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Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.

ABSTRACT: The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. TatC is the largest and most conserved component of the Tat machinery. It forms a multisubunit complex with TatB and binds the signal peptides of Tat substrates. Here we have taken a random mutagenesis approach to identify substitutions in Escherichia coli TatC that inactivate protein transport. We identify 32 individual amino acid substitutions that abolish or severely compromise TatC activity. The majority of the inactivating substitutions fall within the first two periplasmic loops of TatC. These regions are predicted to have conserved secondary structure and results of extensive amino acid insertion and deletion mutagenesis are consistent with these conserved elements being essential for TatC function. Three inactivating substitutions were identified in the fifth transmembrane helix of TatC. The inactive M205R variant could be suppressed by mutations affecting amino acids in the transmembrane helix of TatB. A physical interaction between TatC helix 5 and the TatB transmembrane helix was confirmed by the formation of a site-specific disulphide bond between TatC M205C and TatB L9C variants. This is the first molecular contact site mapped to single amino acid level between these two proteins.

SUBMITTER: Kneuper H

PROVIDER: S-EPMC3712464 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.

Kneuper Holger H Maldonado Barbara B Jäger Franziska F Krehenbrink Martin M Buchanan Grant G Keller Rebecca R Müller Matthias M Berks Ben C BC Palmer Tracy T

Molecular microbiology 20120713 5

The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. TatC is the largest and most conserved component of the Tat machinery. It forms a multisubunit complex with TatB and binds the signal peptides of Tat substrates. Here we have taken a random mutagenesis approach to identify substitutions in Escherichia coli TatC that inactivate protein transport. We identify 32 individual amino acid substitutions that a ...[more]

PMID: 22742417

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Project description:UnlabelledThe twin-arginine translocation (Tat) system, needed to transport folded proteins across biological membranes, has not been characterized in the gastric pathogen Helicobacter pylori. Analysis of all H. pylori genome sequences available thus far reveals the presence of single copies of tatA, tatB, and tatC needed for the synthesis of a fully functional Tat system. Based on the presence of the twin-arginine hallmark in their signal sequence, only four H. pylori proteins appear to be Tat dependent: hydrogenase (HydA), catalase-associated protein (KapA), biotin sulfoxide reductase (BisC), and the ubiquinol cytochrome oxidoreductase Rieske protein (FbcF). In the present study, targeted mutations were aimed at tatA, tatB, tatC, or queA (downstream gene control). While double homologous recombination mutations in tatB and queA were easily obtained, attempts at disrupting tatA proved unsuccessful, while deletion of tatC led to partial mutants following single homologous recombination, with cells retaining a chromosomal copy of tatC. Double homologous recombination tatC mutants were obtained only when a plasmid-borne, isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible copy of tatC was introduced prior to transformation. These conditional tatC mutants could grow only in the presence of IPTG, suggesting that tatC is essential in H. pylori. tatB and tatC mutants had lower hydrogenase and catalase activities than the wild-type strain did, and the ability of tatC mutants to colonize mouse stomachs was severely affected compared to the wild type. Chromosomal complementation of tatC mutants restored hydrogenase and catalase activities to wild-type levels, and additional expression of tatC in wild-type cells resulted in elevated Tat-dependent enzyme activities. Unexpectedly, the tat strains had cell envelope defects.ImportanceThis work reports the first characterization of the twin-arginine translocation (Tat) system in the gastric pathogen Helicobacter pylori. While tatB mutants were easily obtained, only single-crossover partial tatC mutants or conditional tatC mutants could be generated, indicating that tatC is essential in H. pylori, a surprising finding given the fact that only four proteins are predicted to be translocated by the Tat system in this bacterium. The levels of activity of hydrogenase and catalase, two of the predicted Tat-dependent enzymes, were affected in these mutants. In addition, all tat mutants displayed cell envelope defects, and tatC mutants were deficient in mouse colonization.

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Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.

Publications

Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.

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