Twin arginine translocation (Tat)-dependent export in the apparent absence of TatABC or TatA complexes using modified Escherichia coli TatA subunits that substitute for TatB.

The twin arginine translocation pathway exports folded proteins across the cytoplasmic membrane of many bacteria. In Escherichia coli and other Gram-negative bacteria, TatA, TatB, and TatC are all essential for efficient translocation, and current models suggest that separate TatABC and TatA complexes coalesce at the point of translocation. However, other microbes appear only to possess tatA and tatC genes.

The entire N-terminal half of TatC is involved in twin-arginine precursor binding.

Translocation of twin-arginine precursor proteins across the cytoplasmic membrane of Escherichia coli requires the three membrane proteins TatA, TatB, and TatC. TatC and TatB were shown to be involved in precursor binding. We have analyzed in vitro a number of single alanine substitutions in tatC that were previously shown to compromise in vivo the function of the Tat translocase.

Evidence for interactions between domains of TatA and TatB from mutagenesis of the TatABC subunits of the twin-arginine translocase.

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. Three subunits, TatA, B and C, are known to be involved but their modes of action are poorly understood, as are the inter-subunit interactions occurring within Tat complexes. We have generated mutations in the single transmembrane (TM) spans of TatA and TatB, with the aim of generating structural distortions.

Mutations in subunits of the Escherichia coli twin-arginine translocase block function via differing effects on translocation activity or tat complex structure.

We have used a combination of blue-native (BN) gel electrophoresis and protein purification to analyze the effects of TatA or TatC mutations on the structures of the primary TatABC and multimeric TatA complexes in Escherichia coli. Expression of wild-type TatABC leads to the production of a single major TatABC complex of 370 kDa and a heterogeneous set of TatA complexes of <100 kDa to approximately 500 kDa. Two TatC mutations that block translocation have different effects on complex structures.

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.

Quantitative export of a reporter protein, GFP, by the twin-arginine translocation pathway in Escherichia coli.

The Tat system mediates the transport of folded proteins across the bacterial cytoplasmic membrane. To study the properties of the Escherichia coli Tat-system, we used green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). In the presence of arabinose, low levels of this protein rapidly saturate the translocase and cause the accumulation of inactive, membrane-bound TorA-GFP; fluorescence microscopy also showed active TorA-GFP to be distributed throughout the cytoplasm.

Identification of key regions within the Escherichia coli TatAB subunits.

The twin-arginine translocation (Tat) system catalyzes the transport of folded proteins across the bacterial plasma membrane or the chloroplast thylakoid membrane. In Escherichia coli and most other species, three important tat genes have been identified but the structure and mechanism of this system are poorly understood; the role and location of TatA are particularly unclear. In this report we have used site-specific mutagenesis to probe the significance of conserved features of the related TatA/B subunits.

Essential cytoplasmic domains in the Escherichia coli TatC protein.

The twin-arginine translocation (Tat) system mediates the transport of proteins across the bacterial plasma membrane and chloroplast thylakoid membrane. Operating in parallel with Sec-type systems in these membranes, the Tat system is completely different in both structural and mechanistic terms, and is uniquely able to catalyze the translocation of fully folded proteins across coupled membranes. TatC is an essential, multispanning component that has been proposed to form part of the binding site for substrate precursor proteins.