Functional reconstitution of bacterial Tat translocation in vitro

The Tat (twin-arginine translocation) pathway is a Sec-independent mechanism for translocating folded preproteins across or into the inner membrane of Escherichia coli. To study Tat translocation, we sought an in vitro translocation assay using purified inner membrane vesicles and in vitro synthesized substrate protein. While membrane vesicles derived from wild-type cells translocate the Sec-dependent substrate proOmpA, translocation of a Tat-dependent substrate, SufI, was not detected. We established that in vivo overexpression of SufI can saturate the Tat translocase, and that simultaneous overexpression of TatA, B and C relieves this SufI saturation. Using membrane vesicles derived from cells overexpressing TatABC, in vitro translocation of SufI was detected. Like translocation in vivo, translocation of SufI in vitro requires TatABC, an intact membrane potential and the twin-arginine targeting motif within the signal peptide of SUFI: In contrast to Sec translocase, we find that Tat translocase does not require ATP. The development of an in vitro translocation assay is a prerequisite for further biochemical investigations of the mechanism of translocation, substrate recognition and translocase structure.     

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.     

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.     

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.     

Genetic analysis of pathway specificity during posttranslational protein translocation across the Escherichia coli plasma membrane

In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the "Sec avoidance signal," the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.     

The twin-arginine translocation pathway is necessary for correct membrane insertion of the Rieske Fe/S protein in Legionella pneumophila

The twin-arginine translocation (Tat) pathway translocates folded proteins across the cytoplasmic membrane. Proteins transported through this secretion system typically carry two arginine residues in their signal peptide that is cleaved off during translocation. Recently, we demonstrated the presence of the Tat pathway in Legionella pneumophila Philadelphia-1 and the Rieske Fe/S protein PetA was one of the predicted Tat substrates. Because we observed that the signal peptide of PetA is not processed and that this protein is still membrane associated in the tat mutants, correct membrane insertion was assayed using a trypsin sensitivity assay. We conclude that the Tat pathway is necessary for correct membrane insertion of L. pneumophila PetA.     

A putative twin-arginine translocation pathway in Legionella pneumophila

Legionella pneumophila is a facultative intracellular human pathogen causing Legionnaires' disease, a severe form of pneumonia. Because of the importance of secretion pathways in virulence, we were interested in the possible presence of the twin-arginine translocation (Tat) pathway in L. pneumophila. This secretion pathway is used to transport folded proteins, characterized by two arginines in their signal peptide, across the cytoplasmic membrane. We describe here the presence of a putative Tat pathway in L. pneumophila. Three genes encoding Escherichia coli TatA, TatB, and TatC homologues were identified. The tatA and tatB genes were shown to constitute an operon while tatC is monocistronic. RT-PCR analysis revealed expression of the tat genes during both exponential and stationary growth as well as during intracellular growth in Acanthamoeba castellanii. A search for the conserved twin-arginine motif in predicted signal peptides resulted in a list of putative Tat substrates.     

The twin-arginine translocation pathway of Mycobacterium smegmatis is functional and required for the export of mycobacterial beta-lactamases

The twin-arginine translocation (Tat) pathway exports folded proteins across the bacterial cytoplasmic membrane and is responsible for the proper extracytoplasmic localization of proteins involved in a variety of cellular functions, including pathogenesis. The Mycobacterium tuberculosis and Mycobacterium smegmatis genomes contain open reading frames with homology to components of the Tat export system (TatABC) as well as potential Tat-exported proteins possessing N-terminal signal sequences with the characteristic twin-arginine motif. Due to the importance of exported virulence factors in the pathogenesis of M. tuberculosis and the limited understanding of mycobacterial protein export systems, we sought to determine the functional nature of the Tat export pathway in mycobacteria. Here we describe phenotypic analyses of DeltatatA and DeltatatC deletion mutants of M. smegmatis, which demonstrated that tatA and tatC encode components of a functional Tat system capable of exporting characteristic Tat substrates. Both mutants displayed a growth defect on agar medium and hypersensitivity to sodium dodecyl sulfate. The mutants were also defective in the export of active beta-lactamases of M. smegmatis (BlaS) and M. tuberculosis (BlaC), both of which possess twin-arginine signal sequences. The Tat-dependent nature of BlaC was further revealed by mutation of the twin-arginine motif. Finally, we demonstrated that replacement of the native signal sequence of BlaC with the predicted Tat signal sequences of M. tuberculosis phospholipase C proteins (PlcA and PlcB) resulted in the Tat-dependent export of an enzymatically active 'BlaC. Thus, 'BlaC can be used as a genetic reporter for Tat-dependent export in mycobacteria.     

Haloferax volcanii twin-arginine translocation substates include secreted soluble, C-terminally anchored and lipoproteins

Recent in silico and in vivo studies have suggested that the majority of proteins destined for secretion in the haloarchaea are trafficked through the twin-arginine translocation (Tat) pathway. The presence of lipobox motifs in most haloarchaeal Tat signal sequences is intriguing as: (i) bioinformatic searches of archaeal genomes have not identified lipoprotein biogenesis enzymes and (ii) there are no known Tat substrates containing both a twin-arginine and a bona fide lipobox. We have examined six computationally designated Tat substrates in the haloarchaeon Haloferax volcanii to verify previous computational predictions and to initiate studies of lipoprotein biogenesis via the Tat pathway. Our results confirmed that the six candidate proteins were not only Tat substrates, but also belonged to diverse classes of secretory proteins. Analysis of predicted lipoprotein Tat substrates revealed that they are anchored to the archaeal membrane in a cysteine-dependent manner. Interestingly, despite the absence of an archaeal lipoprotein signal peptidase II (SPase II) homologue, the SPase II inhibitor globomycin impeded cell growth and specifically prevented maturation of lipoproteins. Together, this work not only represents the first experimental demonstration of a lipoprotein Tat substrate, but also indicates the presence of an unidentified lipoprotein biogenesis pathway in archaea.     

Secretion of active xylanase C from Streptomyces lividans is exclusively mediated by the Tat protein export system

The bacterial twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane. The precursors targeted to the Tat pathway have signal peptides bearing the consensus motif (S/T-R-R-X-F-L-K). The xylanase C (XlnC) of Streptomyces lividans is a 20-kDa secreted enzyme. The XlnC signal peptide is 49 amino acids long and contains the S-R-R-G-F-L-G sequence, which is similar to the twin-arginine consensus motif. In S. lividans, XlnC secretion was impaired in a tatC insertion mutant, which is unable to secrete proteins that are dependent on the Tat system. When the signal peptide of XlnC was replaced by the Sec-dependent signal peptide of xylanase A, XlnC was secreted as an inactive form and demonstrated rapid proteolytic degradation in the culture supernatant, thus indicating that XlnC was specifically secreted through the Tat system. Deletions of the n-region of the XlnC signal sequence showed that a minimum of six amino acids residues preceding the twin-arginine motif was required to secrete XlnC. Replacement of one or both arginines by lysine residues in the twin arginine motif decreased four- and sevenfold, respectively, the enzyme production but did not abolish it. However, pulse chase experiments showed that the half-life of the precursor was from 2 to 3 h instead of 11 min for the wild- type precursor. Since XlnC is not associated with cofactors to exhibit activity, it is therefore a newly identified prokaryotic non-redox Tat substrate.     

The entire N-terminal half of TatC is involved in twin-arginine precursor binding

Translocation of twin-arginine precursor proteins across the cytoplasmic membrane of Escherichia coli requires the three membrane proteins TatA, TatB, and TatC. TatC and TatB were shown to be involved in precursor binding. We have analyzed in vitro a number of single alanine substitutions in tatC that were previously shown to compromise in vivo the function of the Tat translocase. All tatC mutants that were defective in precursor translocation into cytoplasmic membrane vesicles concomitantly interfered with precursor binding not only to TatC but also to TatB. Hence structural changes of TatC that affect precursor targeting simultaneously abolish engagement of the twin-arginine signal sequence with TatB and block the formation of a functional Tat translocase. Since these phenotypes were observed for tatC mutations spread over the first half of TatC, this entire part of the molecule must globally be involved in precursor binding.     

Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli

The twin-arginine translocation (Tat) machinery of the Escherichia coli inner membrane is dedicated to the export of proteins harboring a conserved SRRxFLK motif in their signal sequence. TatA, TatB, and TatC are the functionally essential constituents of the Tat machinery, but their precise function is unknown. Using site-specific crosslinking, we have analyzed interactions of the twin-arginine precursor preSufI with the Tat proteins upon targeting to inner membrane vesicles. TatA association is observed only in the presence of a transmembrane H(+) gradient. TatB is found in contact with the entire signal sequence and adjacent parts of mature SufI. Interaction of TatC with preSufI is, however, restricted to a discrete area around the consensus motif. The results reveal a hierarchy in targeting of a Tat substrate such that for the primary interaction, TatC is both necessary and sufficient while a subsequent association with TatB likely mediates transfer from TatC to the actual Tat pore.     

The twin-arginine translocation (Tat) system is essential for Rhizobium-legume symbiosis

The Tat (twin-arginine translocation) system mediates export of periplasmic proteins in folded conformation. Proteins transported via Tat contain a characteristic twin-arginine motif in their signal peptide. Genetic determinants (tatABC genes) of the Tat system from Rhizobium leguminosarum bv. viciae were cloned and characterized, and a tatBC deletion mutant was constructed. The mutant lacked the ability for membrane targeting of hydrogenase, a known Tat substrate, and was impaired in hydrogenase activity. Interestingly, in the absence of a functional Tat system, only small, white nodules unable to fix nitrogen were induced in symbiosis with pea plants. Analysis of nodule structure and location of green fluorescent protein (GFP)-tagged bacteria within nodules indicated that the symbiotic process was blocked in the tat mutant at a stage previous to bacteria release into cortical cells. The R. leguminosarum Tat-deficient mutant lacked a functional cytochrome bc1 complex. This was consistent with the fact that R. leguminosarum Rieske protein, a key component of the symbiosis-essential cytochrome bc1 complex, contained a typical twin-arginine signal peptide. However, comparative analyses of nodule structure indicated that nodule development in the tat mutant was arrested at an earlier step than in a cytochrome bc1 mutant. These data indicate that the Tat pathway is also critical for proteins relevant to the initial stages of the symbiotic process.     

Adaptation of protein secretion to extremely high-salt conditions by extensive use of the twin-arginine translocation pathway

Halophilic archaea thrive in environments with salt concentrations approaching saturation. However, little is known about the way in which these organisms stabilize their secreted proteins in such 'hostile' conditions. Here, we present data suggesting that the utilization of protein translocation pathways for protein secretion by the Halobacteriaceae differs significantly from that of non-haloarchaea, and most probably represents an adaptation to the high-salt environment. Although most proteins are secreted via the general secretion (Sec) machinery, the twin-arginine translocation (Tat) pathway is mainly used for the secretion of redox proteins and is distinct from the Sec pathway, in that it allows cytoplasmic folding of secreted proteins. tatfind (developed in this study) was used for systematic whole-genome analysis of Halobacterium sp. NRC-1 and several other prokaryotes to identify putative Tat substrates. Our analyses revealed that the vast majority of haloarchaeal secreted proteins were predicted substrates of the Tat pathway. Strikingly, most of these putative Tat substrates were non-redox proteins, the homologues of which in non-haloarchaea were identified as putative Sec substrates. We confirmed experimentally that the secretion of one such putative Tat substrate depended on the twin-arginine motif in its signal sequence. This extensive utilization of the Tat pathway in haloarchaea suggests an evolutionary adaptation to high-salt conditions by allowing cytoplasmic folding of secreted proteins before their secretion.     

The importance of the twin-arginine translocation pathway for bacterial virulence

The twin-arginine translocation (Tat) pathway is a prokaryotic transport system that enables the transport of folded proteins across the cytoplasmic membrane. The Tat pathway was originally thought to transport only proteins that bind cofactors in the cytoplasm and, thus, fold before transport, like many proteins related to energy metabolism. However, in recent years it has become clear that the Tat pathway has a broader role and is also an important virulence factor in different bacterial pathogens. Because the Tat pathway is well conserved among important bacterial pathogens and absent from mammalian cells, it could be a target for novel antimicrobial compounds. In this review, we highlight the importance of the Tat system for virulence in several human and plant pathogens.     

A facile reporter system for the experimental identification of twin-arginine translocation (Tat) signal peptides from all kingdoms of life

We have developed a reporter protein system for the experimental verification of twin-arginine signal peptides. This reporter system is based on the Streptomyces coelicolor agarase protein, which is secreted into the growth medium by the twin-arginine translocation (Tat) pathway and whose extracellular activity can be assayed colorimetrically in a semiquantitative manner. Replacement of the native agarase signal peptide with previously characterized twin-arginine signal peptides from other Gram-positive and Gram-negative bacteria resulted in efficient Tat-dependent export of agarase. Candidate twin-arginine signal peptides from archaeal proteins as well as plant thylakoid-targeting sequences were also demonstrated to mediate agarase translocation. A naturally occurring variant signal peptide with an arginine-glutamine motif instead of the consensus di-arginine was additionally recognized as a Tat-targeting sequence by Streptomyces. Application of the agarase assay to previously uncharacterized candidate Tat signal peptides from Bacillus subtilis identified two further probable Tat substrates in this organism. This is the first versatile reporter system for Tat signal peptide identification.     

One signal is enough: Stepwise transport of two distinct passenger proteins by the Tat pathway across the thylakoid membrane

The twin-arginine translocation (Tat) pathway, one of four protein transport pathways operating at the thylakoid membrane of chloroplasts, shows remarkable substrate flexibility. Here, we have analyzed the thylakoid transport of chimeric tandem substrates that are composed of two different passenger proteins fused to a single Tat transport signal. The chimera 23/23-EGFP in which the reporter protein EGFP is connected to the C-terminus of the OEC23 precursor shows that a single Tat transport signal is sufficient to mediate transport of two distinct passenger proteins in a row. Replacing the transit peptide of OEC23 in 23/23-EGFP by its homolog from OEC16 yields the chimera 16/23-EGFP, which can likewise be fully translocated by the Tat pathway across the thylakoid membrane. However, transport of 16/23-EGFP is retarded at specific steps in the transport process leading to the temporary and consecutive accumulation of three translocation intermediates with distinct membrane topology. They are associated with two oligomeric membrane complexes presumably representing TatBC-receptor complexes. The composition of the translocation intermediates as determined by immunoprecipitation experiments suggests that the two passenger proteins are translocated in a stepwise manner across the membrane.     

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.     

Twin-Arginine Translocation Pathway in Streptomyces lividans

The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in Streptomyces lividans, a gram-positive organism with a high secretion capacity. The presence of one tatC and two hcf106 homologs in the S. lividans genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the S. lividans secretome. To demonstrate its functionality, a tatC deletion mutant was constructed. This mutation impaired the translocation of the Streptomyces antibioticus tyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in Escherichia coli, could be translocated in wild-type S. lividans but not in the tatC mutant. In contrast, the secretion of the Sec-dependent S. lividans subtilisin inhibitor was not affected. This study therefore demonstrates that also in general in Streptomyces spp. the Tat pathway is functional. Moreover, this Tat pathway can translocate folded proteins, and the E. coli TorA signal peptide can direct Tat-dependent transport in S. lividans.     

Protein targeting by the twin-arginine translocation pathway

The twin-arginine translocation pathway operates in the thylakoid membrane of chloroplasts and in the plasma membrane of most free-living bacteria. Its main function is to transport fully folded proteins across the membrane. Three important tat genes have been identified and the sequences of the encoded proteins, together with the unusual properties of the pathway, indicate that the Tat system is completely different from other protein translocases.