RAN/TC4 mutants identify a common requirement for snRNP and protein import into the nucleus (original) (raw)

Abstract

Kinetic competition experiments have demonstrated that at least some factors required for the nuclear import of proteins and U snRNPs are distinct. Both import processes require energy, and in the case of protein import, the energy requirement is known to be at least partly met by GTP hydrolysis by the Ran GTPase. We have compared the effects of nonhydrolyzable GTP analogues and two mutant Ran proteins on the nuclear import of proteins and U snRNPs in vitro. The mutant Ran proteins have different defects; Q69L (glutamine 69 changed to leucine) is defective in GTP hydrolysis while T24N (threonine 24 changed to asparagine) is defective in binding GTP. Both protein and snRNP import are sensitive either to the presence of the two mutant Ran proteins, which act as dominant negative inhibitors of nuclear import, or to incubation with nonhydrolyzable GTP analogues. This demonstrates that there is a requirement for a GTPase activity for the import of U snRNPs, as well as proteins, into the nucleus. The dominant negative effects of the two mutant Ran proteins indicate that the pathways of protein and snRNP import share at lease one common component.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

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., 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]
  3. Bach M., Bringmann P., Lührmann R. Purification of small nuclear ribonucleoprotein particles with antibodies against modified nucleosides of small nuclear RNAs. Methods Enzymol. 1990;181:232–257. doi: 10.1016/0076-6879(90)81125-e. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. Carberry S. E., Darzynkiewicz E., Stepinski J., Tahara S. M., Rhoads R. E., Goss D. J. A spectroscopic study of the binding of N-7-substituted cap analogues to human protein synthesis initiation factor 4E. Biochemistry. 1990 Apr 3;29(13):3337–3341. doi: 10.1021/bi00465a027. [DOI] [PubMed] [Google Scholar]
  9. Carmo-Fonseca M., Tollervey D., Pepperkok R., Barabino S. M., Merdes A., Brunner C., Zamore P. D., Green M. R., Hurt E., Lamond A. I. Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery. EMBO J. 1991 Jan;10(1):195–206. doi: 10.1002/j.1460-2075.1991.tb07936.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Cortes P., Ye Z. S., Baltimore D. 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):7633–7637. doi: 10.1073/pnas.91.16.7633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cuomo C. A., Kirch S. A., Gyuris J., Brent R., Oettinger M. A. 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):6156–6160. doi: 10.1073/pnas.91.13.6156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dasso M. RCC1 in the cell cycle: the regulator of chromosome condensation takes on new roles. Trends Biochem Sci. 1993 Mar;18(3):96–101. doi: 10.1016/0968-0004(93)90161-f. [DOI] [PubMed] [Google Scholar]
  15. Davanloo P., Rosenberg A. H., Dunn J. J., Studier F. W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2035–2039. doi: 10.1073/pnas.81.7.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Der C. J., Finkel T., Cooper G. M. Biological and biochemical properties of human rasH genes mutated at codon 61. Cell. 1986 Jan 17;44(1):167–176. doi: 10.1016/0092-8674(86)90495-2. [DOI] [PubMed] [Google Scholar]
  17. Drivas G. T., Shih A., Coutavas E., Rush M. G., D'Eustachio P. Characterization of four novel ras-like genes expressed in a human teratocarcinoma cell line. Mol Cell Biol. 1990 Apr;10(4):1793–1798. doi: 10.1128/mcb.10.4.1793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dunn J. J., Krippl B., Bernstein K. E., Westphal H., Studier F. W. Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus. Gene. 1988 Sep 7;68(2):259–266. doi: 10.1016/0378-1119(88)90028-5. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Feig L. A., Cooper G. M. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 1988 Aug;8(8):3235–3243. doi: 10.1128/mcb.8.8.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fischer U., Darzynkiewicz E., Tahara S. M., Dathan N. A., Lührmann R., Mattaj I. W. Diversity in the signals required for nuclear accumulation of U snRNPs and variety in the pathways of nuclear transport. J Cell Biol. 1991 May;113(4):705–714. doi: 10.1083/jcb.113.4.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fischer U., Lührmann R. An essential signaling role for the m3G cap in the transport of U1 snRNP to the nucleus. Science. 1990 Aug 17;249(4970):786–790. doi: 10.1126/science.2143847. [DOI] [PubMed] [Google Scholar]
  23. Fischer U., Sumpter V., Sekine M., Satoh T., Lührmann R. Nucleo-cytoplasmic transport of U snRNPs: definition of a nuclear location signal in the Sm core domain that binds a transport receptor independently of the m3G cap. EMBO J. 1993 Feb;12(2):573–583. doi: 10.1002/j.1460-2075.1993.tb05689.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. Hamm J., Darzynkiewicz E., Tahara S. M., Mattaj I. W. The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal. Cell. 1990 Aug 10;62(3):569–577. doi: 10.1016/0092-8674(90)90021-6. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Izaurralde E., Mattaj I. W. RNA export. Cell. 1995 Apr 21;81(2):153–159. doi: 10.1016/0092-8674(95)90323-2. [DOI] [PubMed] [Google Scholar]
  31. Kadowaki T., Goldfarb D., Spitz L. M., Tartakoff A. M., Ohno M. Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily. EMBO J. 1993 Jul;12(7):2929–2937. doi: 10.1002/j.1460-2075.1993.tb05955.x. [DOI] [PMC free article] [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. Kornbluth S., Dasso M., Newport J. Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression. J Cell Biol. 1994 May;125(4):705–719. doi: 10.1083/jcb.125.4.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Küssel P., Frasch M. Pendulin, a Drosophila protein with cell cycle-dependent nuclear localization, is required for normal cell proliferation. J Cell Biol. 1995 Jun;129(6):1491–1507. doi: 10.1083/jcb.129.6.1491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Leupold C. M., Goody R. S., Wittinghofer A. Stereochemistry of the elongation factor Tu X GTP complex. Eur J Biochem. 1983 Sep 15;135(2):237–241. doi: 10.1111/j.1432-1033.1983.tb07643.x. [DOI] [PubMed] [Google Scholar]
  36. Mattaj I. W., De Robertis E. M. Nuclear segregation of U2 snRNA requires binding of specific snRNP proteins. Cell. 1985 Jan;40(1):111–118. doi: 10.1016/0092-8674(85)90314-9. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Michaud N., Goldfarb D. S. Most nuclear proteins are imported by a single pathway. Exp Cell Res. 1993 Sep;208(1):128–136. doi: 10.1006/excr.1993.1230. [DOI] [PubMed] [Google Scholar]
  39. Michaud N., Goldfarb D. S. Multiple pathways in nuclear transport: the import of U2 snRNP occurs by a novel kinetic pathway. J Cell Biol. 1991 Jan;112(2):215–223. doi: 10.1083/jcb.112.2.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Michaud N., Goldfarb D. Microinjected U snRNAs are imported to oocyte nuclei via the nuclear pore complex by three distinguishable targeting pathways. J Cell Biol. 1992 Feb;116(4):851–861. doi: 10.1083/jcb.116.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Moore M. S., Blobel G. 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):939–950. doi: 10.1016/0092-8674(92)90613-h. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. Newmeyer D. D., Forbes D. J. Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell. 1988 Mar 11;52(5):641–653. doi: 10.1016/0092-8674(88)90402-3. [DOI] [PubMed] [Google Scholar]
  47. Newmeyer D. D., Lucocq J. M., Bürglin T. R., De Robertis E. M. Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation. EMBO J. 1986 Mar;5(3):501–510. doi: 10.1002/j.1460-2075.1986.tb04239.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Powers M. A., Forbes D. J. Cytosolic factors in nuclear transport: what's importin? Cell. 1994 Dec 16;79(6):931–934. doi: 10.1016/0092-8674(94)90024-8. [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. Richardson W. D., Mills A. D., Dilworth S. M., Laskey R. A., Dingwall C. 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):655–664. doi: 10.1016/0092-8674(88)90403-5. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Stenmark H., Parton R. G., Steele-Mortimer O., Lütcke A., Gruenberg J., Zerial M. Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBO J. 1994 Mar 15;13(6):1287–1296. doi: 10.1002/j.1460-2075.1994.tb06381.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sweet D. J., Gerace L. Taking from the cytoplasm and giving to the pore: soluble transport factors in nuclear protein import. Trends Cell Biol. 1995 Dec;5(12):444–447. doi: 10.1016/s0962-8924(00)89108-4. [DOI] [PubMed] [Google Scholar]
  55. 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]
  56. Török I., Strand D., Schmitt R., Tick G., Török T., Kiss I., Mechler B. M. 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):1473–1489. doi: 10.1083/jcb.129.6.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. 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]
  59. van Zee K., Dickmanns A., Fischer U., Lührmann R., Fanning E. A cytoplasmically anchored nuclear protein interferes specifically with the import of nuclear proteins but not U1 snRNA. J Cell Biol. 1993 Apr;121(2):229–240. doi: 10.1083/jcb.121.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]