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. 1997 Sep;17(9):5087–5096. doi: 10.1128/mcb.17.9.5087

Ran-binding protein 5 (RanBP5) is related to the nuclear transport factor importin-beta but interacts differently with RanBP1.

R Deane 1, W Schäfer 1, H P Zimmermann 1, L Mueller 1, D Görlich 1, S Prehn 1, H Ponstingl 1, F R Bischoff 1
PMCID: PMC232359  PMID: 9271386

Abstract

We report the identification and characterization of a novel 124-kDa Ran binding protein, RanBP5. This protein is related to importin-beta, the key mediator of nuclear localization signal (NLS)-dependent nuclear transport. RanBP5 was identified by two independent methods: it was isolated from HeLa cells by using its interaction with RanGTP in an overlay assay to monitor enrichment, and it was also found by the yeast two-hybrid selection method with RanBP1 as bait. RanBP5 binds to RanBP1 as part of a trimeric RanBP1-Ran-RanBP5 complex. Like importin-beta, RanBP5 strongly binds the GTP-bound form of Ran, stabilizing it against both intrinsic and RanGAP1-induced GTP hydrolysis and also against nucleotide exchange. The GAP resistance of the RanBP5-RanGTP complex can be relieved by RanBP1, which might reflect an in vivo role for RanBP1. RanBP5 is a predominantly cytoplasmic protein that can bind to nuclear pore complexes. We propose that RanBP5 is a mediator of a nucleocytoplasmic transport pathway that is distinct from the importin-alpha-dependent import of proteins with a classical NLS.

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Selected References

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  1. 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]
  2. Baum C., Forster P., Hegewisch-Becker S., Harbers K. An optimized electroporation protocol applicable to a wide range of cell lines. Biotechniques. 1994 Dec;17(6):1058–1062. [PubMed] [Google Scholar]
  3. 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]
  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., 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]
  7. 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]
  8. 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]
  9. 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]
  10. Chow T. Y., Ash J. J., Dignard D., Thomas D. Y. Screening and identification of a gene, PSE-1, that affects protein secretion in Saccharomyces cerevisiae. J Cell Sci. 1992 Mar;101(Pt 3):709–719. doi: 10.1242/jcs.101.3.709. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. doi: 10.1101/gad.7.4.555. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  20. Klebe C., Nishimoto T., Wittinghofer F. 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):11923–11928. doi: 10.1021/bi00095a023. [DOI] [PubMed] [Google Scholar]
  21. Klebe C., Prinz H., Wittinghofer A., Goody R. S. The kinetic mechanism of Ran--nucleotide exchange catalyzed by RCC1. Biochemistry. 1995 Oct 3;34(39):12543–12552. doi: 10.1021/bi00039a008. [DOI] [PubMed] [Google Scholar]
  22. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Lounsbury K. M., Beddow A. L., Macara I. G. A family of proteins that stabilize the Ran/TC4 GTPase in its GTP-bound conformation. J Biol Chem. 1994 Apr 15;269(15):11285–11290. [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. 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]
  31. 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]
  32. 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]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. 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]
  39. Rush M. G., Drivas G., D'Eustachio P. The small nuclear GTPase Ran: how much does it run? Bioessays. 1996 Feb;18(2):103–112. doi: 10.1002/bies.950180206. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Sazer S. The search for the primary function of the Ran GTPase continues. Trends Cell Biol. 1996 Mar;6(3):81–85. doi: 10.1016/0962-8924(96)80992-5. [DOI] [PubMed] [Google Scholar]
  42. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. Yaseen N. R., Blobel G. Cloning and characterization of human karyopherin beta3. Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4451–4456. doi: 10.1073/pnas.94.9.4451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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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