Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The structural basis of tail-anchored membrane protein recognition by Get3

Nature volume 461, pages 361–366 (2009)Cite this article

This article has been updated

Abstract

Targeting of newly synthesized membrane proteins to the endoplasmic reticulum is an essential cellular process. Most membrane proteins are recognized and targeted co-translationally by the signal recognition particle. However, nearly 5% of membrane proteins are ‘tail-anchored’ by a single carboxy-terminal transmembrane domain that cannot access the co-translational pathway. Instead, tail-anchored proteins are targeted post-translationally by a conserved ATPase termed Get3. The mechanistic basis for tail-anchored protein recognition or targeting by Get3 is not known. Here we present crystal structures of yeast Get3 in ‘open’ (nucleotide-free) and ‘closed’ (ADP·AlF4--bound) dimer states. In the closed state, the dimer interface of Get3 contains an enormous hydrophobic groove implicated by mutational analyses in tail-anchored protein binding. In the open state, Get3 undergoes a striking rearrangement that disrupts the groove and shields its hydrophobic surfaces. These data provide a molecular mechanism for nucleotide-regulated binding and release of tail-anchored proteins during their membrane targeting by Get3.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Get3 is a dynamic, metal-stabilized homodimer.
Figure 2: A composite hydrophobic groove at the closed dimer interface.
Figure 3: Functional analysis of the hydrophobic groove.
Figure 4: The Get3 nucleotide sensor.
Figure 5: Model for TA protein targeting.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the S. pombe Get3 and Mg2+ ADP·AlF4--bound S. cerevisiae Get3 crystal structures are deposited in the Protein Data Bank under accession codes 2WOO and 2WOJ.

Change history

  • 17 September 2009

    Author affiliations were added for A.S. and M.D. on 17 September 2009.

References

  1. Egea, P. F., Stroud, R. M. & Walter, P. Targeting proteins to membranes: structure of the signal recognition particle. Curr. Opin. Struct. Biol. 15, 213–220 (2005)

    Article  CAS  Google Scholar 

  2. Keenan, R. J., Freymann, D. M., Stroud, R. M. & Walter, P. The signal recognition particle. Annu. Rev. Biochem. 70, 755–775 (2001)

    Article  CAS  Google Scholar 

  3. Rapoport, T. A. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature 450, 663–669 (2007)

    Article  ADS  CAS  Google Scholar 

  4. Beilharz, T., Egan, B., Silver, P. A., Hofmann, K. & Lithgow, T. Bipartite signals mediate subcellular targeting of tail-anchored membrane proteins in Saccharomyces cerevisiae . J. Biol. Chem. 278, 8219–8223 (2003)

    Article  CAS  Google Scholar 

  5. Kalbfleisch, T., Cambon, A. & Wattenberg, B. W. A bioinformatics approach to identifying tail-anchored proteins in the human genome. Traffic 8, 1687–1694 (2007)

    Article  CAS  Google Scholar 

  6. Borgese, N., Brambillasca, S. & Colombo, S. How tails guide tail-anchored proteins to their destinations. Curr. Opin. Cell Biol. 19, 368–375 (2007)

    Article  CAS  Google Scholar 

  7. Wattenberg, B. & Lithgow, T. Targeting of C-terminal (tail)-anchored proteins: understanding how cytoplasmic activities are anchored to intracellular membranes. Traffic 2, 66–71 (2001)

    Article  CAS  Google Scholar 

  8. Kutay, U., Ahnert-Hilger, G., Hartmann, E., Wiedenmann, B. & Rapoport, T. A. Transport route for synaptobrevin via a novel pathway of insertion into the endoplasmic reticulum membrane. EMBO J. 14, 217–223 (1995)

    Article  CAS  Google Scholar 

  9. Favaloro, V., Spasic, M., Schwappach, B. & Dobberstein, B. Distinct targeting pathways for the membrane insertion of tail-anchored (TA) proteins. J. Cell Sci. 121, 1832–1840 (2008)

    Article  CAS  Google Scholar 

  10. Stefanovic, S. & Hegde, R. S. Identification of a targeting factor for posttranslational membrane protein insertion into the ER. Cell 128, 1147–1159 (2007)

    Article  CAS  Google Scholar 

  11. Kurdi-Haidar, B. et al. Isolation of the ATP-binding human homolog of the arsA component of the bacterial arsenite transporter. Genomics 36, 486–491 (1996)

    Article  CAS  Google Scholar 

  12. Kurdi-Haidar, B., Heath, D., Aebi, S. & Howell, S. B. Biochemical characterization of the human arsenite-stimulated ATPase (hASNA-I). J. Biol. Chem. 273, 22173–22176 (1998)

    Article  CAS  Google Scholar 

  13. Rosen, B. P., Bhattacharjee, H., Zhou, T. & Walmsley, A. R. Mechanism of the ArsA ATPase. Biochim. Biophys. Acta 1461, 207–215 (1999)

    Article  CAS  Google Scholar 

  14. Schuldiner, M. et al. The GET complex mediates insertion of tail-anchored proteins into the ER membrane. Cell 134, 634–645 (2008)

    Article  CAS  Google Scholar 

  15. Leipe, D. D., Wolf, Y. I., Koonin, E. V. & Aravind, L. Classification and evolution of P-loop GTPases and related ATPases. J. Mol. Biol. 317, 41–72 (2002)

    Article  CAS  Google Scholar 

  16. Zhou, T., Radaev, S., Rosen, B. P. & Gatti, D. L. Structure of the ArsA ATPase: the catalytic subunit of a heavy metal resistance pump. EMBO J. 19, 4838–4845 (2000)

    Article  CAS  Google Scholar 

  17. Georgiadis, M. M. et al. Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii . Science 257, 1653–1659 (1992)

    Article  ADS  CAS  Google Scholar 

  18. Leonard, T. A., Butler, P. J. & Lowe, J. Bacterial chromosome segregation: structure and DNA binding of the Soj dimer—a conserved biological switch. EMBO J. 24, 270–282 (2005)

    Article  CAS  Google Scholar 

  19. Hayashi, I., Oyama, T. & Morikawa, K. Structural and functional studies of MinD ATPase: implications for the molecular recognition of the bacterial cell division apparatus. EMBO J. 20, 1819–1828 (2001)

    Article  CAS  Google Scholar 

  20. Freymann, D. M., Keenan, R. J., Stroud, R. M. & Walter, P. Structure of the conserved GTPase domain of the signal recognition particle. Nature 385, 361–364 (1997)

    Article  ADS  CAS  Google Scholar 

  21. Montoya, G., Svensson, C., Luirink, J. & Sinning, I. Crystal structure of the NG domain from the signal-recognition particle receptor FtsY. Nature 385, 365–368 (1997)

    Article  ADS  CAS  Google Scholar 

  22. Metz, J., Wachter, A., Schmidt, B., Bujnicki, J. M. & Schwappach, B. The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. J. Biol. Chem. 281, 410–417 (2006)

    Article  CAS  Google Scholar 

  23. Keenan, R. J., Freymann, D. M., Walter, P. & Stroud, R. M. Crystal structure of the signal sequence binding subunit of the signal recognition particle. Cell 94, 181–191 (1998)

    Article  CAS  Google Scholar 

  24. Jiang, Y. et al. Nonequivalence of the nucleotide binding domains of the ArsA ATPase. J. Biol. Chem. 280, 9921–9926 (2005)

    Article  CAS  Google Scholar 

  25. Schindelin, H., Kisker, C., Schlessman, J. L., Howard, J. B. & Rees, D. C. Structure of ADP·AIF4 --stabilized nitrogenase complex and its implications for signal transduction. Nature 387, 370–376 (1997)

    Article  ADS  CAS  Google Scholar 

  26. Sprang, S. R. G protein mechanisms: insights from structural analysis. Annu. Rev. Biochem. 66, 639–678 (1997)

    Article  CAS  Google Scholar 

  27. Gasper, R., Meyer, S., Gotthardt, K., Sirajuddin, M. & Wittinghofer, A. It takes two to tango: regulation of G proteins by dimerization. Nature Rev. Mol. Cell Biol. 10, 423–429 (2009)

    Article  CAS  Google Scholar 

  28. Zhou, T., Radaev, S., Rosen, B. P. & Gatti, D. L. Conformational changes in four regions of the Escherichia coli ArsA ATPase link ATP hydrolysis to ion translocation. J. Biol. Chem. 276, 30414–30422 (2001)

    Article  CAS  Google Scholar 

  29. Bernstein, H. D. et al. Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle. Nature 340, 482–486 (1989)

    Article  ADS  CAS  Google Scholar 

  30. Horie, C., Suzuki, H., Sakaguchi, M. & Mihara, K. Characterization of signal that directs C-tail-anchored proteins to mammalian mitochondrial outer membrane. Mol. Biol. Cell 13, 1615–1625 (2002)

    Article  CAS  Google Scholar 

  31. Kaufmann, T. et al. Characterization of the signal that directs Bcl-xL, but not Bcl-2, to the mitochondrial outer membrane. J. Cell Biol. 160, 53–64 (2003)

    Article  CAS  Google Scholar 

  32. Kuroda, R. et al. Charged amino acids at the carboxyl-terminal portions determine the intracellular locations of two isoforms of cytochrome b5. J. Biol. Chem. 273, 31097–31102 (1998)

    Article  CAS  Google Scholar 

  33. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  34. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D. & Winn, M. D. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  35. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  36. Kiianitsa, K., Solinger, J. A. & Heyer, W. D. NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities. Anal. Biochem. 321, 266–271 (2003)

    Article  CAS  Google Scholar 

  37. Vogel, G. & Steinhart, R. ATPase of Escherichia coli: purification, dissociation, and reconstitution of the active complex from the isolated subunits. Biochemistry 15, 208–216 (1976)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L. & Clardy, J. Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J. Mol. Biol. 229, 105–124 (1993)

    Article  CAS  Google Scholar 

  40. Delano, W. L. The PyMOL Molecular Graphics System 〈http://www.pymol.org〉 (2002)

    Google Scholar 

Download references

Acknowledgements

Data were collected at beamlines 21-IDG and 23-IDD at the Advanced Photon Source (APS), Argonne National Laboratory, and we thank the beamline staff for support. Use of the APS was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. We thank B. Glick and R. Strack for reagents and advice, X. Li for assay characterization, and A. Shiau and T. Steck for comments on the manuscript. This work was supported by a grant from the Edward Mallinckrodt, Jr. Foundation (to R.J.K.) and by the Intramural Research Program of the National Institutes of Health (to R.S.H.).

Author Contributions A.M., A.S. and M.E.D. carried out cloning, protein purification and crystallization. A.M. performed the mutagenesis. M.M. and R.S.H. performed the TA substrate-binding assay. M.D. carried out the ATPase assay. M.E.D. performed the yeast genetic complementation assay. A.M., A.S. and R.J.K. carried out data collection. A.M. and R.J.K. carried out structure determination and model building. R.J.K. supervised the work and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Author information

Authors and Affiliations

  1. Department of Biochemistry & Molecular Biology, The University of Chicago, Gordon Center for Integrative Science, Room W238, 929 East 57th Street, Chicago, Illinois 60637, USA,

    Agnieszka Mateja, Anna Szlachcic, Maureen E. Downing, Malgorzata Dobosz & Robert J. Keenan

  2. Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Room 101, Building 18T, 18 Library Drive, Bethesda, Maryland 20892, USA ,

    Malaiyalam Mariappan & Ramanujan S. Hegde

  3. Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland,

    Anna Szlachcic

  4. Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland ,

    Malgorzata Dobosz

Authors
  1. Agnieszka Mateja
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anna Szlachcic
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maureen E. Downing
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Malgorzata Dobosz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Malaiyalam Mariappan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ramanujan S. Hegde
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Robert J. Keenan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Robert J. Keenan.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-5 with Legends, Supplementary Tables 1-2 and Supplementary References. (PDF 7170 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mateja, A., Szlachcic, A., Downing, M. et al. The structural basis of tail-anchored membrane protein recognition by Get3. Nature 461, 361–366 (2009). https://doi.org/10.1038/nature08319

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08319

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing