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.

  • Article
  • Published:

Structures of the tRNA export factor in the nuclear and cytosolic states

Nature volume 461, pages 60–65 (2009)Cite this article

Abstract

Transfer RNAs are among the most ubiquitous molecules in cells, central to decoding information from messenger RNAs on translating ribosomes. In eukaryotic cells, tRNAs are actively transported from their site of synthesis in the nucleus to their site of function in the cytosol. This is mediated by a dedicated nucleo-cytoplasmic transport factor of the karyopherin-β family (Xpot, also known as Los1 in Saccharomyces cerevisiae). Here we report the 3.2 Å resolution structure of Schizosaccharomyces pombe Xpot in complex with tRNA and RanGTP, and the 3.1 Å structure of unbound Xpot, revealing both nuclear and cytosolic snapshots of this transport factor. Xpot undergoes a large conformational change on binding cargo, wrapping around the tRNA and, in particular, binding to the tRNA 5′ and 3′ ends. The binding mode explains how Xpot can recognize all mature tRNAs in the cell and yet distinguish them from those that have not been properly processed, thus coupling tRNA export to quality control.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Structure of the Xpot–RanGTP–tRNA complex.
Figure 2: The binding of RanGTP to Xpot.
Figure 3: Complementary tRNA-binding surfaces on Xpot.
Figure 4: Recognition of the tRNA 3′ and 5′ ends.
Figure 5: Conformational change of Xpot unbound and in complex.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank under accession codes 3IBV for Xpot and 3ICQ for the Xpot–RanGTP–tRNA complex.

References

  1. Kohler, A. & Hurt, E. Exporting RNA from the nucleus to the cytoplasm. Nature Rev. Mol. Cell Biol. 8, 761–773 (2007)

    Google Scholar 

  2. Rodriguez, M. S., Dargemont, C. & Stutz, F. Nuclear export of RNA. Biol. Cell 96, 639–655 (2004)

    CAS  PubMed  Google Scholar 

  3. Elad, N., Maimon, T., Frenkiel-Krispin, D., Lim, R. Y. & Medalia, O. Structural analysis of the nuclear pore complex by integrated approaches. Curr. Opin. Struct. Biol. 19, 226–232 (2009)

    CAS  PubMed  Google Scholar 

  4. Lim, R. Y., Aebi, U. & Fahrenkrog, B. Towards reconciling structure and function in the nuclear pore complex. Histochem. Cell Biol. 129, 105–116 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Peters, R. Translocation through the nuclear pore: Kaps pave the way. Bioessays 31, 466–477 (2009)

    CAS  PubMed  Google Scholar 

  6. Zasloff, M. tRNA transport from the nucleus in a eukaryotic cell: carrier-mediated translocation process. Proc. Natl Acad. Sci. USA 80, 6436–6440 (1983)

    ADS  CAS  PubMed  Google Scholar 

  7. Arts, G. J., Fornerod, M. & Mattaj, I. W. Identification of a nuclear export receptor for tRNA. Curr. Biol. 8, 305–314 (1998)

    CAS  PubMed  Google Scholar 

  8. Kutay, U. et al. Identification of a tRNA-specific nuclear export receptor. Mol. Cell 1, 359–369 (1998)

    CAS  PubMed  Google Scholar 

  9. Hellmuth, K. et al. Yeast Los1p has properties of an exportin-like nucleocytoplasmic transport factor for tRNA. Mol. Cell. Biol. 18, 6374–6386 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Cook, A., Bono, F., Jinek, M. & Conti, E. Structural biology of nucleocytoplasmic transport. Annu. Rev. Biochem. 76, 647–671 (2007)

    CAS  PubMed  Google Scholar 

  11. Madrid, A. S. & Weis, K. Nuclear transport is becoming crystal clear. Chromosoma 115, 98–109 (2006)

    PubMed  Google Scholar 

  12. Calado, A., Treichel, N., Muller, E. C., Otto, A. & Kutay, U. Exportin-5-mediated nuclear export of eukaryotic elongation factor 1A and tRNA. EMBO J. 21, 6216–6224 (2002)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Shibata, S. et al. Exportin-5 orthologues are functionally divergent among species. Nucleic Acids Res. 34, 4711–4721 (2006)

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Jovine, L., Djordjevic, S. & Rhodes, D. The crystal structure of yeast phenylalanine tRNA at 2.0 Å resolution: cleavage by Mg2+ in 15-year old crystals. J. Mol. Biol. 301, 401–414 (2000)

    CAS  PubMed  Google Scholar 

  15. Shi, H. & Moore, P. B. The crystal structure of yeast phenylalanine tRNA at 1.93 Å resolution: a classic structure revisited. RNA 6, 1091–1105 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Marvin, M. C. & Engelke, D. R. RNase P: increased versatility through protein complexity? RNA Biol. 6, 40–42 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Spath, B., Canino, G. & Marchfelder, A. tRNase Z: the end is not in sight. Cell. Mol. Life Sci. 64, 2404–2412 (2007)

    CAS  PubMed  Google Scholar 

  18. Xiong, Y. & Steitz, T. A. A story with a good ending: tRNA 3′-end maturation by CCA-adding enzymes. Curr. Opin. Struct. Biol. 16, 12–17 (2006)

    CAS  PubMed  Google Scholar 

  19. Lund, E. & Dahlberg, J. E. Proofreading and aminoacylation of tRNAs before export from the nucleus. Science 282, 2082–2085 (1998)

    ADS  CAS  PubMed  Google Scholar 

  20. Bjork, G. R. et al. Transfer RNA modification. Annu. Rev. Biochem. 56, 263–287 (1987)

    CAS  PubMed  Google Scholar 

  21. Iwata-Reuyl, D. An embarrassment of riches: the enzymology of RNA modification. Curr. Opin. Chem. Biol. 12, 126–133 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Arts, G. J., Kuersten, S., Romby, P., Ehresmann, B. & Mattaj, I. W. The role of exportin-t in selective nuclear export of mature tRNAs. EMBO J. 17, 7430–7441 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Lipowsky, G. et al. Coordination of tRNA nuclear export with processing of tRNA. RNA 5, 539–549 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Chook, Y. M. & Blobel, G. Structure of the nuclear transport complex karyopherin-β2–Ran·GppNHp. Nature 399, 230–237 (1999)

    ADS  CAS  PubMed  Google Scholar 

  25. Lee, S. J., Matsuura, Y., Liu, S. M. & Stewart, M. Structural basis for nuclear import complex dissociation by RanGTP. Nature 435, 693–696 (2005)

    ADS  CAS  PubMed  Google Scholar 

  26. Matsuura, Y. & Stewart, M. Structural basis for the assembly of a nuclear export complex. Nature 432, 872–877 (2004)

    ADS  CAS  PubMed  Google Scholar 

  27. Monecke, T. et al. Crystal structure of the nuclear export receptor CRM1 in complex with Snurportin1 and RanGTP. Science 324, 1087–1091 (2009)

    ADS  CAS  PubMed  Google Scholar 

  28. Bischoff, F. R., Klebe, C., Kretschmer, J., Wittinghofer, A. & Ponstingl, H. RanGAP1 induces GTPase activity of nuclear Ras-related Ran. Proc. Natl Acad. Sci. USA 91, 2587–2591 (1994)

    ADS  CAS  PubMed  Google Scholar 

  29. Bloomfield, V. A., Crothers, D. N. & Tinoco, I. Nucleic Acids: Structures, Properties and Functions Ch. 8 298–300 (Macmillan, 1999)

    Google Scholar 

  30. Vetter, I. R., Arndt, A., Kutay, U., Gorlich, D. & Wittinghofer, A. Structural view of the Ran–Importin β interaction at 2.3 Å resolution. Cell 97, 635–646 (1999)

    CAS  PubMed  Google Scholar 

  31. Andrade, M. A., Petosa, C., O’Donoghue, S. I., Muller, C. W. & Bork, P. Comparison of ARM and HEAT protein repeats. J. Mol. Biol. 309, 1–18 (2001)

    CAS  PubMed  Google Scholar 

  32. Cingolani, G., Petosa, C., Weis, K. & Müller, C. W. Structure of importin-β bound to the IBB domain of importin-α. Nature 399, 221–229 (1999)

    ADS  CAS  PubMed  Google Scholar 

  33. Kuersten, S., Arts, G. J., Walther, T. C., Englmeier, L. & Mattaj, I. W. Steady-state nuclear localization of exportin-t involves RanGTP binding and two distinct nuclear pore complex interaction domains. Mol. Cell. Biol. 22, 5708–5720 (2002)

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Sprinzl, M., Horn, C., Brown, M., Ioudovitch, A. & Steinberg, S. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 26, 148–153 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Fukuhara, N., Fernandez, E., Ebert, J., Conti, E. & Svergun, D. Conformational variability of nucleo-cytoplasmic transport factors. J. Biol. Chem. 279, 2176–2181 (2004)

    CAS  PubMed  Google Scholar 

  36. Cook, A. et al. The structure of the nuclear export receptor Cse1 in its cytosolic state reveals a closed conformation incompatible with cargo binding. Mol. Cell 18, 355–367 (2005)

    CAS  PubMed  Google Scholar 

  37. Dong, X., Biswas, A. & Chook, Y. M. Structural basis for assembly and disassembly of the CRM1 nuclear export complex. Nature Struct. Mol. Biol. 16, 558–560 (2009)

    CAS  Google Scholar 

  38. Moras, D. Structural and functional relationships between aminoacyl-tRNA synthetases. Trends Biochem. Sci. 17, 159–164 (1992)

    CAS  PubMed  Google Scholar 

  39. LeMaster, D. M. & Richards, F. M. 1H–15N heteronuclear NMR studies of Escherichia coli thioredoxin in samples isotopically labeled by residue type. Biochemistry 24, 7263–7268 (1985)

    CAS  PubMed  Google Scholar 

  40. Walker, P. A. et al. Efficient and rapid affinity purification of proteins using recombinant fusion proteases. Biotechnology 12, 601–605 (1994)

    CAS  PubMed  Google Scholar 

  41. Doublie, S. et al. Crystallization and preliminary X-ray analysis of the 9 kDa protein of the mouse signal recognition particle and the selenomethionyl-SRP9. FEBS Lett. 384, 219–221 (1996)

    CAS  PubMed  Google Scholar 

  42. Leslie, A. G. W. in Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography No. 26 (CCP4, 1992)

    Google Scholar 

  43. Collaborative Computational Project, Number 4 The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Google Scholar 

  44. Broennimann et al. The PILATUS 1M detector. J. Synchrotron Radiat. 13, 120–130 (2006)

    CAS  PubMed  Google Scholar 

  45. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993)

    CAS  Google Scholar 

  46. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D 58, 1772–1779 (2002)

    PubMed  Google Scholar 

  47. de La Fortelle, E. & Bricogne, G. in Macromolecular Crystallography Part A (eds Sweet, R. M. & Carter, C. W. J.) 472–494 (Academic, 1997)

    Google Scholar 

  48. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Stein, N. CHAINSAW: a program for mutating pdb files used as templates in molecular replacement. J. Appl. Crystallogr. 41, 641–643 (2008)

    CAS  Google Scholar 

  50. Jones, T. A., Bergdoll, M. & Kjekdgaard, M. in Crystallographic and Modeling Methods in Molecular Design (eds Bugg, C. & Ealick, S.) 189–195 (Springer-Verlag, 1990)

    Google Scholar 

  51. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    CAS  PubMed  Google Scholar 

  52. Davis, I. W., Murray, L. W., Richardson, J. S. & Richardson, D. C. MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes. Nucleic Acids Res. 32, W615–W619 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Landau, M. et al. ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res. 33, W299–W302 (2005)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We would like to thank P. Brick for comments and critical reading of the manuscript. We also thank P. Reichelt and J. Ebert for technical assistance, S. Kuersten and I. Mattaj at the initial phase of the project, K. Valer-Saldana, S. Pleyer and J. Basquin of the MPI-Martinsried crystallization facility and the staff at the Swiss Light Source (Villigen, Switzerland) for assistance during data collection. We would like to thank C. Vonrhein (Global Phasing) for optimising the phase calculations of unbound Xpot with SHARP. This study was supported by the Max Planck Gesellschaft, the European Molecular Biology Laboratory (EMBL), the Sonderforschungsbereich SFB646 and the Gottfried Wilhelm Leibniz Program of the Deutsche Forschungsgemeinschaft (DFG), the EU grant 3D Repertoire contract number LSHG-CT-2005-512028.

Author Contributions N.F. identified S. pombe Xpot as the most promising orthologue for crystallisation, created the truncated tRNA construct and obtained initial, low-resolution crystals for the cargo-bound complex. M.J. crystallised unbound Xpot. A.G.C. solved the structure of unbound Xpot, obtained well-diffracting crystals of the ternary complex and solved its structure. E.C. supervised the project. A.G.C. and E.C. wrote the paper.

Author information

Author notes

  1. Noemi Fukuhara & Martin Jinek

    Present address: Present addresses: Division of Cell and Molecular Biology, Biophysics section, Blackett Laboratory, Imperial College London, London SW7 2AZ, UK (N.F.); Department of Molecular Cell Biology, University of California at Berkeley, 731 Stanley Hall, Berkeley, California 94720, USA (M.J.).,

Authors and Affiliations

  1. Structural Cell Biology, MPI for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany,

    Atlanta G. Cook & Elena Conti

  2. EMBL, Meyerhofstrasse 1, Heidelberg D69117, Germany,

    Noemi Fukuhara, Martin Jinek & Elena Conti

Authors
  1. Atlanta G. Cook
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Noemi Fukuhara
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martin Jinek
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elena Conti
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Elena Conti.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-2 and Supplementary Figures 1-10 with Legends and Supplementary References for Figure 8. (PDF 10586 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cook, A., Fukuhara, N., Jinek, M. et al. Structures of the tRNA export factor in the nuclear and cytosolic states. Nature 461, 60–65 (2009). https://doi.org/10.1038/nature08394

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08394

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