Abstract
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.