Extra-cytoplasmic polypeptides are usually synthesized as ‘preproteins’ carrying amino-terminal, cleavable signal peptides1 and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA2,3. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA2,3. Preprotein targeting to SecA is thought to involve signal peptides4 and chaperones like SecB5,6. Here we show that signal peptides have a new role beyond targeting: they are essential allosteric activators of the translocase. On docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, ‘triggering’ that drives the translocase to a lower activation energy state; second, ‘trapping’ that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus; and third, ‘secretion’ during which trapped mature domains undergo several turnovers of translocation in segments7. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases.
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We are grateful to G. Dimitrakopoulos for help with MATLAB; M. Koukaki for constructs and materials; D. Boyd, S. Schulman and V. Koronakis for gifts of strains, plasmids and protocols; A. Kohen for advice on Arrhenius plots; A. Kuhn and K. Tokatlidis for comments. The research leading to these results has received funding from the European Community’s Sixth Framework Programme (FP6/2002–2007) under grant agreement LSHC-CT-2006-037834/Streptomics (to A.E.), the Greek General Secretariat of Research and the European Regional Development Fund (PENED03ED623; to A.E.), the US National Institutes of Health grant GM73854 (to C.G.K.) and a Scientist Development Grant by the American Heart Association (to C.G.K.). G.G. is an Onassis Foundation predoctoral fellow.
Author Contributions G.G. cloned genes, performed in vivo and in vitro secretion experiments, phosphatase assays, membrane binding studies, Arrhenius conversions and developed the in trans reconstitution assay. G.G. and S.K. purified proteins, performed ATPase experiments, analysed data, provided experimental ideas and contributed in writing the paper. S.K. developed thermal-dependence ATPase assay, contributed in assay development, performed preliminary ITC experiments and edited the paper. I.G. purified proteins, performed and analysed ITC experiments. C.G.K. designed, guided and analysed ITC experiments, contributed in experimental ideas and controls and in writing and editing the paper. A.E. conceived, designed and guided experiments, analysed data and wrote the paper. All authors read and commented on the paper.
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Gouridis, G., Karamanou, S., Gelis, I. et al. Signal peptides are allosteric activators of the protein translocase. Nature 462, 363–367 (2009). https://doi.org/10.1038/nature08559
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