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J Cell Biol. 1993 Dec 2; 123(6): 1649–1659.
PMCID: PMC2290879

Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor [published erratum appears in J Cell Biol 1994 Jan;124(1-2):217]

Abstract

We have investigated a possible involvement of GTPases in nuclear protein import using an in vitro transport system involving digitonin- permeabilized cells supplemented with exogenous cytosol. Transport in this system was measured with a novel ELISA-based assay that allows rapid quantitative analysis. GTP gamma S and other nonhydrolyzable analogues of GTP were found to rapidly inhibit the rate of in vitro nuclear import. Transport inhibition by GTP gamma S was dependent on the concentrations of permeabilized cells and cytosol, and was strongly enhanced by a cytosolic factor(s). The predominant cytosolic component responsible for this inhibition was found in a 20-30-kD fraction in molecular sieving chromatography. Furthermore, a component(s) of this 20-30-kD fraction was itself required for efficient nuclear import. Biochemical complementation with bacterially expressed protein demonstrated that this essential GTP gamma S-sensitive transport factor was Ran/TC4, a previously described GTPase of the Ras superfamily found in both nucleus and cytoplasm. Ran/TC4 and its guanine nucleotide release protein RCC1 have previously been implicated in DNA replication, cell cycle checkpoint control, and RNA synthesis, processing and export. Our results suggest that Ran/TC4 serves to integrate nuclear protein import with these other nuclear activities.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adam SA, Gerace L. Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell. 1991 Sep 6;66(5):837–847. [PubMed]
  • Adam SA, Lobl TJ, Mitchell MA, Gerace L. Identification of specific binding proteins for a nuclear location sequence. Nature. 1989 Jan 19;337(6204):276–279. [PubMed]
  • Adam SA, Marr RS, Gerace L. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol. 1990 Sep;111(3):807–816. [PMC free article] [PubMed]
  • Aebi M, Clark MW, 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. [PubMed]
  • Amberg DC, Fleischmann M, Stagljar I, Cole CN, Aebi M. Nuclear PRP20 protein is required for mRNA export. EMBO J. 1993 Jan;12(1):233–241. [PMC free article] [PubMed]
  • Belhumeur P, Lee A, Tam R, DiPaolo T, Fortin N, Clark MW. 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. [PMC free article] [PubMed]
  • Bischoff FR, 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. [PMC free article] [PubMed]
  • Bischoff FR, Ponstingl H. Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1. Nature. 1991 Nov 7;354(6348):80–82. [PubMed]
  • Boman AL, Taylor TC, Melançon P, Wilson KL. A role for ADP-ribosylation factor in nuclear vesicle dynamics. Nature. 1992 Aug 6;358(6386):512–514. [PubMed]
  • Borer RA, Lehner CF, Eppenberger HM, Nigg EA. Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell. 1989 Feb 10;56(3):379–390. [PubMed]
  • Bourne HR, Sanders DA, McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. [PubMed]
  • Clark KL, Ohtsubo M, Nishimoto T, Goebl M, Sprague GF., Jr The yeast SRM1 protein and human RCC1 protein share analogous functions. Cell Regul. 1991 Oct;2(10):781–792. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Dasso M, Nishitani H, Kornbluth S, Nishimoto T, Newport JW. RCC1, a regulator of mitosis, is essential for DNA replication. Mol Cell Biol. 1992 Aug;12(8):3337–3345. [PMC free article] [PubMed]
  • Dingwall C, Laskey RA. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. [PubMed]
  • Drivas GT, Shih A, Coutavas E, Rush MG, 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. [PMC free article] [PubMed]
  • Feldherr CM, Kallenbach E, Schultz N. Movement of a karyophilic protein through the nuclear pores of oocytes. J Cell Biol. 1984 Dec;99(6):2216–2222. [PMC free article] [PubMed]
  • Fischer U, Darzynkiewicz E, Tahara SM, Dathan NA, Lührmann R, Mattaj IW. 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. [PMC free article] [PubMed]
  • Forbes DJ. Structure and function of the nuclear pore complex. Annu Rev Cell Biol. 1992;8:495–527. [PubMed]
  • 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. [PubMed]
  • Gerace L. Molecular trafficking across the nuclear pore complex. Curr Opin Cell Biol. 1992 Aug;4(4):637–645. [PubMed]
  • Goldfarb DS, Gariépy J, Schoolnik G, Kornberg RD. Synthetic peptides as nuclear localization signals. Nature. 1986 Aug 14;322(6080):641–644. [PubMed]
  • Hamm J, Mattaj IW. Monomethylated cap structures facilitate RNA export from the nucleus. Cell. 1990 Oct 5;63(1):109–118. [PubMed]
  • Henkel T, Zabel U, van Zee K, Müller JM, Fanning E, Baeuerle PA. Intramolecular masking of the nuclear location signal and dimerization domain in the precursor for the p50 NF-kappa B subunit. Cell. 1992 Mar 20;68(6):1121–1133. [PubMed]
  • Imamoto N, Matsuoka Y, Kurihara T, Kohno K, Miyagi M, Sakiyama F, Okada Y, Tsunasawa S, Yoneda Y. Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins. J Cell Biol. 1992 Dec;119(5):1047–1061. [PMC free article] [PubMed]
  • Jans DA, Ackermann MJ, Bischoff JR, Beach DH, Peters R. p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV-40 T antigen proteins. J Cell Biol. 1991 Dec;115(5):1203–1212. [PMC free article] [PubMed]
  • Kadowaki T, Goldfarb D, Spitz LM, Tartakoff AM, 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. [PMC free article] [PubMed]
  • Matsumoto T, Beach D. Premature initiation of mitosis in yeast lacking RCC1 or an interacting GTPase. Cell. 1991 Jul 26;66(2):347–360. [PubMed]
  • Meier UT, Blobel G. Nopp140 shuttles on tracks between nucleolus and cytoplasm. Cell. 1992 Jul 10;70(1):127–138. [PubMed]
  • Michaud N, Goldfarb DS. 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. [PMC free article] [PubMed]
  • Moll T, Tebb G, Surana U, Robitsch H, Nasmyth K. The role of phosphorylation and the CDC28 protein kinase in cell cycle-regulated nuclear import of the S. cerevisiae transcription factor SWI5. Cell. 1991 Aug 23;66(4):743–758. [PubMed]
  • Moore MS, 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. [PubMed]
  • Moore MS, Blobel G. The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature. 1993 Oct 14;365(6447):661–663. [PubMed]
  • Newmeyer DD, Forbes DJ. Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell. 1988 Mar 11;52(5):641–653. [PubMed]
  • Newmeyer DD, Forbes DJ. An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore. J Cell Biol. 1990 Mar;110(3):547–557. [PMC free article] [PubMed]
  • Newmeyer DD, Finlay DR, Forbes DJ. In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins. J Cell Biol. 1986 Dec;103(6 Pt 1):2091–2102. [PMC free article] [PubMed]
  • Nigg EA, Baeuerle PA, Lührmann R. Nuclear import-export: in search of signals and mechanisms. Cell. 1991 Jul 12;66(1):15–22. [PubMed]
  • Nishimoto T, Eilen E, Basilico C. Premature of chromosome condensation in a ts DNA- mutant of BHK cells. Cell. 1978 Oct;15(2):475–483. [PubMed]
  • Nishitani H, Ohtsubo M, Yamashita K, Iida H, Pines J, Yasudo H, Shibata Y, Hunter T, Nishimoto T. Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis. EMBO J. 1991 Jun;10(6):1555–1564. [PMC free article] [PubMed]
  • Paine PL, Moore LC, Horowitz SB. Nuclear envelope permeability. Nature. 1975 Mar 13;254(5496):109–114. [PubMed]
  • Peters R. Fluorescence microphotolysis to measure nucleocytoplasmic transport and intracellular mobility. Biochim Biophys Acta. 1986 Dec 22;864(3-4):305–359. [PubMed]
  • Pfeffer SR. GTP-binding proteins in intracellular transport. Trends Cell Biol. 1992 Feb;2(2):41–46. [PubMed]
  • Piñol-Roma S, Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature. 1992 Feb 20;355(6362):730–732. [PubMed]
  • Reichelt R, Holzenburg A, Buhle EL, Jr, Jarnik M, Engel A, Aebi U. Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components. J Cell Biol. 1990 Apr;110(4):883–894. [PMC free article] [PubMed]
  • Ren M, Drivas G, D'Eustachio P, Rush MG. Ran/TC4: a small nuclear GTP-binding protein that regulates DNA synthesis. J Cell Biol. 1993 Jan;120(2):313–323. [PMC free article] [PubMed]
  • Richardson WD, Mills AD, Dilworth SM, Laskey RA, 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. [PubMed]
  • Schmidt-Zachmann MS, Dargemont C, Kühn LC, Nigg EA. Nuclear export of proteins: the role of nuclear retention. Cell. 1993 Aug 13;74(3):493–504. [PubMed]
  • Seydel U, Gerace L. A 28,000-Da GDP/GTP-binding protein specific to the nuclear envelope. J Biol Chem. 1991 Apr 25;266(12):7602–7608. [PubMed]
  • Shi Y, Thomas JO. The transport of proteins into the nucleus requires the 70-kilodalton heat shock protein or its cytosolic cognate. Mol Cell Biol. 1992 May;12(5):2186–2192. [PMC free article] [PubMed]
  • Sterne-Marr R, Blevitt JM, Gerace L. O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import. J Cell Biol. 1992 Jan;116(2):271–280. [PMC free article] [PubMed]

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