Genetic and biochemical analysis of the twin-arginine translocation pathway in halophilic archaea

The twin-arginine translocation (Tat) pathway is present in a wide variety of prokaryotes and is capable of exporting partially or fully folded proteins from the cytoplasm. Although diverse classes of proteins are transported via the Tat pathway, in most organisms it facilitates the secretion of a relatively small number of substrates compared to the Sec pathway. However, computational evidence suggests that haloarchaea route nearly all secreted proteins to the Tat pathway. We have expanded previous computational analyses of the haloarchaeal Tat pathway and initiated in vivo characterization of the Tat machinery in a model haloarchaeon, Haloferax volcanii. Consistent with the predicted usage of the this pathway in the haloarchaea, we determined that three of the four identified tat genes in Haloferax volcanii are essential for viability when grown aerobically in complex medium. This represents the first report of an organism that requires the Tat pathway for viability when grown under such conditions. Deletion of the nonessential gene had no effect on the secretion of a verified substrate of the Tat pathway. The two TatA paralogs TatAo and TatAt were detected in both the membrane and cytoplasm and could be copurified from the latter fraction. Using size exclusion chromatography to further characterize cytoplasmic and membrane TatA proteins, we find these proteins present in high-molecular-weight complexes in both cellular fractions.     

Overlapping transport and chaperone-binding functions within a bacterial twin-arginine signal peptide

The twin-arginine translocation (Tat) pathway is a protein targeting system present in many prokaryotes. The physiological role of the Tat pathway is the transmembrane translocation of fully-folded proteins, which are targeted by N-terminal signal peptides bearing conserved SRRxFLK 'twin-arginine' amino acid motifs. In Escherichia coli the majority of Tat targeted proteins bind redox cofactors and it is important that only mature, cofactor-loaded precursors are presented for export. Cellular processes have been unearthed that sequence these events, for example the signal peptide of the periplasmic nitrate reductase (NapA) is bound by a cytoplasmic chaperone (NapD) that is thought to regulate assembly and export of the enzyme. In this work, genetic, biophysical and structural approaches were taken to dissect the interaction between NapD and the NapA signal peptide. A NapD binding epitope was identified towards the N-terminus of the signal peptide, which overlapped significantly with the twin-arginine targeting motif. NMR spectroscopy revealed that the signal peptide adopted a α-helical conformation when bound by NapD, and substitution of single residues within the NapA signal peptide was sufficient to disrupt the interaction. This work provides an increased level of understanding of signal peptide function on the bacterial Tat pathway.     

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.     

Protein targeting by the bacterial twin-arginine translocation (Tat) pathway

The Tat (twin-arginine translocation) protein export system is found in the cytoplasmic membrane of most prokaryotes and is dedicated to the transport of folded proteins. The Tat system is now known to be essential for many bacterial processes including energy metabolism, cell wall biosynthesis, the nitrogen-fixing symbiosis and bacterial pathogenesis. Recent studies demonstrate that substrate-specific accessory proteins prevent improperly assembled substrates from interacting with the Tat transporter. During the transport cycle itself substrate proteins bind to a receptor complex in the membrane which then recruits a protein-translocating channel to carry out the transport reaction.     

Prokaryotic utilization of the twin-arginine translocation pathway: a genomic survey

The twin-arginine translocation (Tat) pathway, which has been identified in plant chloroplasts and prokaryotes, allows for the secretion of folded proteins. However, the extent to which this pathway is used among the prokaryotes is not known. By using a genomic approach, a comprehensive list of putative Tat substrates for 84 diverse prokaryotes was established. Strikingly, the results indicate that the Tat pathway is utilized to highly varying extents. Furthermore, while many prokaryotes use this pathway predominantly for the secretion of redox proteins, analyses of the predicted substrates suggest that certain bacteria and archaea secrete mainly nonredox proteins via the Tat pathway. While no correlation was observed between the number of Tat machinery components encoded by an organism and the number of predicted Tat substrates, it was noted that the composition of this machinery was specific to phylogenetic taxa.     

Genetic toggling of alkaline phosphatase folding reveals signal peptides for all major modes of transport across the inner membrane of bacteria

Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen.     

Transport and proofreading of proteins by the twin-arginine translocation (Tat) system in bacteria

The twin-arginine translocation (Tat) system operates in plant thylakoid membranes and the plasma membranes of most free-living bacteria. In bacteria, it is responsible for the export of a number of proteins to the periplasm, outer membrane or growth medium, selecting substrates by virtue of cleavable N-terminal signal peptides that contain a key twin-arginine motif together with other determinants. Its most notable attribute is its ability to transport large folded proteins (even oligomeric proteins) across the tightly sealed plasma membrane. In Gram-negative bacteria, TatABC subunits appear to carry out all of the essential translocation functions in the form of two distinct complexes at steady state: a TatABC substrate-binding complex and separate TatA complex. Several studies favour a model in which these complexes transiently coalesce to generate the full translocase. Most Gram-positive organisms possess an even simpler "minimalist" Tat system which lacks a TatB component and contains, instead, a bifunctional TatA component. These Tat systems may involve the operation of a TatAC complex together with a separate TatA complex, although a radically different model for TatAC-type systems has also been proposed. While bacterial Tat systems appear to require the presence of only a few proteins for the actual translocation event, there is increasing evidence for the operation of ancillary components that carry out sophisticated "proofreading" activities. These activities ensure that redox proteins are only exported after full assembly of the cofactor, thereby avoiding the futile export of apo-forms. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.     

Export of Thermus thermophilus cytoplasmic beta-glycosidase via the E. coli Tat pathway

The Tat pathway is distinct from the Sec machinery given its unusual capacity to export folded proteins, which contain a twin-arginine (RR) signal peptide, across the plasma membrane. The functionality of the Tat pathway has been demonstrated for several Gram-negative and Gram-positive mesophilic bacteria. To assess the specificity of the Tat system, and to analyze the capacity of a mesophilic bacterial Tat system to translocate cytoplasmic proteins from hyperthermophilic bacteria, we fused the Thermus thermophilus beta-glycosidase (Glc) to the twin-arginine signal peptide of the E. coli TorA protein. When expressed in E. coli, the thermophilic RR-Glc chimera was successfully synthesized and efficiently translocated into the periplasm of the wild type strain. In contrast, the beta-glycosidase accumulated within the cytoplasm of all the tat mutants analyzed. The beta-glycosidase synthesized in these strains exhibited thermophilic properties. These results demonstrated, for the first time, the capacity of the E. coli Tat system to export cytoplasmic hyperthermophilic protein, implying an important potential of the Tat system for the production of thermostable enzymes used in bioprocessing applications.     

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.     

The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli

In Escherichia coli a subset of periplasmic proteins is exported through the Tat pathway to which substrates are directed by an NH(2)-terminal signal peptide containing a consensus SRRXFLK "twin arginine" motif. The importance of the individual amino acids of the consensus motif for in vivo Tat transport has been assessed by site-directed mutagenesis of the signal peptide of the Tat substrate pre-SufI. Although the invariant arginine residues are crucial for efficient export, we find that slow transport of SufI is still possible if a single arginine is conservatively substituted by a lysine residue. Thus, in at least one signal peptide context there is no absolute dependence of Tat transport on the arginine pair. The consensus phenylalanine residue was found to be a critical determinant for efficient export but could be functionally substituted by leucine, another amino acid with a highly hydrophobic side chain. Unexpectedly, the consensus lysine residue was found to retard Tat transport. These observations and others suggest that the sequence conservation of the Tat consensus motif is a reflection of the functional importance of the consensus residues. Tat signal peptides characteristically have positively charged carboxyl-terminal regions. However, changing the sign of this charge does not affect export of SufI.     

Novel phenotypes of Escherichia coli tat mutants revealed by global gene expression and phenotypic analysis

The Tat protein export system serves to export folded proteins harboring an N-terminal twin arginine signal peptide across the cytoplasmic membrane. In this study, we have used gene expression profiling of Escherichia coli supported by phenotypic analysis to investigate how cells respond to a defect in the Tat pathway. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are defective in the integrity of their cell envelope because of the mislocalization of two amidases involved in cell wall metabolism (Ize, B., Stanley, N. R., Buchanan, G., and Palmer, T. (2003) Mol. Microbiol. 48, 1183-1193). To distinguish between genes that are differentially expressed specifically because of the cell envelope defect and those that result from other effects of the tatC deletion, we also analyzed two different transposon mutants of the DeltatatC strain that have their outer membrane integrity restored. Approximately 50% of the genes that were differentially expressed in the tatC mutant are linked to the envelope defect, with the products of many of these genes involved in self-defense or protection mechanisms, including the production of exopolysaccharide. Among the changes that were not explicitly linked to envelope integrity, we characterized a role for the Tat system in iron acquisition and copper homeostasis. Finally, we have demonstrated that overproduction of the Tat substrate SufI saturates the Tat translocon and produces effects on global gene expression that are similar to those resulting from the DeltatatC mutation.     

TatD is a cytoplasmic protein with DNase activity. No requirement for TatD family proteins in sec-independent protein export

The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Genes known to be involved in this process include tatA, tatB, and tatC that form an operon with a fourth gene, tatD. The tatD gene product has two homologues in E. coli coded by the unlinked ycfH and yjjV genes. An E. coli strain with in-frame chromosomal deletions in all three of tatD, ycfH, and yjjV exhibits no significant defect in the cellular location of five cofactor-containing enzymes that are synthesized with twin arginine signal peptides. Neither these mutations nor overproduction of the TatD protein cause any discernible effect on the export kinetics of an additional E. coli Tat pathway substrate. It is concluded that proteins of the TatD family have no obligate involvement in protein export by the Tat system. TatD is shown to be a cytoplasmic protein. TatD binds to immobilized Ni(2+) or Zn(2+) affinity columns and exhibits magnesium-dependent DNase activity. Features of the tatA operon that may control TatD expression are discussed.     

A bacterial two-hybrid system based on the twin-arginine transporter pathway of E. coli

We have developed a bacterial two-hybrid system for the detection of interacting proteins that capitalizes on the folding quality control mechanism of the Twin Arginine Transporter (Tat) pathway. The Tat export pathway is responsible for the membrane translocation of folded proteins, including proteins consisting of more than one polypeptide, only one of which contains a signal peptide ("hitchhiker export"). Here, one protein (bait) is expressed as a fusion to a Tat signal peptide, whereas the second protein (prey) is fused to a protein reporter that can confer a phenotype only after export into the bacterial periplasmic space. Since the prey-reporter fusion lacks a signal peptide, it can only be exported as a complex with the bait-signal peptide fusion that is capable of targeting the Tat translocon. Using maltose-binding protein as a reporter, clones expressing interacting proteins can be grown on maltose minimal media or on MacConkey plates. In addition, we introduce the use of the cysteine disulfide oxidase DsbA as a reporter. Export of a signal peptide-prey:bait-DsbA complex into the periplasm allows complementation of dsbA(-) mutants and restores the formation of active alkaline phosphatase, which in turn can be detected by a chromogenic assay.     

Conserved signal peptide recognition systems across the prokaryotic domains

The twin-arginine translocation (Tat) pathway is a protein targeting system found in bacteria, archaea, and chloroplasts. Proteins are directed to the Tat translocase by N-terminal signal peptides containing SRRxFLK "twin-arginine" amino acid motifs. The key feature of the Tat system is its ability to transport fully folded proteins across ionically sealed membranes. For this reason the Tat pathway has evolved for the assembly of extracytoplasmic redox enzymes that must bind cofactors, and so fold, prior to export. It is important that only cofactor-loaded, folded precursors are presented for export, and cellular processes have been unearthed that regulate signal peptide activity. One mechanism, termed "Tat proofreading", involves specific signal peptide binding proteins or chaperones. The archetypal Tat proofreading chaperones belong to the TorD family, which are dedicated to the assembly of molybdenum-dependent redox enzymes in bacteria. Here, a gene cluster was identified in the archaeon Archaeoglobus fulgidus that is predicted to encode a putative molybdenum-dependent tetrathionate reductase. The gene cluster also encodes a TorD family chaperone (AF0160 or TtrD) and in this work TtrD is shown to bind specifically to the Tat signal peptide of the TtrA subunit of the tetrathionate reductase. In addition, the 3D crystal structure of TtrD is presented at 1.35 ? resolution and a nine-residue binding epitope for TtrD is identified within the TtrA signal peptide close to the twin-arginine targeting motif. This work suggests that archaea may employ a chaperone-dependent Tat proofreading system that is similar to that utilized by bacteria.     

Selective contribution of the twin-arginine translocation pathway to protein secretion in Bacillus subtilis

The availability of the complete genome sequence of Bacillus subtilis has allowed the prediction of all exported proteins of this Gram-positive eubacterium. Recently, approximately 180 secretory and 114 lipoprotein signal peptides were predicted to direct protein export from the cytoplasm. Whereas most exported proteins appear to use the Sec pathway, 69 of these proteins could potentially use the Tat pathway, as their signal peptides contain RR- or KR-motifs. In the present studies, proteomic techniques were applied to verify how many extracellular B. subtilis proteins follow the Tat pathway. Strikingly, the extracellular accumulation of 13 proteins with potential RR/KR-signal peptides was Tat-independent, showing that their RR/KR-motifs are not recognized by the Tat machinery. In fact, only the phosphodiesterase PhoD was shown to be secreted in a strictly Tat-dependent manner. Sodium azide-inhibition of SecA strongly affected the extracellular appearance of de novo synthesized proteins, including the lipase LipA and two other proteins with predicted RR/KR-signal peptides. The SecA-dependent export of pre-LipA is particularly remarkable, because its RR-signal peptide conforms well to stringent criteria for the prediction of Tat-dependent export in Escherichia coli. Taken together, our observations show that the Tat pathway makes a highly selective contribution to the extracellular proteome of B. subtilis.     

The Canonical Twin-Arginine Translocase Components Are Not Required for Secretion of Folded Green Fluorescent Protein from the Ancestral Strain of Bacillus subtilis

Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.     

Role of the Escherichia coli Tat pathway in outer membrane integrity

The Escherichia coli Tat system serves to export folded proteins harbouring an N-terminal twin-arginine signal peptide across the cytoplasmic membrane. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are highly defective in the integrity of their cell envelope. Here, we report the isolation, by transposon mutagenesis, of tat mutant strains that have their outer membrane integrity restored. This outer membrane repair of the tat mutant arises as a result of upregulation of the amiB gene, which encodes a cell wall amidase. Overexpression of the genes encoding the two additional amidases, amiA and amiC, does not compensate for the outer membrane defect of the tatC strain. Analysis of the amiA and amiC coding sequences indicates that the proteins may be synthesized with plausible twin-arginine signal sequences, and we demonstrate that they are translocated to the periplasm by the Tat pathway. A Tat+ strain that has mislocalized AmiA and AmiC proteins because of deletion of their signal peptides displays an identical defective cell envelope phenotype. The presence of genes encoding amidases with twin-arginine signal sequences in the genomes of other Gram-negative bacteria suggests that a similar cell envelope defect may be a common feature of tat mutant strains.     

Behaviour of topological marker proteins targeted to the Tat protein transport pathway

The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Formate dehydrogenase-N is a three-subunit membrane-bound enzyme, in which localization of the FdnG subunit to the membrane is Tat dependent. FdnG was found in the periplasmic fraction of a mutant lacking the membrane anchor subunit FdnI, confirming that FdnG is located at the periplasmic face of the cytoplasmic membrane. However, the phenotypes of gene fusions between fdnG and the subcellular reporter genes phoA (encoding alkaline phosphatase) or lacZ (encoding beta-galactosidase) were the opposite of those expected for analogous fusions targeted to the Sec translocase. PhoA fusion experiments have previously been used to argue that the peripheral membrane DmsAB subunits of the Tat-dependent enzyme dimethyl sulphoxide reductase are located at the cytoplasmic face of the inner membrane. Biochemical data are presented that instead show DmsAB to be at the periplasmic side of the membrane. The behaviour of reporter proteins targeted to the Tat system was analysed in more detail. These data suggest that the Tat and Sec pathways differ in their ability to transport heterologous passenger proteins. They also suggest that caution should be observed when using subcellular reporter fusions to determine the topological organization of Tat-dependent membrane protein complexes.     

Proteolytic processing of <Emphasis Type="Italic">Escherichia coli</Emphasis> twin-arginine signal peptides by LepB

The twin-arginine translocation (Tat) apparatus is a protein targeting system found in the cytoplasmic membranes of many prokaryotes. Substrate proteins of the Tat pathway are synthesised with signal peptides bearing SRRxFLK ‘twin-arginine’ amino acid motifs. All Tat signal peptides have a common tripartite structure comprising a polar N-terminal region, followed by a hydrophobic region of variable length and a polar C-terminal region. In Escherichia coli, Tat signal peptides are proteolytically cleaved after translocation. The signal peptide C-terminal regions contain conserved AxA motifs, which are possible recognition sequences for leader peptidase I (LepB). In this work, the role of LepB in Tat signal peptide processing was addressed directly. Deliberate repression of lepB expression prevented processing of all Tat substrates tested, including SufI, AmiC, and a TorA-23K reporter protein. In addition, electron microscopy revealed gross defects in cell architecture and membrane integrity following depletion of cellular LepB protein levels.     

Purified components of the Escherichia coli Tat protein transport system form a double-layered ring structure.

The Escherichia coli twin arginine translocation (Tat) system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. The genes tatA, tatB, tatC and tatE code for integral membrane proteins that are components of the Tat pathway. Cells co-overexpressing tatABCDE show an increased rate of export of a signal peptide-defective Tat precursor protein and a complex containing the TatA and TatB proteins can be purified from the membranes of such cells. The purified TatAB complex has an apparent molecular mass of 600 kDa as measured by gel permeation chromatography and, like the membranes of wild-type cells, contains a large molar excess of TatA over TatB. Negative stain electron microscopy of the complex reveals cylindrical structures that may correspond to the Tat protein transport channel.