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. 1996 Jul 9;93(14):7059–7062. doi: 10.1073/pnas.93.14.7059

Nuclear protein import: Ran-GTP dissociates the karyopherin alphabeta heterodimer by displacing alpha from an overlapping binding site on beta.

J Moroianu 1, G Blobel 1, A Radu 1
PMCID: PMC38935  PMID: 8692944

Abstract

The alpha subunit of the karyopherin heterodimer functions in recognition of the protein import substrate and the beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of karyopherin beta's binding sites both for Ran-GTP and for karyopherin alpha. We discovered that karyopherin beta's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and karyopherin alpha bind to overlapping sites on karyopherin beta. Having a higher affinity to the overlapping site, Ran-GTP displaces karyopherin alpha and binds to karyopherin beta. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for karyopherin beta to a cluster of basic residues analogous to those previously shown to constitute karyopherin alpha's binding site to karyopherin beta.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adam E. J., Adam S. A. Identification of cytosolic factors required for nuclear location sequence-mediated binding to the nuclear envelope. J Cell Biol. 1994 May;125(3):547–555. doi: 10.1083/jcb.125.3.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akey C. W., Radermacher M. Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy. J Cell Biol. 1993 Jul;122(1):1–19. doi: 10.1083/jcb.122.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andrade M. A., Bork P. HEAT repeats in the Huntington's disease protein. Nat Genet. 1995 Oct;11(2):115–116. doi: 10.1038/ng1095-115. [DOI] [PubMed] [Google Scholar]
  4. Chi N. C., Adam E. J., Adam S. A. Sequence and characterization of cytoplasmic nuclear protein import factor p97. J Cell Biol. 1995 Jul;130(2):265–274. doi: 10.1083/jcb.130.2.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Coutavas E., Ren M., Oppenheim J. D., D'Eustachio P., Rush M. G. Characterization of proteins that interact with the cell-cycle regulatory protein Ran/TC4. Nature. 1993 Dec 9;366(6455):585–587. doi: 10.1038/366585a0. [DOI] [PubMed] [Google Scholar]
  6. Enenkel C., Blobel G., Rexach M. Identification of a yeast karyopherin heterodimer that targets import substrate to mammalian nuclear pore complexes. J Biol Chem. 1995 Jul 14;270(28):16499–16502. doi: 10.1074/jbc.270.28.16499. [DOI] [PubMed] [Google Scholar]
  7. Floer M., Blobel G. The nuclear transport factor karyopherin beta binds stoichiometrically to Ran-GTP and inhibits the Ran GTPase activating protein. J Biol Chem. 1996 Mar 8;271(10):5313–5316. doi: 10.1074/jbc.271.10.5313. [DOI] [PubMed] [Google Scholar]
  8. Görlich D., Kostka S., Kraft R., Dingwall C., Laskey R. A., Hartmann E., Prehn S. Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope. Curr Biol. 1995 Apr 1;5(4):383–392. doi: 10.1016/s0960-9822(95)00079-0. [DOI] [PubMed] [Google Scholar]
  9. Görlich D., Prehn S., Laskey R. A., Hartmann E. Isolation of a protein that is essential for the first step of nuclear protein import. Cell. 1994 Dec 2;79(5):767–778. doi: 10.1016/0092-8674(94)90067-1. [DOI] [PubMed] [Google Scholar]
  10. Görlich D., Vogel F., Mills A. D., Hartmann E., Laskey R. A. Distinct functions for the two importin subunits in nuclear protein import. Nature. 1995 Sep 21;377(6546):246–248. doi: 10.1038/377246a0. [DOI] [PubMed] [Google Scholar]
  11. Imamoto N., Shimamoto T., Takao T., Tachibana T., Kose S., Matsubae M., Sekimoto T., Shimonishi Y., Yoneda Y. In vivo evidence for involvement of a 58 kDa component of nuclear pore-targeting complex in nuclear protein import. EMBO J. 1995 Aug 1;14(15):3617–3626. doi: 10.1002/j.1460-2075.1995.tb00031.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Imamoto N., Tachibana T., Matsubae M., Yoneda Y. A karyophilic protein forms a stable complex with cytoplasmic components prior to nuclear pore binding. J Biol Chem. 1995 Apr 14;270(15):8559–8565. doi: 10.1074/jbc.270.15.8559. [DOI] [PubMed] [Google Scholar]
  13. Iovine M. K., Watkins J. L., Wente S. R. The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor. J Cell Biol. 1995 Dec;131(6 Pt 2):1699–1713. doi: 10.1083/jcb.131.6.1699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kraemer D. M., Strambio-de-Castillia C., Blobel G., Rout M. P. The essential yeast nucleoporin NUP159 is located on the cytoplasmic side of the nuclear pore complex and serves in karyopherin-mediated binding of transport substrate. J Biol Chem. 1995 Aug 11;270(32):19017–19021. doi: 10.1074/jbc.270.32.19017. [DOI] [PubMed] [Google Scholar]
  15. Melchior F., Paschal B., Evans J., Gerace L. Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor. J Cell Biol. 1993 Dec;123(6 Pt 2):1649–1659. doi: 10.1083/jcb.123.6.1649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Moore M. S., Blobel G. Purification of a Ran-interacting protein that is required for protein import into the nucleus. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10212–10216. doi: 10.1073/pnas.91.21.10212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Moore M. S., Blobel G. The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature. 1993 Oct 14;365(6447):661–663. doi: 10.1038/365661a0. [DOI] [PubMed] [Google Scholar]
  18. Moroianu J., Blobel G., Radu A. Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2008–2011. doi: 10.1073/pnas.92.6.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Moroianu J., Hijikata M., Blobel G., Radu A. Mammalian karyopherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6532–6536. doi: 10.1073/pnas.92.14.6532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nehrbass U., Blobel G. Role of the nuclear transport factor p10 in nuclear import. Science. 1996 Apr 5;272(5258):120–122. doi: 10.1126/science.272.5258.120. [DOI] [PubMed] [Google Scholar]
  21. Paine P. L. Nuclear protein accumulation by facilitated transport and intranuclear binding. Trends Cell Biol. 1993 Oct;3(10):325–329. doi: 10.1016/0962-8924(93)90096-j. [DOI] [PubMed] [Google Scholar]
  22. Paschal B. M., Gerace L. Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62. J Cell Biol. 1995 May;129(4):925–937. doi: 10.1083/jcb.129.4.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Radu A., Blobel G., Moore M. S. Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1769–1773. doi: 10.1073/pnas.92.5.1769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Radu A., Moore M. S., Blobel G. The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex. Cell. 1995 Apr 21;81(2):215–222. doi: 10.1016/0092-8674(95)90331-3. [DOI] [PubMed] [Google Scholar]
  25. Rexach M., Blobel G. Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell. 1995 Dec 1;83(5):683–692. doi: 10.1016/0092-8674(95)90181-7. [DOI] [PubMed] [Google Scholar]
  26. Rout M. P., Wente S. R. Pores for thought: nuclear pore complex proteins. Trends Cell Biol. 1994 Oct;4(10):357–365. doi: 10.1016/0962-8924(94)90085-x. [DOI] [PubMed] [Google Scholar]
  27. Ruediger R., Hentz M., Fait J., Mumby M., Walter G. Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens. J Virol. 1994 Jan;68(1):123–129. doi: 10.1128/jvi.68.1.123-129.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Weis K., Mattaj I. W., Lamond A. I. Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences. Science. 1995 May 19;268(5213):1049–1053. doi: 10.1126/science.7754385. [DOI] [PubMed] [Google Scholar]
  29. Wu J., Matunis M. J., Kraemer D., Blobel G., Coutavas E. Nup358, a cytoplasmically exposed nucleoporin with peptide repeats, Ran-GTP binding sites, zinc fingers, a cyclophilin A homologous domain, and a leucine-rich region. J Biol Chem. 1995 Jun 9;270(23):14209–14213. doi: 10.1074/jbc.270.23.14209. [DOI] [PubMed] [Google Scholar]
  30. Yokoyama N., Hayashi N., Seki T., Panté N., Ohba T., Nishii K., Kuma K., Hayashida T., Miyata T., Aebi U. A giant nucleopore protein that binds Ran/TC4. Nature. 1995 Jul 13;376(6536):184–188. doi: 10.1038/376184a0. [DOI] [PubMed] [Google Scholar]

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