Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Nov 16;17(22):6587–6598. doi: 10.1093/emboj/17.22.6587

NTF2 mediates nuclear import of Ran.

K Ribbeck 1, G Lipowsky 1, H M Kent 1, M Stewart 1, D Görlich 1
PMCID: PMC1171005  PMID: 9822603

Abstract

Importin beta family transport receptors shuttle between the nucleus and the cytoplasm and mediate transport of macromolecules through nuclear pore complexes (NPCs). The interactions between these receptors and their cargoes are regulated by binding RanGTP; all receptors probably exit the nucleus complexed with RanGTP, and so should deplete RanGTP continuously from the nucleus. We describe here the development of an in vitro system to study how nuclear Ran is replenished. Nuclear import of Ran does not rely on simple diffusion as Ran's small size would permit, but instead is stimulated by soluble transport factors. This facilitated import is specific for cytoplasmic RanGDP and employs nuclear transport factor 2 (NTF2) as the actual carrier. NTF2 binds RanGDP initially to NPCs and probably also mediates translocation of the NTF2-RanGDP complex to the nuclear side of the NPCs. A direct NTF2-RanGDP interaction is crucial for this process, since point mutations that disturb the RanGDP-NTF2 interaction also interfere with Ran import. The subsequent nuclear accumulation of Ran also requires GTP, but not GTP hydrolysis. The release of Ran from NTF2 into the nucleus, and thus the directionality of Ran import, probably involves nucleotide exchange to generate RanGTP, for which NTF2 has no detectable affinity, followed by binding of the RanGTP to an importin beta family transport receptor.

Full Text

The Full Text of this article is available as a PDF (599.4 KB).

Selected References

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

  1. Adam S. A., Marr R. S., Gerace L. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol. 1990 Sep;111(3):807–816. doi: 10.1083/jcb.111.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arts G. J., Fornerod M., Mattaj I. W. Identification of a nuclear export receptor for tRNA. Curr Biol. 1998 Mar 12;8(6):305–314. doi: 10.1016/s0960-9822(98)70130-7. [DOI] [PubMed] [Google Scholar]
  3. Becker J., Melchior F., Gerke V., Bischoff F. R., Ponstingl H., Wittinghofer A. RNA1 encodes a GTPase-activating protein specific for Gsp1p, the Ran/TC4 homologue of Saccharomyces cerevisiae. J Biol Chem. 1995 May 19;270(20):11860–11865. doi: 10.1074/jbc.270.20.11860. [DOI] [PubMed] [Google Scholar]
  4. Bischoff F. R., Görlich D. RanBP1 is crucial for the release of RanGTP from importin beta-related nuclear transport factors. FEBS Lett. 1997 Dec 15;419(2-3):249–254. doi: 10.1016/s0014-5793(97)01467-1. [DOI] [PubMed] [Google Scholar]
  5. Bischoff F. R., Klebe C., Kretschmer J., Wittinghofer A., Ponstingl H. RanGAP1 induces GTPase activity of nuclear Ras-related Ran. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2587–2591. doi: 10.1073/pnas.91.7.2587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bischoff F. R., Krebber H., Kempf T., Hermes I., Ponstingl H. Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1749–1753. doi: 10.1073/pnas.92.5.1749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bischoff F. R., Krebber H., Smirnova E., Dong W., Ponstingl H. Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1. EMBO J. 1995 Feb 15;14(4):705–715. doi: 10.1002/j.1460-2075.1995.tb07049.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bischoff F. R., Ponstingl H. Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1. Nature. 1991 Nov 7;354(6348):80–82. doi: 10.1038/354080a0. [DOI] [PubMed] [Google Scholar]
  9. Bischoff F. R., Ponstingl H. Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10830–10834. doi: 10.1073/pnas.88.23.10830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Breeuwer M., Goldfarb D. S. Facilitated nuclear transport of histone H1 and other small nucleophilic proteins. Cell. 1990 Mar 23;60(6):999–1008. doi: 10.1016/0092-8674(90)90348-i. [DOI] [PubMed] [Google Scholar]
  11. Chi N. C., Adam E. J., Visser G. D., Adam S. A. RanBP1 stabilizes the interaction of Ran with p97 nuclear protein import. J Cell Biol. 1996 Nov;135(3):559–569. doi: 10.1083/jcb.135.3.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Clarkson W. D., Corbett A. H., Paschal B. M., Kent H. M., McCoy A. J., Gerace L., Silver P. A., Stewart M. Nuclear protein import is decreased by engineered mutants of nuclear transport factor 2 (NTF2) that do not bind GDP-Ran. J Mol Biol. 1997 Oct 10;272(5):716–730. doi: 10.1006/jmbi.1997.1255. [DOI] [PubMed] [Google Scholar]
  13. Clarkson W. D., Kent H. M., Stewart M. Separate binding sites on nuclear transport factor 2 (NTF2) for GDP-Ran and the phenylalanine-rich repeat regions of nucleoporins p62 and Nsp1p. J Mol Biol. 1996 Nov 8;263(4):517–524. doi: 10.1006/jmbi.1996.0594. [DOI] [PubMed] [Google Scholar]
  14. Corbett A. H., Silver P. A. The NTF2 gene encodes an essential, highly conserved protein that functions in nuclear transport in vivo. J Biol Chem. 1996 Aug 2;271(31):18477–18484. doi: 10.1074/jbc.271.31.18477. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Dahlberg J. E., Lund E. Functions of the GTPase Ran in RNA export from the nucleus. Curr Opin Cell Biol. 1998 Jun;10(3):400–408. doi: 10.1016/s0955-0674(98)80017-3. [DOI] [PubMed] [Google Scholar]
  17. Feldherr C. M., Kallenbach E., Schultz N. Movement of a karyophilic protein through the nuclear pores of oocytes. J Cell Biol. 1984 Dec;99(6):2216–2222. doi: 10.1083/jcb.99.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Feldherr C., Akin D., Moore M. S. The nuclear import factor p10 regulates the functional size of the nuclear pore complex during oogenesis. J Cell Sci. 1998 Jul;111(Pt 13):1889–1896. doi: 10.1242/jcs.111.13.1889. [DOI] [PubMed] [Google Scholar]
  19. Floer M., Blobel G., Rexach M. Disassembly of RanGTP-karyopherin beta complex, an intermediate in nuclear protein import. J Biol Chem. 1997 Aug 1;272(31):19538–19546. doi: 10.1074/jbc.272.31.19538. [DOI] [PubMed] [Google Scholar]
  20. Fornerod M., Ohno M., Yoshida M., Mattaj I. W. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell. 1997 Sep 19;90(6):1051–1060. doi: 10.1016/s0092-8674(00)80371-2. [DOI] [PubMed] [Google Scholar]
  21. Fornerod M., van Deursen J., van Baal S., Reynolds A., Davis D., Murti K. G., Fransen J., Grosveld G. The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88. EMBO J. 1997 Feb 17;16(4):807–816. doi: 10.1093/emboj/16.4.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fridell R. A., Truant R., Thorne L., Benson R. E., Cullen B. R. Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta. J Cell Sci. 1997 Jun;110(Pt 11):1325–1331. doi: 10.1242/jcs.110.11.1325. [DOI] [PubMed] [Google Scholar]
  23. Görlich D., Dabrowski M., Bischoff F. R., Kutay U., Bork P., Hartmann E., Prehn S., Izaurralde E. A novel class of RanGTP binding proteins. J Cell Biol. 1997 Jul 14;138(1):65–80. doi: 10.1083/jcb.138.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Görlich D., Kraft R., Kostka S., Vogel F., Hartmann E., Laskey R. A., Mattaj I. W., Izaurralde E. Importin provides a link between nuclear protein import and U snRNA export. Cell. 1996 Oct 4;87(1):21–32. doi: 10.1016/s0092-8674(00)81319-7. [DOI] [PubMed] [Google Scholar]
  25. Görlich D., Panté N., Kutay U., Aebi U., Bischoff F. R. Identification of different roles for RanGDP and RanGTP in nuclear protein import. EMBO J. 1996 Oct 15;15(20):5584–5594. [PMC free article] [PubMed] [Google Scholar]
  26. Görlich D. Transport into and out of the cell nucleus. EMBO J. 1998 May 15;17(10):2721–2727. doi: 10.1093/emboj/17.10.2721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hopper A. K., Traglia H. M., Dunst R. W. The yeast RNA1 gene product necessary for RNA processing is located in the cytosol and apparently excluded from the nucleus. J Cell Biol. 1990 Aug;111(2):309–321. doi: 10.1083/jcb.111.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Izaurralde E., Adam S. Transport of macromolecules between the nucleus and the cytoplasm. RNA. 1998 Apr;4(4):351–364. [PMC free article] [PubMed] [Google Scholar]
  29. Izaurralde E., Kutay U., von Kobbe C., Mattaj I. W., Görlich D. The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus. EMBO J. 1997 Nov 3;16(21):6535–6547. doi: 10.1093/emboj/16.21.6535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jäkel S., Görlich D. Importin beta, transportin, RanBP5 and RanBP7 mediate nuclear import of ribosomal proteins in mammalian cells. EMBO J. 1998 Aug 3;17(15):4491–4502. doi: 10.1093/emboj/17.15.4491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kent H. M., Clarkson W. D., Bullock T. L., Stewart M. Crystallization and preliminary X-ray diffraction analysis of nuclear transport factor 2. J Struct Biol. 1996 Mar-Apr;116(2):326–329. doi: 10.1006/jsbi.1996.0049. [DOI] [PubMed] [Google Scholar]
  32. Klebe C., Bischoff F. R., Ponstingl H., Wittinghofer A. Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1. Biochemistry. 1995 Jan 17;34(2):639–647. doi: 10.1021/bi00002a031. [DOI] [PubMed] [Google Scholar]
  33. Kose S., Imamoto N., Tachibana T., Shimamoto T., Yoneda Y. Ran-unassisted nuclear migration of a 97-kD component of nuclear pore-targeting complex. J Cell Biol. 1997 Nov 17;139(4):841–849. doi: 10.1083/jcb.139.4.841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kutay U., Bischoff F. R., Kostka S., Kraft R., Görlich D. Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor. Cell. 1997 Sep 19;90(6):1061–1071. doi: 10.1016/s0092-8674(00)80372-4. [DOI] [PubMed] [Google Scholar]
  35. Kutay U., Izaurralde E., Bischoff F. R., Mattaj I. W., Görlich D. Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex. EMBO J. 1997 Mar 17;16(6):1153–1163. doi: 10.1093/emboj/16.6.1153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kutay U., Lipowsky G., Izaurralde E., Bischoff F. R., Schwarzmaier P., Hartmann E., Görlich D. Identification of a tRNA-specific nuclear export receptor. Mol Cell. 1998 Feb;1(3):359–369. doi: 10.1016/s1097-2765(00)80036-2. [DOI] [PubMed] [Google Scholar]
  37. Lounsbury K. M., Macara I. G. Ran-binding protein 1 (RanBP1) forms a ternary complex with Ran and karyopherin beta and reduces Ran GTPase-activating protein (RanGAP) inhibition by karyopherin beta. J Biol Chem. 1997 Jan 3;272(1):551–555. doi: 10.1074/jbc.272.1.551. [DOI] [PubMed] [Google Scholar]
  38. Mahajan R., Delphin C., Guan T., Gerace L., Melchior F. A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell. 1997 Jan 10;88(1):97–107. doi: 10.1016/s0092-8674(00)81862-0. [DOI] [PubMed] [Google Scholar]
  39. Mattaj I. W., Englmeier L. Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem. 1998;67:265–306. doi: 10.1146/annurev.biochem.67.1.265. [DOI] [PubMed] [Google Scholar]
  40. Matunis M. J., Coutavas E., Blobel G. A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol. 1996 Dec;135(6 Pt 1):1457–1470. doi: 10.1083/jcb.135.6.1457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Melchior F., Weber K., Gerke V. A functional homologue of the RNA1 gene product in Schizosaccharomyces pombe: purification, biochemical characterization, and identification of a leucine-rich repeat motif. Mol Biol Cell. 1993 Jun;4(6):569–581. doi: 10.1091/mbc.4.6.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Nakielny S., Dreyfuss G. Import and export of the nuclear protein import receptor transportin by a mechanism independent of GTP hydrolysis. Curr Biol. 1998 Jan 15;8(2):89–95. doi: 10.1016/s0960-9822(98)70039-9. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Ohtsubo M., Okazaki H., Nishimoto T. The RCC1 protein, a regulator for the onset of chromosome condensation locates in the nucleus and binds to DNA. J Cell Biol. 1989 Oct;109(4 Pt 1):1389–1397. doi: 10.1083/jcb.109.4.1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Palacios I., Weis K., Klebe C., Mattaj I. W., Dingwall C. RAN/TC4 mutants identify a common requirement for snRNP and protein import into the nucleus. J Cell Biol. 1996 May;133(3):485–494. doi: 10.1083/jcb.133.3.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Paschal B. M., Delphin C., Gerace L. Nucleotide-specific interaction of Ran/TC4 with nuclear transport factors NTF2 and p97. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7679–7683. doi: 10.1073/pnas.93.15.7679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Paschal B. M., Fritze C., Guan T., Gerace L. High levels of the GTPase Ran/TC4 relieve the requirement for nuclear protein transport factor 2. J Biol Chem. 1997 Aug 22;272(34):21534–21539. doi: 10.1074/jbc.272.34.21534. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Percipalle P., Clarkson W. D., Kent H. M., Rhodes D., Stewart M. Molecular interactions between the importin alpha/beta heterodimer and proteins involved in vertebrate nuclear protein import. J Mol Biol. 1997 Mar 7;266(4):722–732. doi: 10.1006/jmbi.1996.0801. [DOI] [PubMed] [Google Scholar]
  53. Ren M., Drivas G., D'Eustachio P., Rush M. G. Ran/TC4: a small nuclear GTP-binding protein that regulates DNA synthesis. J Cell Biol. 1993 Jan;120(2):313–323. doi: 10.1083/jcb.120.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Richards S. A., Carey K. L., Macara I. G. Requirement of guanosine triphosphate-bound ran for signal-mediated nuclear protein export. Science. 1997 Jun 20;276(5320):1842–1844. doi: 10.1126/science.276.5320.1842. [DOI] [PubMed] [Google Scholar]
  56. Richards S. A., Lounsbury K. M., Carey K. L., Macara I. G. A nuclear export signal is essential for the cytosolic localization of the Ran binding protein, RanBP1. J Cell Biol. 1996 Sep;134(5):1157–1168. doi: 10.1083/jcb.134.5.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Richards S. A., Lounsbury K. M., Macara I. G. The C terminus of the nuclear RAN/TC4 GTPase stabilizes the GDP-bound state and mediates interactions with RCC1, RAN-GAP, and HTF9A/RANBP1. J Biol Chem. 1995 Jun 16;270(24):14405–14411. doi: 10.1074/jbc.270.24.14405. [DOI] [PubMed] [Google Scholar]
  58. Rout M. P., Blobel G., Aitchison J. D. A distinct nuclear import pathway used by ribosomal proteins. Cell. 1997 May 30;89(5):715–725. doi: 10.1016/s0092-8674(00)80254-8. [DOI] [PubMed] [Google Scholar]
  59. Saitoh H., Pu R., Cavenagh M., Dasso M. RanBP2 associates with Ubc9p and a modified form of RanGAP1. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3736–3741. doi: 10.1073/pnas.94.8.3736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Scheffzek K., Klebe C., Fritz-Wolf K., Kabsch W., Wittinghofer A. Crystal structure of the nuclear Ras-related protein Ran in its GDP-bound form. Nature. 1995 Mar 23;374(6520):378–381. doi: 10.1038/374378a0. [DOI] [PubMed] [Google Scholar]
  61. Schlenstedt G., Smirnova E., Deane R., Solsbacher J., Kutay U., Görlich D., Ponstingl H., Bischoff F. R. Yrb4p, a yeast ran-GTP-binding protein involved in import of ribosomal protein L25 into the nucleus. EMBO J. 1997 Oct 15;16(20):6237–6249. doi: 10.1093/emboj/16.20.6237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Schlenstedt G., Wong D. H., Koepp D. M., Silver P. A. Mutants in a yeast Ran binding protein are defective in nuclear transport. EMBO J. 1995 Nov 1;14(21):5367–5378. doi: 10.1002/j.1460-2075.1995.tb00221.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Siomi M. C., Eder P. S., Kataoka N., Wan L., Liu Q., Dreyfuss G. Transportin-mediated nuclear import of heterogeneous nuclear RNP proteins. J Cell Biol. 1997 Sep 22;138(6):1181–1192. doi: 10.1083/jcb.138.6.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Stewart M., Kent H. M., McCoy A. J. Structural basis for molecular recognition between nuclear transport factor 2 (NTF2) and the GDP-bound form of the Ras-family GTPase Ran. J Mol Biol. 1998 Apr 3;277(3):635–646. doi: 10.1006/jmbi.1997.1602. [DOI] [PubMed] [Google Scholar]
  65. Weis K., Dingwall C., Lamond A. I. Characterization of the nuclear protein import mechanism using Ran mutants with altered nucleotide binding specificities. EMBO J. 1996 Dec 16;15(24):7120–7128. [PMC free article] [PubMed] [Google Scholar]
  66. Wong D. H., Corbett A. H., Kent H. M., Stewart M., Silver P. A. Interaction between the small GTPase Ran/Gsp1p and Ntf2p is required for nuclear transport. Mol Cell Biol. 1997 Jul;17(7):3755–3767. doi: 10.1128/mcb.17.7.3755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Xiao Z., McGrew J. T., Schroeder A. J., Fitzgerald-Hayes M. CSE1 and CSE2, two new genes required for accurate mitotic chromosome segregation in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Aug;13(8):4691–4702. doi: 10.1128/mcb.13.8.4691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Zasloff M. tRNA transport from the nucleus in a eukaryotic cell: carrier-mediated translocation process. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6436–6440. doi: 10.1073/pnas.80.21.6436. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES