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PMID: 9003787 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Characterization of the nuclear protein import mechanism using Ran mutants with altered nucleotide binding specificities.

The EMBO journal ·Vol. 15 ·No. 24 ·1996-12-16 ·Pages 7120-8

Weis K, Dingwall C, Lamond AI

Abstract

The small nuclear GTP binding protein Ran is required for transport of nuclear proteins through the nuclear pore complex (NPC). Although it is known that GTP hydrolysis by Ran is essential for this reaction, it has been unclear whether additional energy-consuming steps are also required. To uncouple the energy requirements for Ran from other nucleoside triphosphatases, we constructed a mutant derivative of Ran that has an altered nucleotide specificity from GTP to xanthosine 5' triphosphate. Using this Ran mutant, we demonstrate that nucleotide hydrolysis by Ran is sufficient to promote efficient nuclear protein import in vitro. Under these conditions, protein import could no longer be inhibited with non-hydrolysable nucleotide analogues, indicating that no Ran-independent energy-requiring steps are essential for the protein translocation reaction through the NPC. We further provide evidence that nuclear protein import requires Ran in the GDP form in the cytoplasm. This suggests that a coordinated exchange reaction from Ran-GDP to Ran-GTP at the pore is necessary for translocation into the nucleus.

MeSH Terms
Binding Sites Biological Transport Mutation Nuclear Proteins/genetics,metabolism Nucleotides/metabolism ran GTP-Binding Protein
Chemicals
Nuclear Proteins Nucleotides ran GTP-Binding Protein
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Weis K
Department of Microbiology and Immunology, University of California, San Francisco 94143-0414, USA.
Dingwall C
Lamond A I
References (70)
70 references, click to expand
  1. 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):225-9 PMID: 7816822
  2. Cytosolic factors in nuclear transport: what's importin?
    Cell. 1994 Dec 16;79(6):931-4 PMID: 8001141
  3. RanGAP1 induces GTPase activity of nuclear Ras-related Ran.
    Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2587-91 PMID: 8146159
  4. 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):6532-6 PMID: 7604027
  5. A family of Ran binding proteins that includes nucleoporins.
    Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7525-9 PMID: 7638224
  6. 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):14209-13 PMID: 7775481
  7. 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):1699-713 PMID: 8557738
  8. Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation.
    Cell. 1988 Mar 11;52(5):641-53 PMID: 3345567
  9. Sequence and characterization of cytoplasmic nuclear protein import factor p97.
    J Cell Biol. 1995 Jul;130(2):265-74 PMID: 7615630
  10. The nuclear pore-targeting complex binds to nuclear pores after association with a karyophile.
    FEBS Lett. 1995 Jul 24;368(3):415-9 PMID: 7635189
  11. 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):2357-60 PMID: 8576188
  12. 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):1749-53 PMID: 7878053
  13. RNA export.
    Cell. 1995 Apr 21;81(2):153-9 PMID: 7537634
  14. Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins.
    Cell. 1995 Dec 1;83(5):683-92 PMID: 8521485
  15. The RCC1 protein interacts with Ran, RanBP1, hsc70, and a 340-kDa protein in Xenopus extracts.
    J Biol Chem. 1995 May 5;270(18):10658-63 PMID: 7738003
  16. Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1.
    Biochemistry. 1995 Jan 17;34(2):639-47 PMID: 7819259
  17. Reciprocal stimulation of GTP hydrolysis by two directly interacting GTPases.
    Science. 1995 Sep 8;269(5229):1422-4 PMID: 7660124
  18. A GTPase distinct from Ran is involved in nuclear protein import.
    J Cell Biol. 1996 Jun;133(5):971-83 PMID: 8655588
  19. Nuclear targeting sequences--a consensus?
    Trends Biochem Sci. 1991 Dec;16(12):478-81 PMID: 1664152
  20. GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import.
    J Cell Biol. 1995 Nov;131(3):571-81 PMID: 7593180
  21. 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):309-21 PMID: 2116418
  22. A giant nucleopore protein that binds Ran/TC4.
    Nature. 1995 Jul 13;376(6536):184-8 PMID: 7603572
  23. 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):1649-59 PMID: 8276887
  24. 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):1769-73 PMID: 7878057
  25. 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):2008-11 PMID: 7892216
  26. Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1.
    Nature. 1991 Nov 7;354(6348):80-2 PMID: 1944575
  27. Rapid and efficient site-specific mutagenesis without phenotypic selection.
    Proc Natl Acad Sci U S A. 1985 Jan;82(2):488-92 PMID: 3881765
  28. Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide.
    Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10830-4 PMID: 1961752
  29. A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein.
    J Biol Chem. 1987 Sep 25;262(27):13081-5 PMID: 3308869
  30. Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences.
    Science. 1995 May 19;268(5213):1049-53 PMID: 7754385
  31. RAG-1 interacts with the repeated amino acid motif of the human homologue of the yeast protein SRP1.
    Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7633-7 PMID: 8052633
  32. Pendulin, a Drosophila protein with cell cycle-dependent nuclear localization, is required for normal cell proliferation.
    J Cell Biol. 1995 Jun;129(6):1491-507 PMID: 7790350
  33. The overgrown hematopoietic organs-31 tumor suppressor gene of Drosophila encodes an Importin-like protein accumulating in the nucleus at the onset of mitosis.
    J Cell Biol. 1995 Jun;129(6):1473-89 PMID: 7790349
  34. Structural and functional organization of the nuclear envelope.
    Curr Opin Cell Biol. 1995 Jun;7(3):301-9 PMID: 7662358
  35. RCC1 in the cell cycle: the regulator of chromosome condensation takes on new roles.
    Trends Biochem Sci. 1993 Mar;18(3):96-101 PMID: 8480369
  36. 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):10212-6 PMID: 7937864
  37. Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1.
    EMBO J. 1995 Feb 15;14(4):705-15 PMID: 7882974
  38. 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):3617-26 PMID: 7641681
  39. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors.
    J Cell Biol. 1990 Sep;111(3):807-16 PMID: 2391365
  40. Molecular cloning of a novel mitogen-inducible nuclear protein with a Ran GTPase-activating domain that affects cell cycle progression.
    Mol Cell Biol. 1995 Jan;15(1):552-60 PMID: 7799964
  41. The small nuclear GTPase Ran: how much does it run?
    Bioessays. 1996 Feb;18(2):103-12 PMID: 8851043
  42. NPI-1, the human homolog of SRP-1, interacts with influenza virus nucleoprotein.
    Virology. 1995 Jan 10;206(1):116-25 PMID: 7831767
  43. RAN/TC4 mutants identify a common requirement for snRNP and protein import into the nucleus.
    J Cell Biol. 1996 May;133(3):485-94 PMID: 8636225
  44. Loss of RCC1 leads to suppression of nuclear protein import in living cells.
    J Biol Chem. 1994 Oct 7;269(40):24542-5 PMID: 7929123
  45. Mechanisms of nuclear protein import.
    Curr Opin Cell Biol. 1995 Jun;7(3):310-8 PMID: 7662359
  46. Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores.
    Cell. 1988 Mar 11;52(5):655-64 PMID: 3125984
  47. Distinct functions for the two importin subunits in nuclear protein import.
    Nature. 1995 Sep 21;377(6546):246-8 PMID: 7675110
  48. 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):383-92 PMID: 7627554
  49. Toward a model for the interaction between elongation factor Tu and the ribosome.
    Science. 1993 Feb 26;259(5099):1311-4 PMID: 8446899
  50. Cloning and characterization of SRP1, a suppressor of temperature-sensitive RNA polymerase I mutations, in Saccharomyces cerevisiae.
    Mol Cell Biol. 1992 Dec;12(12):5640-51 PMID: 1448093
  51. Crystal structure of the nuclear Ras-related protein Ran in its GDP-bound form.
    Nature. 1995 Mar 23;374(6520):378-81 PMID: 7885480
  52. Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import.
    J Cell Biol. 1995 Sep;130(5):1017-26 PMID: 7657689
  53. Nucleocytoplasmic transport.
    Science. 1996 Mar 15;271(5255):1513-8 PMID: 8599106
  54. Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import.
    Cell. 1991 Sep 6;66(5):837-47 PMID: 1653647
  55. 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):1810-7 PMID: 8617226
  56. The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import.
    EMBO J. 1996 Apr 15;15(8):1818-25 PMID: 8617227
  57. Identification of a yeast karyopherin heterodimer that targets import substrate to mammalian nuclear pore complexes.
    J Biol Chem. 1995 Jul 14;270(28):16499-502 PMID: 7622450
  58. Structure and function of the nuclear pore complex.
    Annu Rev Cell Biol. 1992;8:495-527 PMID: 1282353
  59. Functional expression in Escherichia coli of the mitotic regulator proteins p24ran and p45rcc1 and fluorescence measurements of their interaction.
    Biochemistry. 1993 Nov 9;32(44):11923-8 PMID: 8218265
  60. Rch1, a protein that specifically interacts with the RAG-1 recombination-activating protein.
    Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6156-60 PMID: 8016130
  61. Role of the nuclear transport factor p10 in nuclear import.
    Science. 1996 Apr 5;272(5258):120-2 PMID: 8600522
  62. Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62.
    J Cell Biol. 1995 May;129(4):925-37 PMID: 7744965
  63. Taking from the cytoplasm and giving to the pore: soluble transport factors in nuclear protein import.
    Trends Cell Biol. 1995 Dec;5(12):444-7 PMID: 14732023
  64. Replacement of Asp333 with Asn by site-directed mutagenesis changes the substrate specificity of Escherichia coli adenylosuccinate synthetase from guanosine 5'-triphosphate to xanthosine 5'-triphosphate.
    J Biol Chem. 1994 Sep 30;269(39):24046-9 PMID: 7929056
  65. The structure of Ras protein: a model for a universal molecular switch.
    Trends Biochem Sci. 1991 Oct;16(10):382-7 PMID: 1785141
  66. The GTP-binding protein Ran/TC4 is required for protein import into the nucleus.
    Nature. 1993 Oct 14;365(6447):661-3 PMID: 8413630
  67. 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):5313-6 PMID: 8621381
  68. 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):569-81 PMID: 8374168
  69. Isolation of a protein that is essential for the first step of nuclear protein import.
    Cell. 1994 Dec 2;79(5):767-78 PMID: 8001116
  70. The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors.
    Cell. 1992 Jun 12;69(6):939-50 PMID: 1606616
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1996-12-16
Pages
7120-8
Language
English
Region
England
NLM ID
8208664
PMCID
PMC452537
Subset
IM
Grants
Wellcome Trust · United Kingdom
External Links
PubMed source PMC Full Text View journal details
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