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.     

Mapping the twin‐arginine protein translocation network of Bacillus subtilis

Bacteria employ twin‐arginine translocation (Tat) pathways for the transport of folded proteins to extracytoplasmic destinations. In recent years, most studies on bacterial Tat pathways addressed the membrane‐bound TatA(B)C subunits of the Tat translocase, and the specific interactions between this translocase and its substrate proteins. In contrast, relatively few studies investigated possible coactors in the TatA(B)C‐dependent protein translocation process. The present studies were aimed at identifying interaction partners of the Tat pathway of Bacillus subtilis, which is a paradigm for studies on protein secretion by Gram‐positive bacteria. Specifically, 36 interaction partners of the TatA and TatC subunits were identified by rigorous application of the yeast two‐hybrid (Y2H) approach. Our Y2H analyses revealed that the three TatA isoforms of B. subtilis can form homo‐ and heterodimers. Subsequently, the secretion of the Tat substrates YwbN and PhoD was tested in mutant strains lacking genes for the TatAC interaction partners identified in our genome‐wide Y2H screens. Our results show that the cell wall‐bound protease WprA is important for YwbN secretion, and that the HemAT and CsbC proteins are required for PhoD secretion under phosphate starvation conditions. Taken together, our findings imply that the Bacillus Tat pathway is embedded in an intricate protein–protein interaction network.     

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.     

Substrate-Dependent Assembly of the Tat Translocase as Observed in Live Escherichia coli Cells

The twin-arginine translocation (Tat) pathway guides fully folded proteins across membranes of bacteria, archaea and plant chloroplasts. In Escherichia coli, Tat-specific transport is executed in a still largely unknown manner by three functionally diverse membrane proteins, termed TatA, TatB, and TatC. In order to follow the intracellular distribution of the TatABC proteins in live E. coli cells, we have individually expressed fluorophore-tagged versions of each Tat protein in addition to a set of chromosomally encoded TatABC proteins. In this way, a Tat translocase could form from the native TatABC proteins and be visualized via the association of a fluorescent Tat variant. A functionally active TatA-green fluorescent protein fusion was found to re-locate from a uniform distribution in the membrane into a few clusters preferentially located at the cell poles. Clustering was absolutely dependent on the co-expression of functional Tat substrates, the proton-motive force, and the cognate TatBC subunits. Likewise, polar cluster formation of a functional TatB-mCherry fusion required TatA and TatC and that of a functional TatC-mCherry fusion a functional Tat substrate. Furthermore we directly demonstrate the co-localization of TatA and TatB in the same fluorescent clusters. Our collective results are consistent with distinct Tat translocation sites dynamically forming in vivo in response to newly synthesized Tat substrates.     

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.     

A TatABC-Type Tat Translocase Is Required for Unimpaired Aerobic Growth of Corynebacterium glutamicum ATCC13032

The twin-arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Escherichia coli and other Gram-negative bacteria possess a TatABC-type Tat translocase in which each of the three inner membrane proteins TatA, TatB, and TatC performs a mechanistically distinct function. In contrast, low-GC Gram-positive bacteria, such as Bacillus subtilis, use a TatAC-type minimal Tat translocase in which the TatB function is carried out by a bifunctional TatA. In high-GC Gram-positive Actinobacteria, such as Mycobacterium tuberculosis and Corynebacterium glutamicum, tatA, tatB, and tatC genes can be identified, suggesting that these organisms, just like E. coli, might use TatABC-type Tat translocases as well. However, since contrary to this view a previous study has suggested that C. glutamicum might in fact use a TatAC translocase with TatB only playing a minor role, we reexamined the requirement of TatB for Tat-dependent protein translocation in this microorganism. Under aerobic conditions, the misassembly of the Rieske iron-sulfur protein QcrA was identified as a major reason for the severe growth defect of Tat-defective C. glutamicum mutant strains. Furthermore, our results clearly show that TatB, besides TatA and TatC, is strictly required for unimpaired aerobic growth. In addition, TatB was also found to be essential for the secretion of a heterologous Tat-dependent model protein into the C. glutamicum culture supernatant. Together with our finding that expression of the C. glutamicum TatB in an E. coli ΔtatB mutant strain resulted in the formation of an active Tat translocase, our results clearly indicate that a TatABC translocase is used as the physiologically relevant functional unit for Tat-dependent protein translocation in C. glutamicum and, most likely, also in other TatB-containing Actinobacteria.     

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.     

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.     

Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways.     

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.     

Two minimal Tat translocases in Bacillus

Activity of the Tat machinery for protein transport across the inner membrane of Escherichia coli and the chloroplast thylakoidal membrane requires the presence of three membrane proteins: TatA, TatB and TatC. Here, we show that the Tat machinery of the Gram-positive bacterium Bacillus subtilis is very different because it contains at least two minimal Tat translocases, each composed of one specific TatA and one specific TatC component. A third, TatB-like component is apparently not required. This implies that TatA proteins of B. subtilis perform the functions of both TatA and TatB of E. coli and thylakoids. Notably, the two B. subtilis translocases named TatAdCd and TatAyCy both function as individual, substrate-specific translocases for the twin-arginine preproteins PhoD and YwbN, respectively. Importantly, these minimal TatAC translocases of B. subtilis are representative for the Tat machinery of the vast majority of Gram-positive bacteria, Streptomycetes being the only known exception with TatABC translocases.     

The Tat system of Gram-positive bacteria

The twin-arginine protein translocation (Tat) system has a unique ability to translocate folded and co-factor-containing proteins across lipid bilayers. The Tat pathway is present in bacteria, archaea and in the thylakoid membranes of chloroplasts and, depending on the organism and environmental conditions, it can be deemed important for cell survival, virulence or bioproduction. This review provides an overview of the current understanding of the Tat system with specific focus on Gram-positive bacteria. The ‘universal minimal Tat system’ is composed of a TatA and a TatC protein. However, this pathway is more commonly composed of two TatA-like proteins and one TatC protein. Often the TatA-like proteins have diverged to have two different functions and, in this case, the second TatA-like protein is usually referred to as TatB. The correct folding and/or incorporation of co-factors are requirements for translocation, and the known quality control mechanisms are examined in this review. A number of examples of crosstalk between the Tat system and other protein transport systems, such as the Sec–YidC translocon and signal peptidases or sheddases are also discussed. Further, an overview of specific Gram-positive bacterial Tat systems found in monoderm and diderm species is detailed. Altogether, this review highlights the unique features of Gram-positive bacterial Tat systems and pinpoints key questions that remain to be addressed in future research. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Subcellular Localization of TatAd of Bacillus subtilis Depends on the Presence of TatCd or TatCy

The gram-positive bacterium Bacillus subtilis contains two minimal Tat translocases, TatAdCd and TatAyCy, which are each involved in the secretion of one or more specific protein substrates. We have investigated the subcellular localization of the TatA components by employing C-terminal green fluorescent protein (GFP) fusions and fluorescence microscopy. When expressed from a xylose-inducible promoter, the TatA-GFP fusion proteins displayed a dual localization pattern, being localized peripherally and showing bright foci which are predominantly located at the division sites and/or poles of the cells. Importantly, the localization of TatAd-GFP was similar when the protein was expressed from its own promoter under phosphate starvation conditions, indicating that these foci are not the result of artificial overexpression. Moreover, the TatAd-GFP fusion protein was shown to be functional in the translocation of its substrate PhoD, provided that TatCd is also present. Furthermore, we demonstrate that the localization of TatAd-GFP in foci depends on the presence of the TatCd component. Remarkably, however, the TatAd-GFP foci can also be observed in the presence of TatCy, indicating that TatAd can interact not only with TatCd but also with TatCy. These results suggest that the formation of TatAd complexes in B. subtilis is controlled by TatC.The bacterial twin-arginine translocation (Tat) machinery is able to transport folded proteins across the cytoplasmic membrane (26). Preproteins translocated by the Tat pathway are characterized by a twin-arginine (RR) motif in their signal sequences.In Escherichia coli, the Tat system consists of three components, the TatA, TatB, and TatC proteins. In the currently favored model for its mode of action, a TatB-TatC complex is involved in initial RR signal peptide recognition and binding of precursor proteins. Multiple TatA subunits then associate with this complex to form a protein-conducting channel (1). TatA, which is homologous to TatB, can be found complexed to TatBC but also forms a wide range of large, homooligomeric complexes (7, 23). In a few cases, the TatB protein can be functionally replaced by the TatA protein, indicating that TatA and TatC are able to form an active, minimal translocase (6, 10).Most gram-positive bacteria contain only two types of Tat subunit, a TatC protein and a TatA protein which has characteristics and the ability to perform the function of both TatA and TatB of E. coli (2, 13). Bacillus subtilis contains two substrate-specific Tat systems: a TatAyCy translocase that is required for translocation of the iron-dependent DyP peroxidase YwbN and a TatAdCd translocase which translocates the phosphodiesterase PhoD (12). In addition, B. subtilis contains a third TatA component, designated TatAc. This protein is dispensable for Tat-dependent translocation of YwbN or PhoD, and its function is currently unknown.TatAd is the most-studied TatA component of B. subtilis, and like TatA of E. coli, it is able to form both homooligomeric complexes and complexes with TatCd (2, 31). Despite the fact that it contains an N-terminal transmembrane segment (17), TatAd was also found in the cytosol, where it appears to interact with its substrate, pre-PhoD, via the signal sequence (24). TatCd was proposed to act as a receptor for the anchoring at and subsequent incorporation into the membrane of this TatAd-PhoD complex (28).The subcellular localization of Tat components in E. coli has been extensively investigated by fluorescence microscopy. Green fluorescent protein (GFP) fusions of TatA were localized at the periphery of the cells, but punctate regions of fluorescence were also reported (4, 25). In these studies, TatB was localized all over the membrane, with some accumulation at the cell poles. TatC was mainly distributed evenly throughout the periphery of the cells, with some small punctate regions. Recently, the oligomeric state of TatA-yellow fluorescent protein (YFP) in living E. coli cells was determined by single-molecule imaging (18). TatA complexes with a broad range of stoichiometries were observed as fluorescent foci, and TatA was also present in a dispersed state in the membrane.For B. subtilis, the subcellular localization of only one Tat component has been reported so far. Both N- and C-terminal fusions of GFP to TatCy were shown to be localized throughout the membrane, with frequent foci at the cell poles and division septa, and this localization pattern was classified as “polar” (20).In this study, we have investigated the subcellular localization of the three TatA proteins of B. subtilis by using GFP fusions, functionality assessments, and fluorescence microscopy. TatAc and TatAd showed a dual localization pattern, with fluorescence in the membrane as well as in foci which were enriched at the cell poles. Notably, the localization of TatAd-GFP in foci was shown to depend on the presence of a TatC component, suggesting that TatC drives complex formation by TatAd.     

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 Basis for TatA Oligomerization: An NMR Study of Escherichia coli TatA Dimeric Structure

Many proteins are transported across lipid membranes by protein translocation systems in living cells. The twin-arginine transport (Tat) system identified in bacteria and plant chloroplasts is a unique system that transports proteins across membranes in their fully-folded states. Up to date, the detailed molecular mechanism of this process remains largely unclear. The Escherichia coli Tat system consists of three essential transmembrane proteins: TatA, TatB and TatC. Among them, TatB and TatC form a tight complex and function in substrate recognition. The major component TatA contains a single transmembrane helix followed by an amphipathic helix, and is suggested to form the translocation pore via self-oligomerization. Since the TatA oligomer has to accommodate substrate proteins of various sizes and shapes, the process of its assembly stands essential for understanding the translocation mechanism. A structure model of TatA oligomer was recently proposed based on NMR and EPR observations, revealing contacts between the transmembrane helices from adjacent subunits. Herein we report the construction and stabilization of a dimeric TatA, as well as the structure determination by solution NMR spectroscopy. In addition to more extensive inter-subunit contacts between the transmembrane helices, we were also able to observe interactions between neighbouring amphipathic helices. The side-by-side packing of the amphipathic helices extends the solvent-exposed hydrophilic surface of the protein, which might be favourable for interactions with substrate proteins. The dimeric TatA structure offers more detailed information of TatA oligomeric interface and provides new insights on Tat translocation mechanism.     

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.     

Affinity of TatCd for TatAd elucidates its receptor function in the Bacillus subtilis twin arginine translocation (Tat) translocase system

Twin arginine translocation (Tat) systems catalyze the transport of folded proteins across the bacterial cytosolic membrane or the chloroplast thylakoid membrane. In the Tat systems of Escherichia coli and many other species TatA-, TatB-, and TatC-like proteins have been identified as essential translocase components. In contrast, the Bacillus subtilis phosphodiesterase PhoD-specific system consists only of a pair of TatA(d)/TatC(d) proteins and involves a TatA(d) protein engaged in a cytosolic and a membrane-embedded localization. Because soluble TatA(d) was able to bind the twin arginine signal peptide of prePhoD prior to membrane integration it could serve to recruit its substrate to the membrane via the interaction with TatC(d). By analyzing the distribution of TatA(d) and studying the mutual affinity with TatC(d) we have shown here that TatC(d) assists the membrane localization of TatA(d). Besides detergent-solubilized TatC(d), membrane-integrated TatC(d) showed affinity for soluble TatA(d). By using a peptide library-specific binding of TatA(d) to cytosolic loops of membrane protein TatC(d) was demonstrated. Depletion of TatC(d) in B. subtilis resulted in a drastic reduction of TatA(d), indicating a stabilizing effect of TatC(d) for TatA(d). In addition, the presence of the substrate prePhoD was the prerequisite for appropriate localization in the cytosolic membrane of B. subtilis as demonstrated by freeze-fracture experiments.     

A mutation leading to super-assembly of twin-arginine translocase (Tat) protein complexes

The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report the analysis of the Bacillus subtilis TatAyCy system, which occurs as separate TatAyCy and TatAy complexes at steady state, using single-particle electron microscopy (EM) and advanced atomic force microscopy (AFM) approaches. We show that a P2A mutation in the TatAy subunit leads to apparent super-assembly of Tat complexes. Purification of TatCy-containing complexes leads to a large increase in the TatA:TatC ratio, suggesting that TatAy^P2A complexes may have attached to the TatAyCy complex. EM and AFM analyses show that the wild-type TatAyCy complex purifies as roughly spherical complexes of 9–16 nm diameter, whereas the P2A mutation leads to accumulation of large (up to 500 nm long) fibrils that are chains of numerous complexes. Time lapsed AFM imaging, recorded on fibrils under liquid, shows that they adopt a variety of tightly curved conformations, with radii of curvature of 10–12 nm comparable to the size of single TatAy^P2A complexes. The combined data indicate that the mutation leads to super-assembly of TatAy^P2A complexes and we propose that an individual TatAy^P2A complex assembles initially with a TatAy^P2ACy complex, after which further TatAy^P2A complexes attach to each other. The data further suggest that the N-terminal extracytoplasmic domain of TatAy plays an essential role in Tat complex interactions.     

The TatC component of the twin‐arginine protein translocase functions as an obligate oligomer

The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate‐bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild‐type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue‐native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild‐type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate‐induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.     

A stromal pool of TatA promotes Tat-dependent protein transport across the thylakoid membrane

In chloroplasts and bacteria, the Tat (twin-arginine translocation) system is engaged in transporting folded passenger proteins across the thylakoid and cytoplasmic membranes, respectively. To date, three membrane proteins (TatA, TatB, and TatC) have been identified to be essential for Tat-dependent protein translocation in the plant system, whereas soluble factors seem not to be required. In contrast, in the bacterial system, several cytosolic chaperones were described to be involved in Tat transport processes. Therefore, we have examined whether stromal or peripherally associated membrane proteins also play a role in Tat transport across the thylakoid membrane. Analyzing both authentic precursors as well as the chimeric 16/23 protein, which allows us to study each step of the translocation process individually, we demonstrate that a soluble form of TatA is present in the chloroplast stroma, which significantly improves the efficiency of Tat-dependent protein transport. Furthermore, this soluble TatA is able to reconstitute the Tat transport properties of thylakoid membranes that are transport-incompetent due to extraction with solutions of chaotropic salts.