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.     

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.     

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.     

Tat subunit stoichiometry in Arabidopsis thaliana challenges the proposed function of TatA as the translocation pore

The twin arginine translocation (Tat) machinery which is capable of transporting folded proteins across lipid bilayers operates in the thylakoid membrane of plant chloroplasts as well as in the cytoplasmic membrane of bacteria. It is composed of three integral membrane proteins (TatA, TatB, and TatC) which form heteromeric complexes of high molecular weight that accomplish binding and transport of substrates carrying Tat pathway-specific signal peptides. Western analyses using affinity purified antibodies showed in both, juvenile and adult tissue from Arabidopsis thaliana, an approximately equimolar ratio of the TatB and TatC components, whereas TatA was detectable only in minor amounts. Upon Blue Native-PAGE, TatB and TatC were found in four heteromeric TatB/C complexes possessing molecular weights of approximately 310, 370, 560 and 620 kDa, respectively, while TatA was detected only in a molecular weight range below 200 kDa. The implications of these findings on the currently existing models explaining the mechanism of Tat transport are discussed.     

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.     

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.     

Sec-independent protein translocation in Escherichia coli. A distinct and pivotal role for the TatB protein

In Escherichia coli, transmembrane translocation of proteins can proceed by a number of routes. A subset of periplasmic proteins are exported via the Tat pathway to which proteins are directed by N-terminal "transfer peptides" bearing the consensus (S/T)RRXFLK "twin-arginine" motif. The Tat system involves the integral membrane proteins TatA, TatB, TatC, and TatE. Of these, TatA, TatB, and TatE are homologues of the Hcf106 component of the DeltapH-dependent protein import system of plant thylakoids. Deletion of the tatB gene alone is sufficient to block the export of seven endogenous Tat substrates, including hydrogenase-2. Complementation analysis indicates that while TatA and TatE are functionally interchangeable, the TatB protein is functionally distinct. This conclusion is supported by the observation that Helicobacter pylori tatA will complement an E. coli tatA mutant, but not a tatB mutant. Analysis of Tat component stability in various tat deletion backgrounds shows that TatC is rapidly degraded in the absence of TatB suggesting that TatC complexes, and is stabilized by, TatB.     

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.     

The TatBC complex formation suppresses a modular TatB-multimerization in Escherichia coli

Twin-arginine translocation (Tat) systems allow the translocation of folded proteins across biological membranes of most prokaryotes. In proteobacteria, a TatBC complex binds Tat substrates and initiates their translocation after recruitment of the component TatA. TatA and TatB belong to one protein family, but only TatB forms stable complexes with TatC. Here we show that TatB builds up TatA-like modular complexes in the absence of TatC. This TatB ladder ranges from about 100 to over 880 kDa with 105+/-10 kDa increments. TatC alone can form a 250 kDa complex which could be a scaffold that can recruit TatB to form defined TatBC complexes.     

Mapping precursor-binding site on TatC subunit of twin arginine-specific protein translocase by site-specific photo cross-linking

A number of secreted precursor proteins of bacteria, archaea, and plant chloroplasts stand out by a conserved twin arginine-containing sequence motif in their signal peptides. Many of these precursor proteins are secreted in a completely folded conformation by specific twin arginine translocation (Tat) machineries. Tat machineries are high molecular mass complexes consisting of two types of membrane proteins, a hexahelical TatC protein, and usually one or two single-spanning membrane proteins, called TatA and TatB. TatC has previously been shown to be involved in the recognition of twin arginine signal peptides. We have performed an extensive site-specific cross-linking analysis of the Escherichia coli TatC protein under resting and translocating conditions. This strategy allowed us to map the recognition site for twin arginine signal peptides to the cytosolic N-terminal region and first cytosolic loop of TatC. In addition, discrete contact sites between TatC, TatB, and TatA were revealed. We discuss a tentative model of how a twin arginine signal sequence might be accommodated in the Tat translocase.     

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.     

Isolation and characterization of bifunctional Escherichia coli TatA mutant proteins that allow efficient tat-dependent protein translocation in the absence of TatB

In Escherichia coli, the Tat system promotes the membrane translocation of a subset of exported proteins across the cytoplasmic membrane. Four genes (tatA, tatB, tatC, and tatE) have been identified that encode the components of the E. coli Tat translocation apparatus. Whereas TatA and TatE can functionally substitute for each other, the TatB and the TatC proteins have been shown to perform distinct functions. In contrast to Tat systems of the ABC(E) type found in E. coli and many other bacteria, some microorganisms possess a TatAC-type translocase that consists of TatA and TatC only, suggesting that, in these systems, TatB is not required or that one of the remaining components (TatA or TatC) additionally takes over the TatB function. We have addressed the molecular basis for the difference in subunit composition between TatABC(E) and TatAC-type systems by using a genetic approach. A plasmid-encoded E. coli minimal Tat translocase consisting solely of TatA and TatC was shown to mediate a low level translocation of a sensitive Tat-dependent reporter protein. Suppressor mutations in the minimal Tat translocase were isolated that compensate for the absence of TatB and that showed substantial increases in translocation activities. All of the mutations mapped to the extreme amino-terminal domain of TatA. No mutations affecting TatC were identified. These results suggest that in TatAC-type systems, the TatA protein represents a bifunctional component fulfilling both the TatA and TatB functions. Furthermore, our results indicate that the structure of the amino-terminal domain of TatA is decisive for whether or not TatB is required.     

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.     

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.     

Characterisation of Tat protein transport complexes carrying inactivating mutations

The Tat system functions to transport folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Tat transport involves a high molecular weight TatBC-containing complex that transiently associates with TatA during protein translocation. Sedimentation equilibrium experiments were used to determine a protein-only molecular mass for the TatBC complex of 630+/-30kDa, suggesting that it contains approximately 13 copies of the TatB and TatC protomers. Point mutations that inactivate Tat transport have previously been identified in each of TatA, TatB, and TatC. Analysis of the TatBC complexes formed by these inactive variants demonstrates that the amino acid substitutions neither affect the composition of the TatBC complex nor cause accumulation of the assembled TatABC translocation site. In addition, the TatA protein is shown not to be required for the assembly or stability of the TatBC complex.     

Substrate-Dependent Assembly of the Tat Translocase as Observed in Live Escherichia coli Cells

The twin-arginine translocation (Tat) pathway guides fully folded proteins across membranes of bacteria, archaea and plant chloroplasts. In Escherichia coli, Tat-specific transport is executed in a still largely unknown manner by three functionally diverse membrane proteins, termed TatA, TatB, and TatC. In order to follow the intracellular distribution of the TatABC proteins in live E. coli cells, we have individually expressed fluorophore-tagged versions of each Tat protein in addition to a set of chromosomally encoded TatABC proteins. In this way, a Tat translocase could form from the native TatABC proteins and be visualized via the association of a fluorescent Tat variant. A functionally active TatA-green fluorescent protein fusion was found to re-locate from a uniform distribution in the membrane into a few clusters preferentially located at the cell poles. Clustering was absolutely dependent on the co-expression of functional Tat substrates, the proton-motive force, and the cognate TatBC subunits. Likewise, polar cluster formation of a functional TatB-mCherry fusion required TatA and TatC and that of a functional TatC-mCherry fusion a functional Tat substrate. Furthermore we directly demonstrate the co-localization of TatA and TatB in the same fluorescent clusters. Our collective results are consistent with distinct Tat translocation sites dynamically forming in vivo in response to newly synthesized Tat substrates.     

TatBC, TatB, and TatC form structurally autonomous units within the twin arginine protein transport system of Escherichia coli

The Tat (twin arginine translocation) system transports folded proteins across bacterial and thylakoid membranes. The integral membrane proteins TatA, TatB, and TatC are the essential components of the Tat pathway in Escherichia coli. We demonstrate that formation of a stable complex between TatB and TatC does not require TatA or other Tat components. We show that the TatB and TatC proteins are each able to a form stable, defined, homomultimeric complexes. These we suggest correspond to structural subcomplexes within the parental TatBC complex. We infer that TatC forms a core to the TatBC complex on to which TatB assembles.     

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.     

A stromal pool of TatA promotes Tat-dependent protein transport across the thylakoid membrane

In chloroplasts and bacteria, the Tat (twin-arginine translocation) system is engaged in transporting folded passenger proteins across the thylakoid and cytoplasmic membranes, respectively. To date, three membrane proteins (TatA, TatB, and TatC) have been identified to be essential for Tat-dependent protein translocation in the plant system, whereas soluble factors seem not to be required. In contrast, in the bacterial system, several cytosolic chaperones were described to be involved in Tat transport processes. Therefore, we have examined whether stromal or peripherally associated membrane proteins also play a role in Tat transport across the thylakoid membrane. Analyzing both authentic precursors as well as the chimeric 16/23 protein, which allows us to study each step of the translocation process individually, we demonstrate that a soluble form of TatA is present in the chloroplast stroma, which significantly improves the efficiency of Tat-dependent protein transport. Furthermore, this soluble TatA is able to reconstitute the Tat transport properties of thylakoid membranes that are transport-incompetent due to extraction with solutions of chaotropic salts.