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Structural basis of signal sequence surveillance and selection by the SRP–FtsY complex

Nature Structural & Molecular Biology volume 20, pages 604–610 (2013)Cite this article

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Abstract

Signal-recognition particle (SRP)-dependent targeting of translating ribosomes to membranes is a multistep quality-control process. Ribosomes that are translating weakly hydrophobic signal sequences can be rejected from the targeting reaction even after they are bound to the SRP. Here we show that the early complex, formed by Escherichia coli SRP and its receptor FtsY with ribosomes translating the incorrect cargo EspP, is unstable and rearranges inefficiently into subsequent conformational states, such that FtsY dissociation is favored over successful targeting. The N-terminal extension of EspP is responsible for these defects in the early targeting complex. The cryo-electron microscopy structure of this 'false' early complex with EspP revealed an ordered M domain of SRP protein Ffh making two ribosomal contacts, and the NG domains of Ffh and FtsY forming a distorted, flexible heterodimer. Our results provide a structural basis for SRP-mediated signal-sequence selection during recruitment of the SRP receptor.

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Figure 1: The N-terminal extension of EspP inhibits co-translational protein targeting but does not affect RNC–SRP binding.
Figure 2: The EspP N-terminal extension leads to a weaker and distorted early targeting complex.
Figure 3: M-domain arrangement during co-translational targeting.
Figure 4: Ffh–FtsY NG-domain arrangement in the 'false' early complex formed with EspP compared to the productive early complex formed with a correct cargo.
Figure 5: The N-terminal extension leads to a less productive early complex and slower assembly of the closed SRP–FtsY complex.
Figure 6: Model of signal sequence surveillance by the SRP and FtsY.

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Acknowledgements

We thank T. Shaikh for advice with Spider refinement, P. Penczek for assistance with SPARX, M. Bacia for excellent technical assistance and members of the protein expression facility at European Molecular Biology Laboratory Heidelberg as well as the Partnership for Structural Biology in Grenoble for support. The Polara microscope is part of the Structural Biology and Dynamics Groupement d'intérêt scientifique–Infrastrutures en Biologie Sante et Agronomie platform of the Institut de Biologie Structurale. C.S. acknowledges support by the Agence Nationale de la Recherche (ANR-09-JCJC-0044), the region Rhône-Alpes (CIBLE_1976) and the European Research Council Starting grant (project 281331). K.K. was supported by a postdoctoral European Molecular Biology Organization fellowship. We thank I. Saraogi in the Shan laboratory for sharing unpublished results and for critical reading of the manuscript. S.S. is supported by US National Institutes of Health grant R01 GM078024, and the Fellowship for science and engineering from the David and Lucile Packard foundation. A.A. was supported by the US National Institute of General Medical Sciences Ruth L. Kirschstein National Research Service Award (F31GM095294) and the National Institutes of Health National Research Service Award Training grant 5T32GM07616. X.Z. is supported by the Howard Hughes Medical Institute Fellowship of the Helen Hay Whitney Foundation.

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Author notes

  1. Kèvin Knoops and Aileen Ariosa: These authors contributed equally to this work.

Authors and Affiliations

  1. European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France

    Ottilie von Loeffelholz, Kèvin Knoops, Manikandan Karuppasamy, Karine Huard, Imre Berger & Christiane Schaffitzel

  2. Unit of Virus–Host Cell Interactions, Université Joseph Fourier, European Molecular Biology Laboratory, Centre National de la Recherche Scientifique, Unité Mixte Internationale 3265, Grenoble, France

    Ottilie von Loeffelholz, Kèvin Knoops, Manikandan Karuppasamy, Karine Huard, Guy Schoehn, Imre Berger & Christiane Schaffitzel

  3. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA

    Aileen Ariosa, Xin Zhang & Shu-ou Shan

  4. Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA

    Xin Zhang

  5. Centre National de la Recherche Scientifique, Commissariat à l'énergie atomique et aux énergies alternatives, Université Joseph Fourier, Institut de Biologie Structurale–Jean-Pierre Ebel, Unité Mixte de Recherche 5075, Grenoble, France

    Guy Schoehn

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Contributions

C.S., I.B., X.Z. and S.S. designed experiments; C.S., K.H., O.v.L., A.A. and X.Z. prepared samples; A.A. and X.Z. carried out biochemical experiments; K.K., G.S. and M.K. performed the electron microscopy; O.v.L., M.K. and C.S. performed image analysis and model building; C.S., O.v.L., A.A., X.Z. and S.S. prepared the manuscript.

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Correspondence to Shu-ou Shan or Christiane Schaffitzel.

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von Loeffelholz, O., Knoops, K., Ariosa, A. et al. Structural basis of signal sequence surveillance and selection by the SRP–FtsY complex. Nat Struct Mol Biol 20, 604–610 (2013). https://doi.org/10.1038/nsmb.2546

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