title: Coordinating assembly and export of complex bacterial proteins.
abstract: The Escherichia coli twin-arginine protein transport (Tat) system is a molecular machine dedicated to the translocation of fully folded substrate proteins across the energy-transducing inner membrane. Complex cofactor-containing Tat substrates, such as the model (NiFe) hydrogenase-2 and trimethylamine N-oxide reductase (TorA) systems, acquire their redox cofactors prior to export from the cell and require to be correctly assembled before transport can proceed. It is likely, therefore, that cellular mechanisms exist to prevent premature export of immature substrates. Using a combination of genetic and biochemical approaches including gene knockouts, signal peptide swapping, complementation, and site-directed mutagenesis, we highlight here this crucial 'proofreading' or 'quality control' activity in operation during assembly of complex endogenous Tat substrates. Our experiments successfully uncouple the Tat transport and cofactor-insertion activities of the TorA-specific chaperone TorD and demonstrate unequivocally that TorD recognises the TorA twin-arginine signal peptide. It is proposed that some Tat signal peptides operate in tandem with cognate binding chaperones to orchestrate the assembly and transport of complex enzymes.
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich, UK.
authors.forename: Rachael L
authors.initials: RL
authors.lastname: Jack
authors.forename: Grant
authors.initials: G
authors.lastname: Buchanan
authors.forename: Alexandra
authors.initials: A
authors.lastname: Dubini
authors.forename: Kostas
authors.initials: K
authors.lastname: Hatzixanthis
authors.forename: Tracy
authors.initials: T
authors.lastname: Palmer
authors.forename: Frank
authors.initials: F
authors.lastname: Sargent
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006864:Hydrogenase / Q000378:metabolism; D008744:Methylamines / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D017510:Protein Folding; D021382:Protein Sorting Signals; D021381:Protein Transport; D013347:Subcellular Fractions; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008744:Methylamines; D018832:Molecular Chaperones; D021382:Protein Sorting Signals; D010088:Oxidoreductases; D006864:Hydrogenase; C023336:trimethylamine