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 core TatABC complex of the twin-arginine translocase in Escherichia coli: TatC drives assembly whereas TatA is essential for stability

Current models for the action of the twin-arginine translocation (Tat) system propose that substrates bind initially to the TatBC subunits, after which a separate TatA complex is recruited to form an active translocon. Here, we have studied the roles of individual subunits in the assembly and stability of the core TatBC-containing substrate-binding complex. Previous studies have shown that TatB and TatC are active when fused together; we show here that deletion of the entire TatB transmembrane span from this Tat(BC) fusion inactivates the Tat system but does not affect assembly of the core complex. In this mutated complex, TatA is present but more loosely bound, indicating a role for TatB in the correct binding of TatA. In the absence of TatA, the truncated TatBC fusion protein still assembles into a complex of the correct magnitude, demonstrating that the transmembrane spans of TatC are the only determinants within the membrane bilayer that specify assembly of this complex. Further studies on both the Tat(BC) construct and the wild-type TatBC subunits show that the TatBC complex is unstable in the absence of TatA, and we show that TatA stabilises the TatB subunit specifically within this complex. The results demonstrate a dual role and location for TatA: in the functioning/maintenance of the core complex, and as a separate homo-oligomeric complex.     

The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC complex, spectrum of modular TatA complexes and minor TatAB complex

The Tat system transports folded proteins across bacterial plasma and plant thylakoid membranes. To date, three key Tat subunits have been identified and mechanistic studies indicate the presence of two types of complex: a TatBC-containing substrate-binding unit and a separate TatA complex. Here, we used blue-native gel electrophoresis and affinity purification to study the nature of these complexes in Escherichia coli. Analysis of solubilized membrane shows that the bulk of TatB and essentially all of the TatC is found in a single 370kDa TatABC complex. TatABC was purified to homogeneity using an affinity tag on TatC and this complex runs apparently as an identical band. We conclude that this is the primary core complex, predicted to contain six or seven copies of TatBC together with a similar number of TatA subunits. However, the data indicate the presence of an additional form of Tat complex containing TatA and TatB, but not TatC; we speculate that this may be an assembly or disassembly intermediate of the translocator. The vast majority of TatA is found in separate complexes that migrate in blue-native gels as a striking ladder of bands with sizes ranging from under 100 kDa to over 500 kDa. Further analysis shows that the bands differ by an average of 34 kDa, indicating that TatA complexes are built largely, but possibly not exclusively, from modules of three or four TatA molecules. The range and nature of these complexes are similar in a TatC mutant that is totally inactive, indicating that the ladder of bands does not stem from ongoing translocation activity, and we show that purified TatA can self-assemble in vitro to form similar complexes. This spectrum of TatA complexes may provide the flexibility required to generate a translocon capable of transporting substrates of varying sizes across the plasma membrane in a folded state.     

Comparing system-specific chaperone interactions with their Tat dependent redox enzyme substrates

Redox enzyme substrates of the twin-arginine translocation (Tat) system contain a RR-motif in their leader peptide and require the assistance of chaperones, redox enzyme maturation proteins (REMPs). Here various regions of the RR-containing oxidoreductase subunit (leader peptide, full preprotein with and without a leader cleavage site, mature protein) were assayed for interaction with their REMPs. All REMPs bound their preprotein substrates independent of the cleavage site. Some showed binding to either the leader or mature region, whereas in one case only the preprotein bound its REMP. The absence of Tat also influenced the amount of chaperone-substrate interaction.

Structured summary

MINT-8047497: FdhE (uniprotkb:P13024) and FdoG (uniprotkb:P32176) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046441: HybO (uniprotkb:P69741) and HybE (uniprotkb:P0AAN1) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046375: DmsA (uniprotkb:P18775) and DmsD (uniprotkb:P69853) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046425: TorA (uniprotkb:P33225) and TorD (uniprotkb:P36662) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046393: NarJ (uniprotkb:P0AF26) and NarG (uniprotkb:P09152) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046409: NapD (uniprotkb:P0A9I5) and NapA (uniprotkb:P33937) physically interact (MI:0915) by two hybrid (MI:0018)     

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.     

Contributions of the Transmembrane Domain and a Key Acidic Motif to Assembly and Function of the TatA Complex

The twin-arginine translocase (Tat) pathway transports folded proteins across bacterial and thylakoid membranes. In Escherichia coli, a membrane-bound TatA complex, which oligomerizes to form complexes of less than 100 to more than 500 kDa, is considered essential for translocation. We have studied the contributions of various TatA domains to the assembly and function of this heterogeneous TatA complex. The TOXCAT assay was used to analyze the potential contribution of the TatA transmembrane (TM) domain. We observed relatively weak interactions between TatA TM domains, suggesting that the TM domain is not the sole driving force behind oligomerization. A potential hydrogen-bonding role for a TM domain glutamine was also investigated, and it was found that mutation blocks transport at low expression levels, while assembly is unaffected at higher expression levels. Analysis of truncated TatA proteins instead highlighted an acidic motif directly following the TatA amphipathic helix. Mutating these negatively charged residues to apolar uncharged residues completely blocks activity, even at high levels of TatA, and appears to disrupt ordered complex formation.     

Pathfinders and trailblazers: a prokaryotic targeting system for transport of folded proteins

The twin-arginine (Tat) protein translocase is a highly unusual protein transport machine that is dedicated to the movement of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat pathway by means of N-terminal signal peptides harbouring a distinctive twin-arginine motif. In this minireview, we describe our current knowledge of the Tat system, paying particular attention to the function of the TatA protein and to the often overlooked step of signal peptide cleavage.     

The tatC gene cluster is essential for viability in halophilic archaea

In prokaryotes the twin-arginine translocase (Tat) is a unique transport system for the export of folded proteins. The Tat pathway is usually involved in the export of a small proportion of extracytoplasmic proteins. An exception is found in halophilic archaea, in which the majority of secretory proteins have been predicted to be Tat-dependent. All haloarchaea analysed to date contain two genes encoding homologues of the Tat-component TatC. In all of these cases both genes are located adjacently on the chromosome, indicating that they form a functional unit. We show that this gene cluster is essential for viability in haloarchaea, which is in complete contrast to all other prokaryotes that have been tested thus far.     

Evolution of mitochondrial protein biogenesis

Mitochondria and the nucleus are key features that distinguish eukaryotic cells from prokaryotic cells. Mitochondria originated from a bacterium that was endosymbiotically taken up by another cell more than a billion years ago. Subsequently, most mitochondrial genes were transferred and integrated into the host cell's genome, making the evolution of pathways for specific import of mitochondrial proteins necessary. The mitochondrial protein translocation machineries are composed of numerous subunits. Interestingly, many of these subunits are at least in part derived from bacterial proteins, although only few of them functioned in bacterial protein translocation. We propose that the primitive α-proteobacterium, which was once taken up by the eukaryote ancestor cell, contained a number of components that were utilized for the generation of mitochondrial import machineries. Many bacterial components of seemingly unrelated pathways were integrated to form the modern cooperative mitochondria-specific protein translocation system.