Essential Cytoplasmic Domains in the Escherichia coli TatC Protein

Abstract

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. In this study we have tested the importance of conserved residues on the periplasmic and cytoplasmic face of the Escherichia coli protein. We find that many of the mutations on the cytoplasmic face have little or no effect. However, substitution at several positions in the extreme N-terminal cytoplasmic region or the predicted first cytoplasmic loop lead to a significant or complete loss of Tat-dependent export. The mutated strains are unable to grow anaerobically on trimethylamine N-oxide minimal media and are unable to export trimethylamine-N-oxide reductase (TorA). The same mutants are completely unable to export a chimeric protein, comprising the TorA signal peptide linked to green fluorescent protein, indicating that translocation is blocked rather than cofactor insertion into the TorA mature protein. The data point to two essential cytoplasmic domains on the TatC protein that are essential for export.

Affinity of TatCd for TatAd Elucidates Its Receptor Function in the Bacillus subtilis Twin Arginine Translocation (Tat) Translocase System

“…This group of proteins shares a similar orientation in the membrane with N and C termini exposed to the cytoplasmic or stromal side of the membrane (20,44,50,51). Particularly, alignment of TatC proteins revealed that conserved residues are preferably localized in the region exposed to the cytosolic site (52,53). Mutagenesis of conserved positions of E. coli TatC has revealed that some of these residues are critical for its function, which would be consistent with a role in substrate binding (52,53).…”

“…Particularly, alignment of TatC proteins revealed that conserved residues are preferably localized in the region exposed to the cytosolic site (52,53). Mutagenesis of conserved positions of E. coli TatC has revealed that some of these residues are critical for its function, which would be consistent with a role in substrate binding (52,53). Elucidated site-specific affinity of For E. coli TatC a dual topology has been suggested: Its calculated 6 transmembrane-spanning domains were confirmed by Behrendt et al (54).…”

Dual Topology of the Escherichia coli TatA Protein

“…Bolhuis et al (9) have constructed a TatB-TatC fusion protein by connecting the C terminus of TatB to the N terminus of TatC. Because the N terminus of TatC is located in the cytoplasm (20,35,36), the fusion should prohibit the C terminus of TatB changing the cellular location. Interestingly, the TatB-TatC fusion protein functionally complements a null mutant lacking both TatB and TatC in E. coli.…”

Mapping the Signal Peptide Binding and Oligomer Contact Sites of the Core Subunit of the Pea Twin Arginine Protein Translocase

“…One approach to address this combines mutagenesis with biochemical characterization and protein-protein interaction studies. This approach has been conducted to some extent with the Escherichia coli Tat system (Allen et al, 2002;Buchanan et al, 2002;Barrett et al, 2005;McDevitt et al, 2006;Holzapfel et al, 2007), but not with the plant Tat system owing to the relative difficulty of mutagenesis and gene replacement.…”