Skip to main content
PMC home page
Molecular Biology of the Cell logoLink to Molecular Biology of the Cell
. 1997 Jun;8(6):945–956. doi: 10.1091/mbc.8.6.945

Functional domains in nuclear import factor p97 for binding the nuclear localization sequence receptor and the nuclear pore.

N C Chi 1, S A Adam 1
PMCID: PMC305705  PMID: 9201707

Abstract

The interaction of the nuclear protein import factor p97 with the nuclear localization sequence (NLS) receptor, the nuclear pore complex, and Ran/TC4 is important for coordinating the events of protein import to the nucleus. We have mapped the binding domains on p97 for the NLS receptor and the nuclear pore. The NLS receptor-binding domain of p97 maps to the C-terminal 60% of the protein between residues 356 and 876. The pore complex-binding domain of p97 maps to residues 152-352. The pore complex-binding domain overlaps the Ran-GTP- and Ran-GDP-binding domains on p97, but only Ran-GTP competes for docking in permeabilized cells. The N-ethylmaleimide sensitivity of the p97 for docking was investigated and found to be due to inhibition of p97 binding to the pore complex and to the NLS receptor. Site-directed mutagenesis of conserved cysteine residues in the pore- and receptor-binding domains identified two cysteines, C223 and C228, that were required for p97 to bind the nuclear pore. Inhibition studies on docking and accumulation of a NLS protein provided additional evidence that the domains identified biochemically are the functional domains involved in protein import. Together, these results suggest that Ran-GTP dissociates the receptor complex and prevents p97 binding to the pore by inducing a conformational change in the structure of p97 rather than simple competition for binding sites.

Full text

PDF
945

Images in this article

948Image
on p.948
949Image
on p.949
951Image
on p.951
952Image
on p.952
953Image
on p.953

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. Adam S. A., Adam E. J., Chi N. C., Visser G. D. Cytoplasmic factors in NLS-mediated targeting to the nuclear pore complex. Cold Spring Harb Symp Quant Biol. 1995;60:687–694. doi: 10.1101/sqb.1995.060.01.074. [DOI] [PubMed] [Google Scholar]
  3. Adam S. A., Gerace L. Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell. 1991 Sep 6;66(5):837–847. doi: 10.1016/0092-8674(91)90431-w. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Aitchison J. D., Blobel G., Rout M. P. Kap104p: a karyopherin involved in the nuclear transport of messenger RNA binding proteins. Science. 1996 Oct 25;274(5287):624–627. doi: 10.1126/science.274.5287.624. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Beddow A. L., Richards S. A., Orem N. R., Macara I. G. The Ran/TC4 GTPase-binding domain: identification by expression cloning and characterization of a conserved sequence motif. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3328–3332. doi: 10.1073/pnas.92.8.3328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. Bullock T. L., Clarkson W. D., Kent H. M., Stewart M. The 1.6 angstroms resolution crystal structure of nuclear transport factor 2 (NTF2). J Mol Biol. 1996 Jul 19;260(3):422–431. doi: 10.1006/jmbi.1996.0411. [DOI] [PubMed] [Google Scholar]
  12. Carey K. L., Richards S. A., Lounsbury K. M., Macara I. G. Evidence using a green fluorescent protein-glucocorticoid receptor chimera that the Ran/TC4 GTPase mediates an essential function independent of nuclear protein import. J Cell Biol. 1996 Jun;133(5):985–996. doi: 10.1083/jcb.133.5.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chi N. C., Adam E. J., Adam S. A. Different binding domains for Ran-GTP and Ran-GDP/RanBP1 on nuclear import factor p97. J Biol Chem. 1997 Mar 7;272(10):6818–6822. doi: 10.1074/jbc.272.10.6818. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Corbett A. H., Koepp D. M., Schlenstedt G., Lee M. S., Hopper A. K., Silver P. A. Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import. J Cell Biol. 1995 Sep;130(5):1017–1026. doi: 10.1083/jcb.130.5.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
  17. Distel B., Erdmann R., Gould S. J., Blobel G., Crane D. I., Cregg J. M., Dodt G., Fujiki Y., Goodman J. M., Just W. W. A unified nomenclature for peroxisome biogenesis factors. J Cell Biol. 1996 Oct;135(1):1–3. doi: 10.1083/jcb.135.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Guan T., Müller S., Klier G., Panté N., Blevitt J. M., Haner M., Paschal B., Aebi U., Gerace L. Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex. Mol Biol Cell. 1995 Nov;6(11):1591–1603. doi: 10.1091/mbc.6.11.1591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Görlich D., Henklein P., Laskey R. A., Hartmann E. A 41 amino acid motif in importin-alpha confers binding to importin-beta and hence transit into the nucleus. EMBO J. 1996 Apr 15;15(8):1810–1817. [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Görlich D., Mattaj I. W. Nucleocytoplasmic transport. Science. 1996 Mar 15;271(5255):1513–1518. doi: 10.1126/science.271.5255.1513. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  27. Hu T., Guan T., Gerace L. Molecular and functional characterization of the p62 complex, an assembly of nuclear pore complex glycoproteins. J Cell Biol. 1996 Aug;134(3):589–601. doi: 10.1083/jcb.134.3.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Imamoto N., Shimamoto T., Kose S., Takao T., Tachibana T., Matsubae M., Sekimoto T., Shimonishi Y., Yoneda Y. The nuclear pore-targeting complex binds to nuclear pores after association with a karyophile. FEBS Lett. 1995 Jul 24;368(3):415–419. doi: 10.1016/0014-5793(95)00699-a. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. Koepp D. M., Silver P. A. A GTPase controlling nuclear trafficking: running the right way or walking RANdomly? Cell. 1996 Oct 4;87(1):1–4. doi: 10.1016/s0092-8674(00)81315-x. [DOI] [PubMed] [Google Scholar]
  33. Koepp D. M., Wong D. H., Corbett A. H., Silver P. A. Dynamic localization of the nuclear import receptor and its interactions with transport factors. J Cell Biol. 1996 Jun;133(6):1163–1176. doi: 10.1083/jcb.133.6.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lounsbury K. M., Richards S. A., Perlungher R. R., Macara I. G. Ran binding domains promote the interaction of Ran with p97/beta-karyopherin, linking the docking and translocation steps of nuclear import. J Biol Chem. 1996 Feb 2;271(5):2357–2360. doi: 10.1074/jbc.271.5.2357. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Melchior F., Guan T., Yokoyama N., Nishimoto T., Gerace L. GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import. J Cell Biol. 1995 Nov;131(3):571–581. doi: 10.1083/jcb.131.3.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. 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]
  41. Moroianu J., Blobel G., Radu A. Nuclear protein import: Ran-GTP dissociates the karyopherin alphabeta heterodimer by displacing alpha from an overlapping binding site on beta. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7059–7062. doi: 10.1073/pnas.93.14.7059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Newmeyer D. D., Forbes D. J. An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore. J Cell Biol. 1990 Mar;110(3):547–557. doi: 10.1083/jcb.110.3.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Panté N., Aebi U. Sequential binding of import ligands to distinct nucleopore regions during their nuclear import. Science. 1996 Sep 20;273(5282):1729–1732. doi: 10.1126/science.273.5282.1729. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Pollard V. W., Michael W. M., Nakielny S., Siomi M. C., Wang F., Dreyfuss G. A novel receptor-mediated nuclear protein import pathway. Cell. 1996 Sep 20;86(6):985–994. doi: 10.1016/s0092-8674(00)80173-7. [DOI] [PubMed] [Google Scholar]
  49. Prieve M. G., Guttridge K. L., Munguia J. E., Waterman M. L. The nuclear localization signal of lymphoid enhancer factor-1 is recognized by two differentially expressed Srp1-nuclear localization sequence receptor proteins. J Biol Chem. 1996 Mar 29;271(13):7654–7658. doi: 10.1074/jbc.271.13.7654. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. 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]
  53. Saitoh H., Cooke C. A., Burgess W. H., Earnshaw W. C., Dasso M. Direct and indirect association of the small GTPase ran with nuclear pore proteins and soluble transport factors: studies in Xenopus laevis egg extracts. Mol Biol Cell. 1996 Sep;7(9):1319–1334. doi: 10.1091/mbc.7.9.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Schlenstedt G., Saavedra C., Loeb J. D., Cole C. N., Silver P. A. The GTP-bound form of the yeast Ran/TC4 homologue blocks nuclear protein import and appearance of poly(A)+ RNA in the cytoplasm. Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):225–229. doi: 10.1073/pnas.92.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Siomi H., Dreyfuss G. A nuclear localization domain in the hnRNP A1 protein. J Cell Biol. 1995 May;129(3):551–560. doi: 10.1083/jcb.129.3.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tachibana T., Imamoto N., Seino H., Nishimoto T., Yoneda Y. Loss of RCC1 leads to suppression of nuclear protein import in living cells. J Biol Chem. 1994 Oct 7;269(40):24542–24545. [PubMed] [Google Scholar]
  58. 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]
  59. Weis K., Ryder U., Lamond A. I. The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import. EMBO J. 1996 Apr 15;15(8):1818–1825. [PMC free article] [PubMed] [Google Scholar]
  60. 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]
  61. Yano R., Oakes M., Yamaghishi M., Dodd J. A., Nomura M. Cloning and characterization of SRP1, a suppressor of temperature-sensitive RNA polymerase I mutations, in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Dec;12(12):5640–5651. doi: 10.1128/mcb.12.12.5640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. 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]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology

RESOURCES