked translocation of two Tat substrates when present in combination. Finally, we have re-examined the effects of previously analyzed TatABC mutations under conditions of high translocation activity. Among numerous TatA or TatB mutations tested, TatA F39A alone blocked translocation, and only substitutions of P48 and F94 in TatC blocked translocation activity.     

The Escherichia coli twin-arginine translocase: conserved residues of TatA and TatB family components involved in protein transport

The Escherichia coli Tat system serves to export folded proteins harbouring an N-terminal twin-arginine signal peptide across the cytoplasmic membrane. In this report we have studied the functions of conserved residues within the structurally related TatA and TatB proteins. Our results demonstrate that there are two regions within each protein of high sequence conservation that are critical for efficient Tat translocase function. The first region is the interdomain hinge between the transmembrane and the amphipathic alpha-helices of TatA and TatB proteins. The second region is within the amphipathic helices of TatA and TatB. In particular an invariant phenylalanine residue within TatA proteins is essential for activity, whereas a string of glutamic acid residues on the same face of the amphipathic helix of TatB is important for function.     

Charge Composition Features of Model Single-span Membrane Proteins That Determine Selection of YidC and SecYEG Translocase Pathways in Escherichia coli

We have investigated the features of single-span model membrane proteins based upon leader peptidase that determines whether the proteins insert by a YidC/Sec-independent, YidC-only, or YidC/Sec mechanism. We find that a protein with a highly hydrophobic transmembrane segment that inserts into the membrane by a YidC/Sec-independent mechanism becomes YidC-dependent if negatively charged residues are inserted into the translocated periplasmic domain or if the hydrophobicity of the transmembrane segment is reduced by substituting polar residues for nonpolar ones. This suggests that charged residues in the translocated domain and the hydrophobicity within the transmembrane segment are important determinants of the insertion pathway. Strikingly, the addition of a positively charged residue to either the translocated region or the transmembrane region can switch the insertion requirements such that insertion requires both YidC and SecYEG. To test conclusions from the model protein studies, we confirmed that a positively charged residue is a SecYEG determinant for the endogenous proteins ATP synthase subunits a and b and the TatC subunit of the Tat translocase. These findings provide deeper insights into how pathways are selected for the insertion of proteins into the Escherichia coli inner membrane.     

TatBC-Independent TatA/Tat Substrate Interactions Contribute to Transport Efficiency

The Tat system can transport folded, signal peptide-containing proteins (Tat substrates) across energized membranes of prokaryotes and plant plastids. A twin-arginine motif in the signal peptide of Tat substrates is recognized by TatC-containing complexes, and TatA permits the membrane passage. Often, as in the model Tat systems of Escherichia coli and plant plastids, a third component – TatB – is involved that resembles TatA but has a higher affinity to TatC. It is not known why most TatA dissociates from TatBC complexes in vivo and distributes more evenly in the membrane. Here we show a TatBC-independent substrate-binding to TatA from Escherichia coli, and we provide evidence that this binding enhances Tat transport. First hints came from in vivo cross-linking data, which could be confirmed by affinity co-purification of TatA with the natural Tat substrates HiPIP and NrfC. Two positions on the surface of HiPIP could be identified that are important for the TatA interaction and transport efficiency, indicating physiological relevance of the interaction. Distributed TatA thus may serve to accompany membrane-interacting Tat substrates to the few TatBC spots in the cells.     

Twin-arginine-dependent translocation of folded proteins

Twin-arginine translocation (Tat) denotes a protein transport pathway in bacteria, archaea and plant chloroplasts, which is specific for precursor proteins harbouring a characteristic twin-arginine pair in their signal sequences. Many Tat substrates receive cofactors and fold prior to translocation. For a subset of them, proofreading chaperones coordinate maturation and membrane-targeting. Tat translocases comprise two kinds of membrane proteins, a hexahelical TatC-type protein and one or two members of the single-spanning TatA protein family, called TatA and TatB. TatC- and TatA-type proteins form homo- and hetero-oligomeric complexes. The subunits of TatABC translocases are predominantly recovered from two separate complexes, a TatBC complex that might contain some TatA, and a homomeric TatA complex. TatB and TatC coordinately recognize twin-arginine signal peptides and accommodate them in membrane-embedded binding pockets. Advanced binding of the signal sequence to the Tat translocase requires the proton-motive force (PMF) across the membranes and might involve a first recruitment of TatA. When targeted in this manner, folded twin-arginine precursors induce homo-oligomerization of TatB and TatA. Ultimately, this leads to the formation of a transmembrane protein conduit that possibly consists of a pore-like TatA structure. The translocation step again is dependent on the PMF.     

The TatC component of the twin‐arginine protein translocase functions as an obligate oligomer

The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate‐bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild‐type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue‐native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild‐type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate‐induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.     

Solution structure of the TatB component of the twin-arginine translocation system

The twin-arginine protein transport (Tat) system translocates fully folded proteins across lipid membranes. In Escherichia coli, the Tat system comprises three essential components: TatA, TatB and TatC. The protein translocation process is proposed to initiate by signal peptide recognition and substrate binding to the TatBC complex. Upon formation of the TatBC–substrate protein complex, the TatA subunits are recruited and form the protein translocation pore. Experimental evidences suggest that TatB forms a tight complex with TatC at 1:1 molar ratio and the TatBC complex contains multiple copies of both proteins. Cross-linking experiments demonstrate that TatB functions in tetrameric units and interacts with both TatC and substrate proteins. However, structural information of the TatB protein is still lacking, and its functional mechanism remains elusive. Herein, we report the solution structure of TatB in DPC micelles determined by Nuclear Magnetic Resonance (NMR) spectroscopy. Overall, the structure shows an extended ‘L-shape’ conformation comprising four helices: a transmembrane helix (TMH) α1, an amphipathic helix (APH) α2, and two solvent exposed helices α3 and α4. The packing of TMH and APH is relatively rigid, whereas helices α3 and α4 display notably higher mobility. The observed floppiness of helices α3 and α4 allows TatB to sample a large conformational space, thus providing high structural plasticity to interact with substrate proteins of different sizes and shapes.     

Contributions of the Transmembrane Domain and a Key Acidic Motif to Assembly and Function of the TatA Complex

The twin-arginine translocase (Tat) pathway transports folded proteins across bacterial and thylakoid membranes. In Escherichia coli, a membrane-bound TatA complex, which oligomerizes to form complexes of less than 100 to more than 500 kDa, is considered essential for translocation. We have studied the contributions of various TatA domains to the assembly and function of this heterogeneous TatA complex. The TOXCAT assay was used to analyze the potential contribution of the TatA transmembrane (TM) domain. We observed relatively weak interactions between TatA TM domains, suggesting that the TM domain is not the sole driving force behind oligomerization. A potential hydrogen-bonding role for a TM domain glutamine was also investigated, and it was found that mutation blocks transport at low expression levels, while assembly is unaffected at higher expression levels. Analysis of truncated TatA proteins instead highlighted an acidic motif directly following the TatA amphipathic helix. Mutating these negatively charged residues to apolar uncharged residues completely blocks activity, even at high levels of TatA, and appears to disrupt ordered complex formation.     

The core TatABC complex of the twin-arginine translocase in Escherichia coli: TatC drives assembly whereas TatA is essential for stability

Current models for the action of the twin-arginine translocation (Tat) system propose that substrates bind initially to the TatBC subunits, after which a separate TatA complex is recruited to form an active translocon. Here, we have studied the roles of individual subunits in the assembly and stability of the core TatBC-containing substrate-binding complex. Previous studies have shown that TatB and TatC are active when fused together; we show here that deletion of the entire TatB transmembrane span from this Tat(BC) fusion inactivates the Tat system but does not affect assembly of the core complex. In this mutated complex, TatA is present but more loosely bound, indicating a role for TatB in the correct binding of TatA. In the absence of TatA, the truncated TatBC fusion protein still assembles into a complex of the correct magnitude, demonstrating that the transmembrane spans of TatC are the only determinants within the membrane bilayer that specify assembly of this complex. Further studies on both the Tat(BC) construct and the wild-type TatBC subunits show that the TatBC complex is unstable in the absence of TatA, and we show that TatA stabilises the TatB subunit specifically within this complex. The results demonstrate a dual role and location for TatA: in the functioning/maintenance of the core complex, and as a separate homo-oligomeric complex.     

The Tat pathway in bacteria and chloroplasts (Review)

Both in prokaryotic organisms and in chloroplasts, a specialized protein transport pathway exists which is capable of translocating proteins in a fully folded conformation. Transport is mediated in both instances by signal peptides harbouring a twin-arginine consensus motif (twin-arginine translocation (Tat) pathway). The Tat translocase comprises the three functionally different membrane proteins TatA, TatB, and TatC. While TatB and TatC are involved in the specific recognition of the substrate, TatA might be the major pore-forming component. Current evidence suggests that a functional Tat translocase is assembled from separate TatBC and TatA assemblies only on demand, i.e., in the presence of transport substrate and a transmembrane H⁺-motive force.     

An early event in the transport mechanism of LacY protein: interaction between helices V and I

Helix V in LacY, which abuts and crosses helix I in the N-terminal helix bundle of LacY, contains Arg¹⁴⁴ and Trp¹⁵¹, two residues that play direct roles in sugar recognition and binding, as well as Cys¹⁵⁴, which is important for conformational flexibility. In this study, paired Cys replacement mutants in helices V and I were strategically constructed with tandem factor Xa protease cleavage sites in the loop between the two helices to test cross-linking. None of the mutants form disulfides spontaneously; however, three mutants (Pro²⁸ → Cys/Cys¹⁵⁴, Pro²⁸ → Cys/Val¹⁵⁸ → Cys, and Phe²⁹ → Cys/Val¹⁵⁸ → Cys) exhibit cross-linking after treatment with copper/1,10-phenanthroline (Cu/Ph) or 1,1-methanediyl bismethanethiosulfonate ((MTS)₂-1), 3–4 Å), and cross-linking is quantitative in the presence of ligand. Remarkably, with one mutant, complete cross-linking with (MTS)₂-1 has no effect on lactose transport, whereas quantitative disulfide cross-linking catalyzed by Cu/Ph markedly inhibits transport activity. The findings are consistant with a number of previous conclusions suggesting that sugar binding to LacY causes a localized scissors-like movement between helices V and I near the point where the two helices cross in the middle of the membrane. This ligand-induced movement may act to initiate the global conformational change resulting from sugar binding.     

The TatAd component of the Bacillus subtilis twin-arginine protein transport system forms homo-multimeric complexes in its cytosolic and membrane embedded localisation

The twin arginine translocation (Tat) system has the capacity to transfer completely folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The most abundant TatA protein of this system has been suggested to form the protein conducting channel. Here, the molecular organisation of soluble and membrane embedded Bacillus subtilis TatA_d was analysed using negative contrast and freeze-fractured electron microscopy. In both compartments, the protein showed homo-oligomerisation. In aqueous solution, TatA_d formed homo-multimeric micelle-like complexes. Freeze-fracture analysis of proteoliposomes revealed self association of membrane-integrated TatA_d independent from TatC_d, the second component of this transport system. Immunogold labelling demonstrated that the substrate prePhoD was co-localised with membrane-integrated TatA_d complexes.     

Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels

The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Substrates are targeted to the Tat pathway by signal peptides containing a pair of consecutive arginine residues. The membrane proteins TatA, TatB and TatC are the essential components of this pathway in Escherichia coli. The complexes that these proteins form at native levels of expression have been investigated by the use of affinity tag-coding sequences fused to chromosomal tat genes. Distinct TatA and TatBC complexes were identified using size-exclusion chromatography and shown to have apparent molecular masses of approximately 700 and 500 kDa, respectively. Following in vivo expression, the Tat substrate protein SufI was found to copurify with the TatBC, but not the TatA, complex. This binding required the SufI signal peptide. Substitution of the twin-arginine residues in the SufI signal peptide by either twin lysine or twin alanine residues abolished export. However, both variant SufI proteins still copurified with the TatBC complex. These data show that the twin-arginine residues of the Tat consensus motif are not essential for binding of precursor to the TatBC complex but are required for the successful entry of the precursor into the transport cycle. The effect on substrate binding of single amino acid substitutions in TatC that affect Tat transport were studied using TatC variants Phe94Ala, Glu103Ala, Glu103Arg and Asp211Ala. Only variant Glu103Arg showed reduced copurification of SufI with TatBC. The transport defects associated with the other TatC variants do not, therefore, arise from an inability to bind substrate proteins.     

The TatA subunit of Escherichia coli twin-arginine translocase has an N-in topology

The twin-arginine translocase (Tat) system is used by many bacteria to translocate folded proteins across the cytoplasmic membrane. The TatA subunit is the predicted pore-forming subunit and has been shown to form a homo-oligomeric complex. Through accessibility experiments using the thiol-reactive reagents 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid and Nalpha-(3-maleimidylproprionyl)biocytin toward site-specific cysteine mutants in TatA, we show that the N-terminus of TatA is located in the cytoplasm rather than the previously assumed periplasm. We also confirm previous observations that the C-terminus has a dual topology. By treatment with the membrane uncoupler carbonyl cyanide-m-chlorophenyl hydrazone, we show that the topological state of the C-terminus is dependent on the membrane potential. These results suggest two architectures of TatA in the membrane: one with a single transmembrane helix and the other with two transmembrane helices. Molecular models of both topologies were used to develop and cartoon a homo-oligomeric complex as a channel with a diameter of approximately 50 A and suggest that the double transmembrane helix topology might be the building block for the translocation channel. Additionally, in vivo cross-linking experiments of Gly2Cys and Thr22Cys mutants showed that Gly2, at the beginning of transmembrane helix-1, is in close proximity with Gly2 of a neighboring TatA, as Cys2 cross-linked immediately upon the addition of copper phenanthroline. On the other hand, Cys22, at the other end of the transmembrane helix, took at least 10 min to cross-link, suggesting that a possible movement or reorientation is required to bring this residue into proximity with a neighboring TatA subunit.