Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.     

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.     

In vivo assessment of the Tat signal peptide specificity in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Tat- and Sec-targeting signal peptides are specific for the cognate Tat or Sec pathways. Using two reporter proteins, the specificity and convertibility of a Tat signal peptide were assessed in vivo. The specific substitutions by RK, KR and KK for the RR motif of the TorA signal peptide had no effect on the exclusive Tat-dependent export of colicin V (ColV). By introducing multiple substitutions in a typical Tat signal peptide, altered signal peptides lacking the twin-arginine motif were obtained. Interestingly, some of these signal peptides preserved Tat-pathway targeting capacity, but resulted in a loss of exclusivity. In addition, further increasing the hydrophobicity of the n-region without modifying the h-region converted the Tat signal peptides to Sec signal peptides in the ColV transport. Replacement of positively charged residues in the c-region also abolished the Tat-exclusive targeting of ColV or green fluorescent protein (GFP), but the folded GFP could be transported only through the Tat pathway. These results strongly suggest that the overall hydrophobicity of the n-region is one of the determinants of Tat-targeting exclusivity.     

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.     

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.     

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

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

Structural organization of the twin-arginine translocation system in Streptomyces lividans

The twin-arginine translocation (Tat) system exports folded proteins across bacterial cytoplasmic membranes. Recently, genes encoding TatA, TatB and TatC homologues were identified in Streptomyces lividans and the functionality of the Tat pathway was demonstrated. Here, we have examined the localization and structural organization of the Tat components in S. lividans. Interestingly, besides being membrane-associated proteins, S. lividans TatA and TatB were also detected in the cytoplasm. TatC could only be detected in isolated membrane fractions. Whereas all TatC was found to be stably inserted in the membrane, part of membrane-associated TatA and TatB could be extracted following high salt, sodium carbonate or urea treatment suggesting a more loose association with the membrane. Finally, we have analyzed Tat complexes that could be purified from an S. lividans TatABC overproducing strain. From the cytoplasmic membrane, two types of high molecular mass Tat complexes could be isolated having a similar composition as those isolated from Escherichia coli. In the cytoplasm, TatA and TatB were detected as monomer or as homo-oligomeric complexes.     

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.     

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

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

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

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

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.     

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.     

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

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

Functional analysis of the twin-arginine translocation pathway in Corynebacterium glutamicum ATCC 13869

Compared to those of other gram-positive bacteria, the genetic structure of the Corynebacterium glutamicum Tat system is unique in that it contains the tatE gene in addition to tatA, tatB, and tatC. The tatE homologue has been detected only in the genomes of gram-negative enterobacteria. To assess the function of the C. glutamicum Tat pathway, we cloned the tatA, tatB, tatC, and tatE genes from C. glutamicum ATCC 13869 and constructed mutants carrying deletions of each tat gene or of both the tatA and tatE genes. Using green fluorescent protein (GFP) fused with the twin-arginine signal peptide of the Escherichia coli TorA protein, we demonstrated that the minimal functional Tat system required TatA and TatC. TatA and TatE provide overlapping function. Unlike the TatB proteins from gram-negative bacteria, C. glutamicum TatB was dispensable for Tat function, although it was required for maximal efficiency of secretion. The signal peptide sequence of the isomaltodextranase (IMD) of Arthrobacter globiformis contains a twin-arginine motif. We showed that both IMD and GFP fused with the signal peptide of IMD were secreted via the C. glutamicum Tat pathway. These observations indicate that IMD is a bona fide Tat substrate and imply great potential of the C. glutamicum Tat system for industrial production of heterologous folded proteins.     

Characterisation of Tat protein transport complexes carrying inactivating mutations

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

利用荧光蛋白对大肠杆菌蛋白质Tat转运系统的研究

探讨了荧光蛋白作为报告蛋白用于蛋白质转运系统研究的可行性 ,结果表明海葵红色荧光蛋白聚集在细胞质内 ,不能转运至周质空间。而水母绿色荧光蛋白在Tat信号肽和Tat转运酶的共同作用下 ,以折叠形式转运至周质空间。通过荧光定量分析表明信号肽保守序列中的双精氨酸是保证绿色荧光蛋白转运及转运效率所必需的 ,且第二个精氨酸比第一个精氨酸更为重要。同时 ,揭示了Tat信号肽需要一定的高级结构才能行使功能 ;Tat信号肽不仅引导蛋白质的转运 ,而且也参与蛋白质的折叠。因此 ,绿色荧光蛋白是非常理想的报告蛋白 ,可用于研究Tat系统 ,但是海葵红色荧光蛋白易于聚集而不适合于此目的。     

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

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

TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli

In Escherichia coli, a subset of periplasmic proteins is exported via the twin-arginine translocation (Tat) pathway. In the present study, we have purified the Tat complex from E. coli, and we show that it contains only TatA, TatB, and TatC. Within the purified complex, TatB and TatC are present in a strict 1:1 ratio, suggesting a functional association. This has been confirmed by expression of a translational fusion between TatB and TatC. This Tat(BC) chimera supports efficient Tat-dependent export, indicating that TatB and TatC act as a unit in both structural and functional terms. The purified Tat complex contains varying levels of TatA, suggesting a gradual loss during isolation and a looser association. The molecular mass of the complex is approximately 600 kDa, demonstrating the presence of multiple copies of TatA, B, and C. Co-immunoprecipitation experiments show that TatC is required for the interaction of TatA with TatB, suggesting that TatA may interact with the complex via binding to TatC.     

Salt sensitivity of minimal twin arginine translocases

Bacterial twin arginine translocation (Tat) pathways have evolved to facilitate transport of folded proteins across membranes. Gram-negative bacteria contain a TatABC translocase composed of three subunits named TatA, TatB, and TatC. In contrast, the Tat translocases of most Gram-positive bacteria consist of only TatA and TatC subunits. In these minimal TatAC translocases, a bifunctional TatA subunit fulfils the roles of both TatA and TatB. Here we have probed the importance of conserved residues in the bifunctional TatAy subunit of Bacillus subtilis by site-specific mutagenesis. A set of engineered TatAy proteins with mutations in the cytoplasmic hinge and amphipathic helix regions were found to be inactive in protein translocation under standard growth conditions for B. subtilis or when heterologously expressed in Escherichia coli. Nevertheless, these mutated TatAy proteins did assemble into TatAy and TatAyCy complexes, and they facilitated membrane association of twin arginine precursor proteins in E. coli. Interestingly, most of the mutated TatAyCy translocases were salt-sensitive in B. subtilis. Similarly, the TatAC translocases of Bacillus cereus and Staphylococcus aureus were salt-sensitive when expressed in B. subtilis. Taken together, our present observations imply that salt-sensitive electrostatic interactions have critical roles in the preprotein translocation activity of certain TatAC type translocases from Gram-positive bacteria.     

Solution structure of the TatB component of the twin-arginine translocation system

The twin-arginine protein transport (Tat) system translocates fully folded proteins across lipid membranes. In Escherichia coli, the Tat system comprises three essential components: TatA, TatB and TatC. The protein translocation process is proposed to initiate by signal peptide recognition and substrate binding to the TatBC complex. Upon formation of the TatBC–substrate protein complex, the TatA subunits are recruited and form the protein translocation pore. Experimental evidences suggest that TatB forms a tight complex with TatC at 1:1 molar ratio and the TatBC complex contains multiple copies of both proteins. Cross-linking experiments demonstrate that TatB functions in tetrameric units and interacts with both TatC and substrate proteins. However, structural information of the TatB protein is still lacking, and its functional mechanism remains elusive. Herein, we report the solution structure of TatB in DPC micelles determined by Nuclear Magnetic Resonance (NMR) spectroscopy. Overall, the structure shows an extended ‘L-shape’ conformation comprising four helices: a transmembrane helix (TMH) α1, an amphipathic helix (APH) α2, and two solvent exposed helices α3 and α4. The packing of TMH and APH is relatively rigid, whereas helices α3 and α4 display notably higher mobility. The observed floppiness of helices α3 and α4 allows TatB to sample a large conformational space, thus providing high structural plasticity to interact with substrate proteins of different sizes and shapes.