pmc.ncbi.nlm.nih.gov

A nuclear export signal is essential for the cytosolic localization of the Ran binding protein, RanBP1

Abstract

RanBP1 is a Ran/TC4 binding protein that can promote the interaction between Ran and beta-importin /beta-karyopherin, a component of the docking complex for nuclear protein cargo. This interaction occurs through a Ran binding domain (RBD). Here we show that RanBP1 is primarily cytoplasmic, but the isolated RBD accumulates in the nucleus. A region COOH-terminal to the RBD is responsible for this cytoplasmic localization. This domain acts heterologously, localizing a nuclear cyclin B1 mutant to the cytoplasm. The domain contains a nuclear export signal that is necessary but not sufficient for the nuclear export of a functional RBD In transiently transfected cells, epitope-tagged RanBP1 promotes dexamethasone-dependent nuclear accumulation of a glucocorticoid receptor-green fluorescent protein fusion, but the isolated RBD potently inhibits this accumulation. The cytosolic location of RanBP1 may therefore be important for nuclear protein import. RanBP1 may provide a key link between the nuclear import and export pathways.

Full Text

The Full Text of this article is available as a PDF (2.7 MB).

Selected References

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

  1. Aebi M., Clark M. W., Vijayraghavan U., Abelson J. A yeast mutant, PRP20, altered in mRNA metabolism and maintenance of the nuclear structure, is defective in a gene homologous to the human gene RCC1 which is involved in the control of chromosome condensation. Mol Gen Genet. 1990 Oct;224(1):72–80. doi: 10.1007/BF00259453. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Belhumeur P., Lee A., Tam R., DiPaolo T., Fortin N., Clark M. W. GSP1 and GSP2, genetic suppressors of the prp20-1 mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the maintenance of nuclear organization. Mol Cell Biol. 1993 Apr;13(4):2152–2161. doi: 10.1128/mcb.13.4.2152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Bogerd H. P., Fridell R. A., Madore S., Cullen B. R. Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins. Cell. 1995 Aug 11;82(3):485–494. doi: 10.1016/0092-8674(95)90437-9. [DOI] [PubMed] [Google Scholar]
  8. Bressan A., Somma M. P., Lewis J., Santolamazza C., Copeland N. G., Gilbert D. J., Jenkins N. A., Lavia P. Characterization of the opposite-strand genes from the mouse bidirectionally transcribed HTF9 locus. Gene. 1991 Jul 22;103(2):201–209. doi: 10.1016/0378-1119(91)90274-f. [DOI] [PubMed] [Google Scholar]
  9. Clark K. L., Sprague G. F., Jr Yeast pheromone response pathway: characterization of a suppressor that restores mating to receptorless mutants. Mol Cell Biol. 1989 Jun;9(6):2682–2694. doi: 10.1128/mcb.9.6.2682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clarke P. R., Klebe C., Wittinghofer A., Karsenti E. Regulation of Cdc2/cyclin B activation by Ran, a Ras-related GTPase. J Cell Sci. 1995 Mar;108(Pt 3):1217–1225. doi: 10.1242/jcs.108.3.1217. [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. Dasso M., Nishitani H., Kornbluth S., Nishimoto T., Newport J. W. RCC1, a regulator of mitosis, is essential for DNA replication. Mol Cell Biol. 1992 Aug;12(8):3337–3345. doi: 10.1128/mcb.12.8.3337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Davis L. I. The nuclear pore complex. Annu Rev Biochem. 1995;64:865–896. doi: 10.1146/annurev.bi.64.070195.004245. [DOI] [PubMed] [Google Scholar]
  14. Eckert R. L., Katzenellenbogen B. S. Physical properties of estrogen receptor complexes in MCF-7 human breast cancer cells. Differences with anti-estrogen and estrogen. J Biol Chem. 1982 Aug 10;257(15):8840–8846. [PubMed] [Google Scholar]
  15. Fantozzi D. A., Harootunian A. T., Wen W., Taylor S. S., Feramisco J. R., Tsien R. Y., Meinkoth J. L. Thermostable inhibitor of cAMP-dependent protein kinase enhances the rate of export of the kinase catalytic subunit from the nucleus. J Biol Chem. 1994 Jan 28;269(4):2676–2686. [PubMed] [Google Scholar]
  16. Fischer U., Huber J., Boelens W. C., Mattaj I. W., Lührmann R. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell. 1995 Aug 11;82(3):475–483. doi: 10.1016/0092-8674(95)90436-0. [DOI] [PubMed] [Google Scholar]
  17. Forrester W., Stutz F., Rosbash M., Wickens M. Defects in mRNA 3'-end formation, transcription initiation, and mRNA transport associated with the yeast mutation prp20: possible coupling of mRNA processing and chromatin structure. Genes Dev. 1992 Oct;6(10):1914–1926. doi: 10.1101/gad.6.10.1914. [DOI] [PubMed] [Google Scholar]
  18. Gerace L. Nuclear export signals and the fast track to the cytoplasm. Cell. 1995 Aug 11;82(3):341–344. doi: 10.1016/0092-8674(95)90420-4. [DOI] [PubMed] [Google Scholar]
  19. Guddat U., Bakken A. H., Pieler T. Protein-mediated nuclear export of RNA: 5S rRNA containing small RNPs in xenopus oocytes. Cell. 1990 Feb 23;60(4):619–628. doi: 10.1016/0092-8674(90)90665-2. [DOI] [PubMed] [Google Scholar]
  20. Hayashi N., Yokoyama N., Seki T., Azuma Y., Ohba T., Nishimoto T. RanBP1, a Ras-like nuclear G protein binding to Ran/TC4, inhibits RCC1 via Ran/TC4. Mol Gen Genet. 1995 Jun 25;247(6):661–669. doi: 10.1007/BF00290397. [DOI] [PubMed] [Google Scholar]
  21. Heim R., Cubitt A. B., Tsien R. Y. Improved green fluorescence. Nature. 1995 Feb 23;373(6516):663–664. doi: 10.1038/373663b0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. 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]
  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. Matsumoto T., Beach D. Premature initiation of mitosis in yeast lacking RCC1 or an interacting GTPase. Cell. 1991 Jul 26;66(2):347–360. doi: 10.1016/0092-8674(91)90624-8. [DOI] [PubMed] [Google Scholar]
  28. Mattingly R. R., Sorisky A., Brann M. R., Macara I. G. Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain. Mol Cell Biol. 1994 Dec;14(12):7943–7952. doi: 10.1128/mcb.14.12.7943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Melchior F., Gerace L. Mechanisms of nuclear protein import. Curr Opin Cell Biol. 1995 Jun;7(3):310–318. doi: 10.1016/0955-0674(95)80084-0. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Michael W. M., Choi M., Dreyfuss G. A nuclear export signal in hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export pathway. Cell. 1995 Nov 3;83(3):415–422. doi: 10.1016/0092-8674(95)90119-1. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Moroianu J., Blobel G. Protein export from the nucleus requires the GTPase Ran and GTP hydrolysis. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4318–4322. doi: 10.1073/pnas.92.10.4318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ouspenski I. I., Mueller U. W., Matynia A., Sazer S., Elledge S. J., Brinkley B. R. Ran-binding protein-1 is an essential component of the Ran/RCC1 molecular switch system in budding yeast. J Biol Chem. 1995 Feb 3;270(5):1975–1978. doi: 10.1074/jbc.270.5.1975. [DOI] [PubMed] [Google Scholar]
  36. Pines J., Hunter T. The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B. EMBO J. 1994 Aug 15;13(16):3772–3781. doi: 10.1002/j.1460-2075.1994.tb06688.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Piñol-Roma S., Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature. 1992 Feb 20;355(6362):730–732. doi: 10.1038/355730a0. [DOI] [PubMed] [Google Scholar]
  38. Ren M., Coutavas E., D'Eustachio P., Rush M. G. Effects of mutant Ran/TC4 proteins on cell cycle progression. Mol Cell Biol. 1994 Jun;14(6):4216–4224. doi: 10.1128/mcb.14.6.4216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Rudt F., Pieler T. Cytoplasmic retention and nuclear import of 5S ribosomal RNA containing RNPs. EMBO J. 1996 Mar 15;15(6):1383–1391. [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Stutz F., Neville M., Rosbash M. Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast. Cell. 1995 Aug 11;82(3):495–506. doi: 10.1016/0092-8674(95)90438-7. [DOI] [PubMed] [Google Scholar]
  44. Wen W., Meinkoth J. L., Tsien R. Y., Taylor S. S. Identification of a signal for rapid export of proteins from the nucleus. Cell. 1995 Aug 11;82(3):463–473. doi: 10.1016/0092-8674(95)90435-2. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. 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]