Hydrophobic mismatch is a key factor in protein transport across lipid bilayer membranes via the Tat pathway

The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoids, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits: TatA, TatB, and TatC. TatA and TatB possess short transmembrane alpha helices (TMHs), both of which are only 15 residues long in Escherichia coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer, although the functional significance of this mismatch is unclear. Here, we sought to address the functional importance of the hydrophobic mismatch in the Tat transport mechanism in E. coli. We conducted three different assays to evaluate the effect of TMH length mutants on Tat activity and observed that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off following any modification of this length. Surprisingly, TatA and TatB with as few as 11 residues in their TMHs can still insert into the membrane bilayer, albeit with a decline in membrane integrity. These findings support a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, we conclude that the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes.     

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.     

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.     

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.     

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 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.     

Truncation analysis of TatA and TatB defines the minimal functional units required for protein translocation

The TatA and TatB proteins are essential components of the twin arginine protein translocation pathway in Escherichia coli. C-terminal truncation analysis of the TatA protein revealed that a plasmid-expressed TatA protein shortened by 40 amino acids is still fully competent to support protein translocation. Similar truncation analysis of TatB indicated that the final 30 residues of TatB are dispensable for function. Further deletion experiments with TatB indicated that removal of even 70 residues from its C terminus still allowed significant transport. These results imply that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity. A chimeric protein comprising the N-terminal region of TatA fused to the amphipathic and C-terminal domains of TatB supports a low level of Tat activity in a strain in which the wild-type copy of either tatA or tatB (but not both) is deleted.     

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.     

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 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.     

Membrane interactions and self-association of the TatA and TatB components of the twin-arginine translocation pathway

The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin-arginine signal peptides. The essential Tat pathway components TatA, TatB and TatC are shown to be integral membrane proteins. Upon removal of the predicted N-terminal transmembrane helix TatA becomes a water-soluble protein. In contrast the homologous TatB protein retains weak peripheral interactions with the cytoplasmic membrane when the analogous helix is deleted. Chemical crosslinking studies indicate that TatA forms at least homotrimers, and TatB minimally homodimers, in the native membrane environment. The presence of such homo-oligomeric interactions is supported by size exclusion chromatography.     

Cysteine-scanning mutagenesis and disulfide mapping studies of the conserved domain of the twin-arginine translocase TatB component

The cytoplasmic membrane protein TatB is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. Together with the TatC component it forms a complex that functions as a membrane receptor for substrate proteins. Structural predictions suggest that TatB is anchored to the membrane via an N-terminal transmembrane alpha-helix that precedes an amphipathic alpha-helical section of the protein. From truncation analysis it is known that both these regions of the protein are essential for function. Here we construct 31 unique cysteine substitutions in the first 42 residues of TatB. Each of the substitutions results in a TatB protein that is competent to support Tat-dependent protein translocation. Oxidant-induced disulfide cross-linking shows that both the N-terminal and amphipathic helices form contacts with at least one other TatB protomer. For the transmembrane helix these contacts are localized to one face of the helix. Molecular modeling and molecular dynamics simulations provide insight into the possible structural basis of the transmembrane helix interactions. Using variants with double cysteine substitutions in the transmembrane helix, we were able to detect cross-links between up to five TatB molecules. Protein purification showed that species containing at least four cross-linked TatB molecules are found in correctly assembled TatBC complexes. Our results suggest that the transmembrane helices of TatB protomers are in the center rather than the periphery of the TatBC complex.     

Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context

In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.     

TatB functions as an oligomeric binding site for folded Tat precursor proteins

Twin-arginine-containing signal sequences mediate the transmembrane transport of folded proteins. The cognate twin-arginine translocation (Tat) machinery of Escherichia coli consists of the membrane proteins TatA, TatB, and TatC. Whereas Tat signal peptides are recognized by TatB and TatC, little is known about molecular contacts of the mature, folded part of Tat precursor proteins. We have placed a photo-cross-linker into Tat substrates at sites predicted to be either surface-exposed or hidden in the core of the folded proteins. On targeting of these variants to the Tat machinery of membrane vesicles, all surface-exposed sites were found in close proximity to TatB. Correspondingly, incorporation of the cross-linker into TatB revealed multiple precursor-binding sites in the predicted transmembrane and amphipathic helices of TatB. Large adducts indicative of TatB oligomers contacting one precursor molecule were also obtained. Cross-linking of Tat substrates to TatB required an intact twin-arginine signal peptide and disappeared upon transmembrane translocation. Our collective data are consistent with TatB forming an oligomeric binding site that transiently accommodates folded Tat precursors.     

Subunit Organization in the TatA Complex of the Twin Arginine Protein Translocase: A SITE-DIRECTED EPR SPIN LABELING STUDY*

The Tat system is used to transport folded proteins across the cytoplasmic membrane in bacteria and archaea and across the thylakoid membrane of plant chloroplasts. Multimers of the integral membrane TatA protein are thought to form the protein-conducting element of the Tat pathway. Nitroxide radicals were introduced at selected positions within the transmembrane helix of Escherichia coli TatA and used to probe the structure of detergent-solubilized TatA complexes by EPR spectroscopy. A comparison of spin label mobilities allowed classification of individual residues as buried within the TatA complex or exposed at the surface and suggested that residues Ile¹² and Val¹⁴ are involved in interactions between helices. Analysis of inter-spin distances suggested that the transmembrane helices of TatA subunits are arranged as a single-walled ring containing a contact interface between Ile¹² on one subunit and Val¹⁴ on an adjacent subunit. Experiments in which labeled and unlabeled TatA samples were mixed demonstrate that TatA subunits are exchanged between TatA complexes. This observation is consistent with the TatA dynamic polymerization model for the mechanism of Tat transport.     

The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC complex, spectrum of modular TatA complexes and minor TatAB complex

The Tat system transports folded proteins across bacterial plasma and plant thylakoid membranes. To date, three key Tat subunits have been identified and mechanistic studies indicate the presence of two types of complex: a TatBC-containing substrate-binding unit and a separate TatA complex. Here, we used blue-native gel electrophoresis and affinity purification to study the nature of these complexes in Escherichia coli. Analysis of solubilized membrane shows that the bulk of TatB and essentially all of the TatC is found in a single 370kDa TatABC complex. TatABC was purified to homogeneity using an affinity tag on TatC and this complex runs apparently as an identical band. We conclude that this is the primary core complex, predicted to contain six or seven copies of TatBC together with a similar number of TatA subunits. However, the data indicate the presence of an additional form of Tat complex containing TatA and TatB, but not TatC; we speculate that this may be an assembly or disassembly intermediate of the translocator. The vast majority of TatA is found in separate complexes that migrate in blue-native gels as a striking ladder of bands with sizes ranging from under 100 kDa to over 500 kDa. Further analysis shows that the bands differ by an average of 34 kDa, indicating that TatA complexes are built largely, but possibly not exclusively, from modules of three or four TatA molecules. The range and nature of these complexes are similar in a TatC mutant that is totally inactive, indicating that the ladder of bands does not stem from ongoing translocation activity, and we show that purified TatA can self-assemble in vitro to form similar complexes. This spectrum of TatA complexes may provide the flexibility required to generate a translocon capable of transporting substrates of varying sizes across the plasma membrane in a folded state.     

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.     

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.     

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.     

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.