An essential role for the DnaK molecular chaperone in stabilizing over-expressed substrate proteins of the bacterial twin-arginine translocation pathway

All secreted proteins in Escherichia coli must be maintained in an export-competent state before translocation across the inner membrane. In the case of the Sec pathway, this function is carried out by the dedicated SecB chaperone and the general chaperones DnaK-DnaJ-GrpE and GroEL-GroES, whose job collectively is to render substrate proteins partially or entirely unfolded before engagement of the translocon. To determine whether these or other general molecular chaperones are similarly involved in the translocation of folded proteins through the twin-arginine translocation (Tat) system, we screened a collection of E. coli mutant strains for their ability to transport a green fluorescent protein (GFP) reporter through the Tat pathway. We found that the molecular chaperone DnaK was essential for cytoplasmic stability of GFP bearing an N-terminal Tat signal peptide, as well as for numerous other recombinantly expressed endogenous and heterologous Tat substrates. Interestingly, the stability conferred by DnaK did not require a fully functional Tat signal as substrates bearing translocation defective twin lysine substitutions in the consensus Tat motif were equally unstable in the absence of DnaK. These findings were corroborated by crosslinking experiments that revealed an in vivo association between DnaK and a truncated version of the Tat substrate trimethylamine N-oxide reductase (TorA502) bearing an RR or a KK signal peptide. Since TorA502 lacks nine molybdo-cofactor ligands essential for cofactor attachment, the involvement of DnaK is apparently independent of cofactor acquisition. Finally, we show that the stabilizing effects of DnaK can be exploited to increase the expression and translocation of Tat substrates under conditions where the substrate production level exceeds the capacity of the Tat translocase. This latter observation is expected to have important consequences for the use of the Tat system in biotechnology applications where high levels of periplasmic expression are desirable.     

Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane

Nascent polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytosolic, integral membrane or exported proteins. Extensive genetic and biochemical analyses have significantly expanded our knowledge of chaperone tasking throughout this process. In bacteria, it is known that the folding of newly-synthesized cytosolic proteins is mainly orchestrated by three highly conserved molecular chaperones, namely Trigger Factor (TF), DnaK (HSP70) and GroEL (HSP60). Yet, it has been reported that these major chaperones are strongly involved in protein translocation pathways as well. This review describes such essential molecular chaperone functions, with emphasis on both the biogenesis of inner membrane proteins and the post-translational targeting of presecretory proteins to the Sec and the twin-arginine translocation (Tat) pathways. Critical interplay between TF, DnaK, GroEL and other molecular chaperones and targeting factors, including SecB, SecA, the signal recognition particle (SRP) and the redox enzyme maturation proteins (REMPs) is also discussed. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

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.     

Large-scale translocation reversal within the thylakoid Tat system in vivo

In vitro import assays have shown that the thylakoid twin-arginine translocase (Tat) system transports folded proteins in a unidirectional manner. Here, we expressed a natural substrate, pre-23K, and a 23K presequence-green fluorescent protein (GFP) chimera in vivo in tobacco protoplasts. Both are imported into chloroplasts, targeted to the thylakoids, and processed to the mature size by the lumen-facing processing peptidase. However, the vast majority of mature GFP and about half of the 23K are then returned to the stroma. Mutations in the twin-arginine motif block thylakoid targeting and maturation, confirming an involvement of the Tat apparatus. Mutation of the processing site yields membrane-associated intermediate-size protein in vivo, indicating a delayed reversal of translocation to the stroma and suggesting a longer lived interaction with the Tat machinery. We conclude that, in vivo, the Tat system can reject substrates at a late stage in translocation and on a very large scale, indicating the influence of factors that are absent in reconstitution assays.     

Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway

Overexpression of either heterologous or homologous proteins that are routed to the periplasm via the twin-arginine translocation (Tat) pathway results in a block of export and concomitant accumulation of the respective protein precursor in the cytoplasm. Screening of a plasmid-encoded genomic library for mutants that confer enhanced export of a TorA signal sequence (ssTorA)-GFP-SsrA fusion protein, and thus result in higher cell fluorescence, yielded the pspA gene encoding phage shock protein A. Coexpression of pspA relieved the secretion block observed with ssTorA-GFP-SsrA or upon overexpression of the native Tat proteins SufI and CueO. A similar effect was observed with the Synechocystis sp. strain PCC6803 PspA homologue, VIPP1, indicating that the role of PspA in Tat export may be phylogenetically conserved. Mutations in Tat components that completely abolish export result in a marked induction of PspA protein synthesis, consistent with its proposed role in enhancing protein translocation via Tat.     

Membrane targeting of a folded and cofactor-containing protein.

Targeting of proteins to and translocation across the membranes is a fundamental biological process in all organisms. In bacteria, the twin arginine translocation (Tat) system can transport folded proteins. Here, we demonstrate in vivo that the high potential iron-sulfur protein (HiPIP) from Allochromatium vinosum is translocated into the periplasmic space by the Tat system of Escherichia coli. In vitro, reconstituted HiPIP precursor (preHoloHiPIP) was targeted to inverted membrane vesicles from E. coli by a process requiring ATP when the Tat substrate was properly folded. During membrane targeting, the protein retained its cofactor, indicating that it was targeted in a folded state. Membrane targeting did not require a twin arginine motif and known Tat system components. On the basis of these findings, we propose that a pathway exists for the insertion of folded cofactor-containing proteins such as HiPIP into the bacterial cytoplasmic membrane.     

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 system proofreads FeS protein substrates and directly initiates the disposal of rejected molecules

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane, including FeS proteins that receive their cofactors in the cytoplasm. We have studied two Escherichia coli Tat substrates, NrfC and NapG, to examine how, or whether, the system exports only correctly folded and assembled FeS proteins. With NrfC, substitutions in even one of four predicted FeS centres completely block export, indicating an effective proofreading activity. The FeS mutants are rapidly degraded but only if they interact with the Tat translocon; they are stable in a tat deletion strain and equally stable in wild-type cells if the signal peptide twin-arginine motif is removed to block targeting. Basically similar results are obtained with NapG. The Tat apparatus thus proofreads these substrates and directly initiates the turnover of rejected molecules. Turnover of mutated FeS substrates is completely dependent on the TatA/E subunits that are believed to be involved in the late stages of translocation, and we propose that partial translocation triggers substrate turnover within an integrated quality control system for FeS proteins.     

Separate analysis of twin-arginine translocation (Tat)-specific membrane binding and translocation in Escherichia coli

The twin-arginine translocation (Tat) pathway exports those precursor proteins to the periplasmic space of bacteria that harbor a twin-arginine (RR) consensus motif in their signal sequences. We have reproduced translocation of several Tat substrates into inside-out plasma membrane vesicles from Escherichia coli. Translocation proceeding at an efficiency of up to 20% occurs specifically via the Tat pathway as indicated by (i) its requirement for elevated levels of the TatABC proteins in the membrane vesicles, (ii) competition by an intact twin-arginine signal peptide, and (iii) susceptibility toward dissipation of the transmembrane H(+) gradient. The latter treatment, while blocking translocation, still allows for functional membrane association of Tat precursors. This is shown by the finding that translocation of isolated membrane-bound Tat precursor is restored upon re-energization of the vesicles.     

DmsD,a Tat system specific chaperone,interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis

Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin-arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential interacting partners of DmsD by using several in vitro protein–protein interaction screening approaches, including affinity chromatography, co-precipitation, and cross-linking. Candidate hits from these in vitro findings were analyzed by in vivo methods of bacterial two-hybrid (BACTH) and bimolecular fluorescence complementation (BiFC). From these data, DmsD was confirmed to interact with the general molecular chaperones DnaK, DnaJ, GrpE, GroEL, Tig and Ef-Tu. In addition, DmsD was also found to interact with proteins involved in the molybdenum cofactor biosynthesis pathway. Our data suggests that DmsD may play a role as a “node” in escorting its substrate through a cascade of chaperone assisted protein-folding maturation events.     

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.     

Sequence-specific binding of prePhoD to soluble TatAd indicates protein-mediated targeting of the Tat export in Bacillus subtilis

The Tat (twin-arginine protein translocation) system initially discovered in the thylakoid membrane of chloroplasts has been described recently for a variety of eubacterial organisms. Although in Escherichia coli four Tat proteins with calculated membrane spanning domains have been demonstrated to mediate Tat-dependent transport, a specific transport system for twin-arginine signal peptide containing phosphodiesterase PhoD of Bacillus subtilis consists of one TatA/TatC (TatAd/TatCd) pair of proteins. Here, we show that TatAd was found beside its membrane-integrated localization in the cytosol were it interacted with prePhoD. prePhoD was efficiently co-immunoprecipitated by TatAd. Inefficient co-immunoprecipitation of mature PhoD and missing interaction to Sec-dependent and cytosolic peptides by TatAd demonstrated a particular role of the twin-arginine signal peptide for this interaction. Affinity of prePhoD to TatAd was interfered by peptides containing the twin-arginine motif but remained active when the arginine residues were substituted. The selective binding of TatAd to peptides derived from the signal peptide of PhoD elucidated the function of the twin-arginine motif as a target site for pre-protein TatAd interaction. Substitution of the binding motif demonstrated the pivotal role of basic amino acid residues for TatA binding. These features suggest that TatA interacts prior to membrane integration with its pre-protein substrate and could therefore assist targeting of twin-arginine pre-proteins.     

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.     

Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels

The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Substrates are targeted to the Tat pathway by signal peptides containing a pair of consecutive arginine residues. The membrane proteins TatA, TatB and TatC are the essential components of this pathway in Escherichia coli. The complexes that these proteins form at native levels of expression have been investigated by the use of affinity tag-coding sequences fused to chromosomal tat genes. Distinct TatA and TatBC complexes were identified using size-exclusion chromatography and shown to have apparent molecular masses of approximately 700 and 500 kDa, respectively. Following in vivo expression, the Tat substrate protein SufI was found to copurify with the TatBC, but not the TatA, complex. This binding required the SufI signal peptide. Substitution of the twin-arginine residues in the SufI signal peptide by either twin lysine or twin alanine residues abolished export. However, both variant SufI proteins still copurified with the TatBC complex. These data show that the twin-arginine residues of the Tat consensus motif are not essential for binding of precursor to the TatBC complex but are required for the successful entry of the precursor into the transport cycle. The effect on substrate binding of single amino acid substitutions in TatC that affect Tat transport were studied using TatC variants Phe94Ala, Glu103Ala, Glu103Arg and Asp211Ala. Only variant Glu103Arg showed reduced copurification of SufI with TatBC. The transport defects associated with the other TatC variants do not, therefore, arise from an inability to bind substrate proteins.     

TatBC-Independent TatA/Tat Substrate Interactions Contribute to Transport Efficiency

The Tat system can transport folded, signal peptide-containing proteins (Tat substrates) across energized membranes of prokaryotes and plant plastids. A twin-arginine motif in the signal peptide of Tat substrates is recognized by TatC-containing complexes, and TatA permits the membrane passage. Often, as in the model Tat systems of Escherichia coli and plant plastids, a third component – TatB – is involved that resembles TatA but has a higher affinity to TatC. It is not known why most TatA dissociates from TatBC complexes in vivo and distributes more evenly in the membrane. Here we show a TatBC-independent substrate-binding to TatA from Escherichia coli, and we provide evidence that this binding enhances Tat transport. First hints came from in vivo cross-linking data, which could be confirmed by affinity co-purification of TatA with the natural Tat substrates HiPIP and NrfC. Two positions on the surface of HiPIP could be identified that are important for the TatA interaction and transport efficiency, indicating physiological relevance of the interaction. Distributed TatA thus may serve to accompany membrane-interacting Tat substrates to the few TatBC spots in the cells.     

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.     

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 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 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.     

Novel GFP expression using a short N-terminal polypeptide through the defined twin-arginine translocation (Tat) pathway

Escherichia coli is frequently used as a convenient host organism for soluble recombinant protein expression. However, additional strategies are needed for proteins with complex folding characteristics. Here, we suggested that the acidic, neutral, and alkaline isoelectric point (pI) range curves correspond to the channels of the E. coli type-II cytoplasmic membrane translocation (periplasmic translocation) pathways of twin-arginine translocation (Tat), Yid, and general secretory pathway (Sec), respectively, for unfolded and folded target proteins by examining the characteristic pI values of the N-termini of the signal sequences or the leader sequences, matching with the known diameter of the translocation channels, and analyzing the N-terminal pI value of the signal sequences of the Tat substrates. To confirm these proposed translocation pathways, we investigated the soluble expression of the folded green fluorescent protein (GFP) with short N-terminal polypeptides exhibiting pI and hydrophilicity separately or collectively. This, in turn, revealed the existence of an anchor function with a specific directionality based on the N-terminal pI value (termed as N-terminal pI-specific directionality) and distinguished the presence of the E. coli type-II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for the unfolded and folded target proteins. We concluded that the pI value and hydrophilicity of the short N-terminal polypeptide, and the total translational efficiency of the target proteins based on the ΔGRNA value of the N-terminal coding regions are important factors for promoting more efficient translocation (secretion) through the largest diameter of the Tat channel. These results show that the short N-terminal polypeptide could substitute for the Tat signal sequence with improved efficiency.