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Structure and function of a membrane component SecDF that enhances protein export

Nature volume 474, pages 235–238 (2011)Cite this article

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Abstract

Protein translocation across the bacterial membrane, mediated by the secretory translocon SecYEG and the SecA ATPase1,2,3,4, is enhanced by proton motive force5,6 and membrane-integrated SecDF7,8,9, which associates with SecYEG. The role of SecDF has remained unclear, although it is proposed to function in later stages of translocation as well as in membrane protein biogenesis4,10,11,12,13. Here, we determined the crystal structure of Thermus thermophilus SecDF at 3.3 Å resolution, revealing a pseudo-symmetrical, 12-helix transmembrane domain belonging to the RND superfamily and two major periplasmic domains, P1 and P4. Higher-resolution analysis of the periplasmic domains suggested that P1, which binds an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and proton motive force. Electrophysiological analyses revealed that SecDF conducts protons in a manner dependent on pH and the presence of an unfolded protein, with conserved Asp and Arg residues at the transmembrane interface between SecD and SecF playing essential roles in the movements of protons and preproteins. Therefore, we propose that SecDF functions as a membrane-integrated chaperone, powered by proton motive force, to achieve ATP-independent protein translocation.

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Figure 1: Structures of T. thermophilus SecDF.
Figure 2: SecDF-dependent translocation completion.
Figure 3: Functional charged residues and proton conduction of SecDF.
Figure 4: A working model of the PMF-driven translocation enhancement by SecDF.

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Accession codes

Primary accessions

Biological Magnetic Resonance Data Bank

Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited in the Protein Data Bank under the accession codes 3AQP for the entire TtSecDF protein and 3AQO for the P1 domain. The PDB and BMRB codes for the deposited P4 domain are 2RRN and 11426 respectively.

References

  1. van den Berg, B. et al. X-ray structure of a protein-conducting channel. Nature 427, 36–44 (2004)

    Article  CAS  PubMed  Google Scholar 

  2. Zimmer, J., Nam, Y. & Rapoport, T. A. Structure of a complex of the ATPase SecA and the protein-translocation channel. Nature 455, 936–943 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  3. Tsukazaki, T. et al. Conformational transition of Sec machinery inferred from bacterial SecYE structures. Nature 455, 988–991 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. du Plessis, D. J., Nouwen, N. & Driessen, A. J. The Sec translocase. Biochim. Biophys. Acta 1808, 851–865 (2011)

    Article  CAS  PubMed  Google Scholar 

  5. Driessen, A. J. & Wickner, W. Proton transfer is rate-limiting for translocation of precursor proteins by the Escherichia coli translocase. Proc. Natl Acad. Sci. USA 88, 2471–2475 (1991)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shiozuka, K., Tani, K., Mizushima, S. & Tokuda, H. The proton motive force lowers the level of ATP required for the in vitro translocation of a secretory protein in Escherichia coli . J. Biol. Chem. 265, 18843–18847 (1990)

    CAS  PubMed  Google Scholar 

  7. Pogliano, J. A. & Beckwith, J. SecD and SecF facilitate protein export in Escherichia coli . EMBO J. 13, 554–561 (1994)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sagara, K., Matsuyama, S. & Mizushima, S. SecF stabilizes SecD and SecY, components of the protein translocation machinery of the Escherichia coli cytoplasmic membrane. J. Bacteriol. 176, 4111–4116 (1994)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hand, N. J., Klein, R., Laskewitz, A. & Pohlschroder, M. Archaeal and bacterial SecD and SecF homologs exhibit striking structural and functional conservation. J. Bacteriol. 188, 1251–1259 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Matsuyama, S., Fujita, Y. & Mizushima, S. SecD is involved in the release of translocated secretory proteins from the cytoplasmic membrane of Escherichia coli . EMBO J. 12, 265–270 (1993)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Economou, A., Pogliano, J. A., Beckwith, J., Oliver, D. B. & Wickner, W. SecA membrane cycling at SecYEG is driven by distinct ATP binding and hydrolysis events and is regulated by SecD and SecF. Cell 83, 1171–1181 (1995)

    Article  CAS  PubMed  Google Scholar 

  12. Arkowitz, R. A. & Wickner, W. SecD and SecF are required for the proton electrochemical gradient stimulation of preprotein translocation. EMBO J. 13, 954–963 (1994)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Duong, F. & Wickner, W. The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling. EMBO J. 16, 4871–4879 (1997)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Nouwen, N., Piwowarek, M., Berrelkamp, G. & Driessen, A. J. The large first periplasmic loop of SecD and SecF plays an important role in SecDF functioning. J. Bacteriol. 187, 5857–5860 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Pogliano, K. J. & Beckwith, J. Genetic and molecular characterization of the Escherichia coli secD operon and its products. J. Bacteriol. 176, 804–814 (1994)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Tseng, T. T. et al. The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins. J. Mol. Microbiol. Biotechnol. 1, 107–125 (1999)

    CAS  PubMed  Google Scholar 

  17. Tsukazaki, T. et al. Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus. Acta Crystallogr. F 62, 376–380 (2006)

    Article  CAS  Google Scholar 

  18. Uchida, K., Mori, H. & Mizushima, S. Stepwise movement of preproteins in the process of translocation across the cytoplasmic membrane of Escherichia coli . J. Biol. Chem. 270, 30862–30868 (1995)

    Article  CAS  PubMed  Google Scholar 

  19. Schiebel, E., Driessen, A. J., Hartl, F. U. & Wickner, W. ΔμH+ and ATP function at different steps of the catalytic cycle of preprotein translocase. Cell 64, 927–939 (1991)

    Article  CAS  PubMed  Google Scholar 

  20. Murakami, S., Nakashima, R., Yamashita, E., Matsumoto, T. & Yamaguchi, A. Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 443, 173–179 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

  21. Seeger, M. A., von Ballmoos, C., Verrey, F. & Pos, K. M. Crucial role of Asp408 in the proton translocation pathway of multidrug transporter AcrB: evidence from site-directed mutagenesis and carbodiimide labeling. Biochemistry 48, 5801–5812 (2009)

    Article  CAS  PubMed  Google Scholar 

  22. Häse, C. C. & Barquera, B. Role of sodium bioenergetics in Vibrio cholerae . Biochim. Biophys. Acta 1505, 169–178 (2001)

    Article  PubMed  Google Scholar 

  23. Sasaki, M., Takagi, M. & Okamura, Y. A voltage sensor-domain protein is a voltage-gated proton channel. Science 312, 589–592 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

  24. Hattori, M. et al. Mg2+-dependent gating of bacterial MgtE channel underlies Mg2+ homeostasis. EMBO J. 28, 3602–3612 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Vassylyev, D. G. et al. Structural basis for substrate loading in bacterial RNA polymerase. Nature 448, 163–168 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  26. Inaba, K. et al. Crystal structure of the DsbB-DsbA complex reveals a mechanism of disulfide bond generation. Cell 127, 789–801 (2006)

    Article  CAS  PubMed  Google Scholar 

  27. Matsuo, E., Mori, H., Shimoike, T. & Ito, K. Syd, a SecY-interacting protein, excludes SecA from the SecYE complex with an altered SecY24 subunit. J. Biol. Chem. 273, 18835–18840 (1998)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Y. Akiyama, R. Suno, Y. Morimoto, T. Minamino, K. Namba, K. Inaba, M. Hattori and H. Nishimasu for suggestions; T. Sakamoto and A. Kurabayashi for assistance with sample preparation; R. Yamasaki, M. Sano, K. Mochizuki, K. Yoshikaie, K. Imayoshi and T. Adachi for technical support; M. Homma and S. Kojima for providing the Vibrio genomic DNA; the beamline staff members at BL41XU of SPring-8 (Hyogo, Japan) and at NW12 of KEK PF-AR (Tsukuba, Japan) for technical help during data collection and M. Ibba for comments on our manuscript. This work was supported by a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to O.N., by a CREST grant from JST to K.I., by a BIRD grant from JST to H.M. and R.I., by a grant for the National Project on Protein Structural and Functional Analyses to O.N., by NIH grants to D.G.V. and by grants from MEXT to T.Tsukazaki, H.M., R.I., S.F. and K.I.

Author information

Author notes

  1. Tomoya Tsukazaki and Hiroyuki Mori: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan,

    Tomoya Tsukazaki, Yuka Echizen, Ryuichiro Ishitani & Osamu Nureki

  2. Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan ,

    Hiroyuki Mori

  3. Life Science Division, Structural Biology Laboratory, Synchrotron Radiation Research Organization, The University of Tokyo, Tokyo 113-0032, Japan,

    Shuya Fukai

  4. Laboratory of Macromolecular Complexes, Center for Structural Biology of Challenging Proteins, The University of Tokyo, Tokyo 113-0032, Japan ,

    Shuya Fukai

  5. Mitsubishi Kagaku Institute of Life Sciences, Machida-shi, Tokyo 194-8511, Japan ,

    Takeshi Tanaka & Toshiyuki Kohno

  6. Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, 35294, Alabama, USA

    Anna Perederina & Dmitry G. Vassylyev

  7. Bioengineering Department, Nagaoka University of Technology, Niigata 940-2188, Japan,

    Andrés D. Maturana

  8. Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan ,

    Koreaki Ito

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  1. Tomoya Tsukazaki
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Contributions

T.Tsukazaki performed the structural determination and the biochemical experiments with SecDF. H.M. performed the functional analyses of SecDF. Y.E. solved the crystal structure of the SecDF P1 domain and assisted with the functional analysis of SecDF. R.I., S.F., D.G.V. and O.N. assisted with the structural determination. A.D.M. performed patch clamp and pH fluorescence experiments. T.Tanaka and T.K. solved the structure of the P4 domain by NMR. A.P. and D.G.V. assisted with the crystallization and data collection of SecDF. All authors discussed the results and commented on the manuscript. O.N. and K.I. supervised the work and wrote and edited the manuscript.

Corresponding authors

Correspondence to Koreaki Ito or Osamu Nureki.

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The authors declare no competing financial interests.

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This file contains a Supplementary Discussion, Supplementary Methods, Supplementary References, Supplementary Figures 1- 9 with legends and Supplementary Tables 1-2. (PDF 1906 kb)

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Tsukazaki, T., Mori, H., Echizen, Y. et al. Structure and function of a membrane component SecDF that enhances protein export. Nature 474, 235–238 (2011). https://doi.org/10.1038/nature09980

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