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. 1996 Jun 1;133(5):985–996. doi: 10.1083/jcb.133.5.985

Evidence using a green fluorescent protein-glucocorticoid receptor chimera that the Ran/TC4 GTPase mediates an essential function independent of nuclear protein import

PMCID: PMC2120855  PMID: 8655589

Abstract

The Ran/TC4 GTPase is required for the nuclear accumulation of artificial karyophiles in permeabilized cell assays. To investigate Ran function in a physiologically intact setting using mammalian cells, we examined the effects of several Ran mutants on cell growth and on the nuclear translocation of a glucocorticoid receptor-green fluorescent protein fusion (GR-GFP). Glucocorticoid receptor is cytosolic in the absence of ligand, but translocates to the nucleus on binding the agonist dexamethasone. After transfection into baby hamster kidney cells (BHK21), GR-GFP was detectable in living cells by direct fluorescence microscopy. Addition of dexamethasone caused a rapid translocation of the chimeric protein from the cytosol into the nucleus (t1/2 approximately 5 min). Cotransfection with epitope-tagged, wild- type Ran led to expression of HA1-Ran that was approximately 1.6-fold higher than the level of the endogenous protein, but it had no deleterious effect on nuclear import of the GR-GFP. However, expression of the Ran mutants G19V, T24N, or a COOH-terminal deletion (delta C) mutant dramatically reduced the accumulation of GR-GFP in the nuclei. An L43E mutant of Ran was without significant effect on nuclear GR-GFP import. Identical results were obtained following micro-injection of recombinant Ran mutants into cells expressing GR-GFP. Significantly, all of the Ran mutants, including L43E, strongly inhibited cell growth. These results demonstrate the use of GR-GFP in real-time imaging of nuclear transport. They also show that multiple types of Ran mutant exert dominant effects on this process, and that normal Ran function requires cycling between the GTP- and GDP-bound states of the protein. Most importantly, the results with the L43E Ran mutant provide strong evidence that Ran mediates a function essential to cell viability that is independent of nuclear protein import.

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Selected References

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  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. 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]
  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., 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]
  5. 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]
  6. Bloom E., Matulich D. T., Lan N. C., Higgins S. J., Simons S. S., Baxter J. D. Nuclear binding of glucocorticoid receptors: relations between cytosol binding, activation and the biological response. J Steroid Biochem. 1980 Jan;12:175–184. doi: 10.1016/0022-4731(80)90267-8. [DOI] [PubMed] [Google Scholar]
  7. Bourne H. R. Do GTPases direct membrane traffic in secretion? Cell. 1988 Jun 3;53(5):669–671. doi: 10.1016/0092-8674(88)90081-5. [DOI] [PubMed] [Google Scholar]
  8. Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802–805. doi: 10.1126/science.8303295. [DOI] [PubMed] [Google Scholar]
  9. Cheng Y., Dahlberg J. E., Lund E. Diverse effects of the guanine nucleotide exchange factor RCC1 on RNA transport. Science. 1995 Mar 24;267(5205):1807–1810. doi: 10.1126/science.7534442. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Cserpán I., Udvardy A. The mechanism of nuclear transport of natural or artificial transport substrates in digitonin-permeabilized cells. J Cell Sci. 1995 May;108(Pt 5):1849–1861. doi: 10.1242/jcs.108.5.1849. [DOI] [PubMed] [Google Scholar]
  12. Dascher C., Balch W. E. Dominant inhibitory mutants of ARF1 block endoplasmic reticulum to Golgi transport and trigger disassembly of the Golgi apparatus. J Biol Chem. 1994 Jan 14;269(2):1437–1448. [PubMed] [Google Scholar]
  13. Dasso M., Seki T., Azuma Y., Ohba T., Nishimoto T. A mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a regulator of chromosome condensation. EMBO J. 1994 Dec 1;13(23):5732–5744. doi: 10.1002/j.1460-2075.1994.tb06911.x. [DOI] [PMC free article] [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. 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]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. 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]
  26. 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]
  27. 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]
  28. Picard D., Yamamoto K. R. Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J. 1987 Nov;6(11):3333–3340. doi: 10.1002/j.1460-2075.1987.tb02654.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Qi M., Hamilton B. J., DeFranco D. v-mos oncoproteins affect the nuclear retention and reutilization of glucocorticoid receptors. Mol Endocrinol. 1989 Aug;3(8):1279–1288. doi: 10.1210/mend-3-8-1279. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Ren M., Villamarin A., Shih A., Coutavas E., Moore M. S., LoCurcio M., Clarke V., Oppenheim J. D., D'Eustachio P., Rush M. G. Separate domains of the Ran GTPase interact with different factors to regulate nuclear protein import and RNA processing. Mol Cell Biol. 1995 Apr;15(4):2117–2124. doi: 10.1128/mcb.15.4.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Satoh T., Nakafuku M., Kaziro Y. Function of Ras as a molecular switch in signal transduction. J Biol Chem. 1992 Dec 5;267(34):24149–24152. [PubMed] [Google Scholar]
  36. Sazer S., Nurse P. A fission yeast RCC1-related protein is required for the mitosis to interphase transition. EMBO J. 1994 Feb 1;13(3):606–615. doi: 10.1002/j.1460-2075.1994.tb06298.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. 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]
  40. 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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