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

• Tomoya Tsukazaki,
• Hiroyuki Mori,
• Yuka Echizen,
• Ryuichiro Ishitani,
• Shuya Fukai,
• Takeshi Tanaka,
• Anna Perederina,
• Dmitry G. Vassylyev,
• Toshiyuki Kohno,
• Andrés D. Maturana,
• Koreaki Ito
• & Osamu Nureki

• Affiliations
• Contributions
• Corresponding authors

Nature

 

(2011)

 

doi:10.1038/nature09980

Received

 

11 January 2010

 

Accepted

 

09 March 2011

 

Published online

 

11 May 2011

Protein translocation across the bacterial membrane, mediated by the secretory translocon SecYEG and the SecA ATPase^{1, 2, 3, 4}, is enhanced by proton motive force^5, 6 and membrane-integrated SecDF^7, 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 biogenesis^{4, 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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Accession codes

• Accession codes
• Author information
 
• Supplementary information
 
• Comments

Primary accessions

Biological Magnetic Resonance Data Bank

• 11426

Protein Data Bank

• 3AQP
• 3AQO
• 2RRN

• 3AQP
• 3AQO
• 2RRN

Author information

• Accession codes
 
• Author information
• Supplementary information
 
• Comments

Primary authors

1. These authors contributed equally to this work.

   • Tomoya Tsukazaki & 
   • Hiroyuki Mori

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. Structural Biology Laboratory, Life Science Division, 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, Alabama 35294, 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

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to: 

• Osamu Nureki or
 
• Koreaki Ito

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.

Supplementary