Tat Machinery Research Articles

Identification of novel tail-anchored membrane proteins integrated by the bacterial twin-arginine translocase.

The twin-arginine protein transport (Tat) system exports folded proteins across the cytoplasmic membranes of prokaryotes and the energy transducing-membranes of plant thylakoids and mitochondria. Proteins are targeted to the Tat machinery by N-terminal signal peptides with a conserved twin-arginine motif, and some substrates are exported as heterodimers where the signal peptide is present on one of the partner proteins. A subset of Tat substrates is found in the membrane. Tat-dependent membrane proteins usually have large globular domains and a single transmembrane helix present at the N- or C-terminus. Five Tat substrates that have C-terminal transmembrane helices have previously been characterized in the model bacterium Escherichia coli. Each of these is an iron-sulfur cluster-containing protein involved in electron transfer from hydrogen or formate. Here we have undertaken a bioinformatic search to identify further tail-anchored Tat substrates encoded in bacterial genomes. Our analysis has revealed additional tail-anchored iron-sulfur proteins associated in modules with either a b-type cytochrome or a quinol oxidase. We also identified further candidate tail-anchored Tat substrates, particularly among members of the actinobacterial phylum, that are not predicted to contain cofactors. Using reporter assays, we show experimentally that six of these have both N-terminal Tat signal peptides and C-terminal transmembrane helices. The newly identified proteins include a carboxypeptidase and a predicted protease, and four sortase substrates for which membrane integration is a prerequisite for covalent attachment to the cell wall.

Characterization of a TatA/TatB binding site on the TatC component of the Escherichia coli twin arginine translocase.

The twin arginine transport (Tat) pathway exports folded proteins across the cytoplasmic membranes of prokaryotes and the thylakoid membranes of chloroplasts. In Escherichia coli and other Gram-negative bacteria, the Tat machinery comprises TatA, TatB and TatC components. A Tat receptor complex, formed from all three proteins, binds Tat substrates, which triggers receptor organization and recruitment of further TatA molecules to form the active Tat translocon. The polytopic membrane protein TatC forms the core of the Tat receptor and harbours two binding sites for the sequence-related TatA and TatB proteins. A 'polar' cluster binding site, formed by TatC transmembrane helices (TMH) 5 and 6 is occupied by TatB in the resting receptor and exchanges for TatA during receptor activation. The second binding site, lying further along TMH6, is occupied by TatA in the resting state, but its functional relevance is unclear. Here we have probed the role of this second binding site through a programme of random and targeted mutagenesis. Characterization of three stably produced TatC variants, P221R, M222R and L225P, each of which is inactive for protein transport, demonstrated that the substitutions did not affect assembly of the Tat receptor. Moreover, the substitutions that we analysed did not abolish TatA or TatB binding to either binding site. Using targeted mutagenesis we introduced bulky substitutions into the TatA binding site. Molecular dynamics simulations and crosslinking analysis indicated that TatA binding at this site was substantially reduced by these amino acid changes, but TatC retained function. While it is not clear whether TatA binding at the TMH6 site is essential for Tat activity, the isolation of inactivating substitutions indicates that this region of the protein has a critical function.

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.

Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation.

The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitro transport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD > 50 μM), and this monomer binds reversibly to spTorA (KD ≈ 1 μM). While TorD binds to membranes (KD ≈ 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.

Double trouble: Bacillus depends on a functional Tat machinery to avoid severe oxidative stress and starvation upon entry into a NaCl-depleted environment

The widely conserved twin-arginine translocases (Tat) allow the transport of fully folded cofactor-containing proteins across biological membranes. In doing so, these translocases serve different biological functions ranging from energy conversion to cell division. In the Gram-positive soil bacterium Bacillus subtilis, the Tat machinery is essential for effective growth in media lacking iron or NaCl. It was previously shown that this phenomenon relates to the Tat-dependent export of the heme-containing peroxidase EfeB, which converts Fe2+ to Fe3+ at the expense of hydrogen peroxide. However, the reasons why the majority of tat mutant bacteria perish upon dilution in NaCl-deprived medium and how, after several hours, a sub-population adapts to this condition was unknown. Here we show that, upon growth in the absence of NaCl, the bacteria face two major problems, namely severe oxidative stress at the membrane and starvation leading to death. The tat mutant cells can overcome these challenges if they are fed with arginine, which implies that severe arginine depletion is a major cause of death and resumed arginine synthesis permits their survival. Altogether, our findings show that the Tat system of B. subtilis is needed to preclude severe oxidative stress and starvation upon sudden drops in the environmental Na+ concentration as caused by flooding or rain.

Targeting of proteins to the twin-arginine translocation pathway.

The twin‐arginine protein transport (Tat pathway) is found in prokaryotes and plant organelles and transports folded proteins across membranes. Targeting of substrates to the Tat system is mediated by the presence of an N‐terminal signal sequence containing a highly conserved twin‐arginine motif. The Tat machinery comprises membrane proteins from the TatA and TatC families. Assembly of the Tat translocon is dynamic and is triggered by the interaction of a Tat substrate with the Tat receptor complex. This review will summarise recent advances in our understanding of Tat transport, focusing in particular on the roles played by Tat signal peptides in protein targeting and translocation.

Functional reconstitution of TatB into the thylakoidal Tat translocase

We have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twin-arginine protein transport (Tat) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analysed with the authentic Tat substrate pOEC16, reached routinely 20–25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutated TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional analyses of protein variants. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport.

Mechanistic analysis of the minimalistic twin-arginine translocation system found in Bacillus subtilis

The twin-arginine translocation (Tat) machinery mediates the transport of folded proteins across the cytoplasmic membranes of prokaryotes and thylakoid membranes of chloroplasts. In Gram-negative bacteria three integral membrane components, TatA, TatB and TatC, are essential for generating an active complex. Most Gram-positive bacteria however, have a minimalist Tat complex formed solely from TatA and TatC subunits. Bacillus subtilis encodes two TatAC systems; TatAdCd and TatAyCy. These complexes operate in parallel but have differing substrate specificities. To date, little is known about how the B. subtilis TatAC-complexes assemble, however, the Escherichia coli TatABC complex is well studied and a substrate-triggered positional exchange of TatA and TatB has been identified as the first step in the assembly of an active complex. In this study, we aim to identify site specific interactions taking place between the B. subtilis Tat components and how these interactions differ whilst the complex is engaged in transport of a substrate. Molecular modelling was used to identify likely interaction interfaces between TatA and TatC, and site-directed mutagenesis was used to generate single cysteine variants of both proteins. Subsequently in vivo disulfide crosslinking was undertaken in both E. coli and B.subtilis, in the presence and absence of overproduced Tat substrates, to determine changes that occur between resting state and activated complexes. Overall, this work will enable us to outline a possible mechanism of assembly in TatAC-complexes of Gram-positive bacteria and identify whether the two distinct translocases assemble in a similar fashion.

Evolution of mitochondrial TAT translocases illustrates the loss of bacterial protein transport machines in mitochondria

BackgroundBacteria and mitochondria contain translocases that function to transport proteins across or insert proteins into their inner and outer membranes. Extant mitochondria retain some bacterial-derived translocases but have lost others. While BamA and YidC were integrated into general mitochondrial protein transport pathways (as Sam50 and Oxa1), the inner membrane TAT translocase, which uniquely transports folded proteins across the membrane, was retained sporadically across the eukaryote tree.ResultsWe have identified mitochondrial TAT machinery in diverse eukaryotic lineages and define three different types of eukaryote-encoded TatABC-derived machineries (TatAC, TatBC and TatC-only). Here, we investigate TatAC and TatC-only machineries, which have not been studied previously. We show that mitochondria-encoded TatAC of the jakobid Andalucia godoyi represent the minimal functional pathway capable of substituting for the Escherichia coli TatABC complex and can transport at least one substrate. However, selected TatC-only machineries, from multiple eukaryotic lineages, were not capable of supporting the translocation of this substrate across the bacterial membrane. Despite the multiple losses of the TatC gene from the mitochondrial genome, the gene was never transferred to the cell nucleus. Although the major constraint preventing nuclear transfer of mitochondrial TatC is likely its high hydrophobicity, we show that in chloroplasts, such transfer of TatC was made possible due to modifications of the first transmembrane domain.ConclusionsAt its origin, mitochondria inherited three inner membrane translocases Sec, TAT and Oxa1 (YidC) from its bacterial ancestor. Our work shows for the first time that mitochondrial TAT has likely retained its unique function of transporting folded proteins at least in those few eukaryotes with TatA and TatC subunits encoded in the mitochondrial genome. However, mitochondria, in contrast to chloroplasts, abandoned the machinery multiple times in evolution. The overall lower hydrophobicity of the Oxa1 protein was likely the main reason why this translocase was nearly universally retained in mitochondrial biogenesis pathways.

The Tat protein transport system: intriguing questions and conundrums.

The Tat machinery catalyzes the transport of folded proteins across the cytoplasmic membrane in bacteria and the thylakoid membrane in plants. Transport occurs only in the presence of an electric field (Δψ) and/or a pH (ΔpH) gradient, and thus, Tat transport is considered to be dependent on the proton motive force (pmf). This presents a fundamental and major challenge, namely, that the Tat system catalyzes the movement of large folded protein cargos across a membrane without collapse of ion gradients. Current models argue that the active translocon assembles de novo for each cargo transported, thus providing an effective gating mechanism to minimize ion leakage. A limited structural understanding of the intermediates occurring during transport and the role of the pmf in stabilizing and/or driving this process have hindered the development of more detailed models. A fundamental question that remains unanswered is whether the pmf is actually 'consumed', providing an energetic driving force for transport, or alternatively, whether its presence is instead necessary to provide the appropriate environment for the translocon components to become active. Including addressing this issue in greater detail, we explore a series of additional questions that challenge current models, and, hopefully, motivate future work.

Probing the quality control mechanism of the Escherichia coli twin-arginine translocase with folding variants of a de novo–designed heme protein

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo–designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b–induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system's quality-control mechanism.

Plant Mitochondrial Inner Membrane Protein Insertion.

During the biogenesis of the mitochondrial inner membrane, most nuclear-encoded inner membrane proteins are laterally released into the membrane by the TIM23 and the TIM22 machinery during their import into mitochondria. A subset of nuclear-encoded mitochondrial inner membrane proteins and all the mitochondrial-encoded inner membrane proteins use the Oxa machinery—which is evolutionarily conserved from the endosymbiotic bacterial ancestor of mitochondria—for membrane insertion. Compared to the mitochondria from other eukaryotes, plant mitochondria have several unique features, such as a larger genome and a branched electron transport pathway, and are also involved in additional cellular functions such as photorespiration and stress perception. This review focuses on the unique aspects of plant mitochondrial inner membrane protein insertion machinery, which differs from that in yeast and humans, and includes a case study on the biogenesis of Cox2 in yeast, humans, two plant species, and an algal species to highlight lineage-specific similarities and differences. Interestingly, unlike mitochondria of other eukaryotes but similar to bacteria and chloroplasts, plant mitochondria appear to use the Tat machinery for membrane insertion of the Rieske Fe/S protein.

A Hinged Signal Peptide Hairpin Enables Tat-Dependent Protein Translocation

The Tat machinery catalyzes the transport of folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane in plants. Using fluorescence quenching and cross-linking approaches, we demonstrate that the Escherichia coli TatBC complex catalyzes insertion of a pre-SufI signal peptide hairpin that penetrates about halfway across the membrane bilayer. Analysis of 512 bacterial Tat signal peptides using secondary structure prediction and docking algorithms suggest that this hairpin interaction mode is generally conserved. An internal cross-link in the signal peptide that blocks transport but does not affect binding indicates that a signal peptide conformational change is required during translocation. These results suggest, to our knowledge, a novel hairpin-hinge model in which the signal peptide hairpin unhinges during movement of the mature domain across the membrane. Thus, in addition to enabling the necessary recognition, the interaction of Tat signal peptides with the receptor complex plays a critical role in the transport process itself.

How to achieve Tat transport with alien TatA

TatA is an essential and structurally conserved component of all known Twin-arginine transport (Tat) machineries which are able to catalyse membrane transport of fully folded proteins. Here we have investigated if bacterial TatA, or chimeric pea/E. coli TatA derivatives, are capable of replacing thylakoidal TatA in function. While authentic E. coli TatA does not show any transport activity in thylakoid transport experiments, TatA chimeras comprising the transmembrane helix (TMH) of pea TatA are fully active. For minimal catalytic activity it is even sufficient to replace three residues within TMH of E. coli TatA by the corresponding pea residues. Almost any further substitution within TMH gradually raises transport activity in the thylakoid system, while functional characterization of the same set of TatA derivatives in E. coli yields essentially inverse catalytic activities. Closer inspection of the substituted residues suggests that the two transport systems have deviating demands with regard to the hydrophobicity of the transmembrane helix.

Signal Peptide Hydrophobicity Modulates Interaction with the Twin-Arginine Translocase.

ABSTRACTThe general secretory pathway (Sec) and twin-arginine translocase (Tat) operate in parallel to export proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Substrates are targeted to their respective machineries by N-terminal signal peptides that share a tripartite organization; however, Tat signal peptides harbor a conserved and almost invariant arginine pair that is critical for efficient targeting to the Tat machinery. Tat signal peptides interact with a membrane-bound receptor complex comprised of TatB and TatC components, with TatC containing the twin-arginine recognition site. Here, we isolated suppressors in the signal peptide of the Tat substrate, SufI, that restored Tat transport in the presence of inactivating substitutions in the TatC twin-arginine binding site. These suppressors increased signal peptide hydrophobicity, and copurification experiments indicated that they restored binding to the variant TatBC complex. The hydrophobic suppressors could also act in cis to suppress substitutions at the signal peptide twin-arginine motif that normally prevent targeting to the Tat pathway. Highly hydrophobic variants of the SufI signal peptide containing four leucine substitutions retained the ability to interact with the Tat system. The hydrophobic signal peptides of two Sec substrates, DsbA and OmpA, containing twin lysine residues, were shown to mediate export by the Tat pathway and to copurify with TatBC. These findings indicate that there is unprecedented overlap between Sec and Tat signal peptides and that neither the signal peptide twin-arginine motif nor the TatC twin-arginine recognition site is an essential mechanistic feature for operation of the Tat pathway.

Substrate-triggered position switching of TatA and TatB during Tat transport in Escherichia coli.

The twin-arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. The active translocase is assembled on demand, with substrate-binding at a TatABC receptor complex triggering recruitment and assembly of multiple additional copies of TatA; however, the molecular interactions mediating translocase assembly are poorly understood. A ‘polar cluster’ site on TatC transmembrane (TM) helix 5 was previously identified as binding to TatB. Here, we use disulfide cross-linking and molecular modelling to identify a new binding site on TatC TM helix 6, adjacent to the polar cluster site. We demonstrate that TatA and TatB each have the capacity to bind at both TatC sites, however in vivo this is regulated according to the activation state of the complex. In the resting-state system, TatB binds the polar cluster site, with TatA occupying the TM helix 6 site. However when the system is activated by overproduction of a substrate, TatA and TatB switch binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

Single-Molecule Fluorescence Imaging to Determine the Stoichiometry of the Twin-Arginine Translocase

The twin-arginine translocation (Tat) machinery transports folded substrate proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. This mechanistically challenging machinery deploys three essential proteins, TatA, TatB, and TatC. TatA is known to form the protein translocating element of the Tat system. The size and the stoichiometry of the Tat translocation complex are thought to be dependent on the size of the substrate. Current models for Tat transport only predict the oligomeric state of TatA and if, and whether, this state changes during the transport cycle. We use single-molecule fluorescence techniques to probe into the stoichiometry of the translocase.

C-terminal truncation of a Tat passenger protein affects its membrane translocation by interfering with receptor binding.

During thylakoid transport of the chimeric model twin-arginine translocation (Tat) substrate 16/23, two consecutive translocation intermediates with different membrane topology are observed. The early translocation intermediate Ti-1 is bound to the membrane such that almost half of the protein is protected against proteolysis and it was concluded that not only the signal peptide but also part of the passenger protein participates in membrane binding. However, topology studies using a membrane-impermeable thiol-reactive reagent show that most of the passenger remains accessible from the stromal side in Ti-1 conformation. Establishment of such Ti-1 topology at the membrane apparently requires the fully folded passenger protein, as it was not observed with 16/23 truncation derivatives lacking the C-terminal 20, 40, 60, or 88 residues. Thylakoid transport of these mutants, which depends on a fully functional Tat machinery, is progressively reduced with increasing size of the truncated passenger polypeptide. The same holds true also for the interaction with the thylakoidal TatBC complexes, suggesting that in this case receptor binding, which is apparently impaired by extended unfolded or malfolded passenger polypeptides, is the rate-limiting step of Tat-dependent membrane transport.

Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation

Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery.

The twin-arginine protein translocation pathway.

The twin-arginine translocation (Tat) system, found in prokaryotes, chloroplasts, and some mitochondria, allows folded proteins to be moved across membranes. How this transport is achieved without significant ion leakage is an intriguing mechanistic question. Tat transport is mediated by complexes formed from small integral membrane proteins from just two protein families. Atomic-resolution structures have recently been determined for representatives of both these protein families, providing the first molecular-level glimpse of the Tat machinery. I review our current understanding of the mechanism of Tat transport in light of these new structural data.