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J Cell Biol. 1990 March 1; 110(3): 547–557.
PMCID: PMC2116052

An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore

Abstract

We described previously an assay for authentic nuclear protein import in vitro. In this assay, exogenous nuclei are placed in an extract of Xenopus eggs; a rhodamine-labeled protein possessing a nuclear localization signal is added, and fluorescence microscopy is used to measure nuclear uptake. The requirement in this system for a cytosolic extract suggests that nuclear import is dependent on at least one cytosolic factor. We now confirm this hypothesis. Treatment of the cytosol with N-ethylmaleimide (NEM) abolishes nuclear protein import; readdition of a cytosolic fraction to the NEM-inactivated extract rescues transport. Thus, at least one NEM-sensitive factor required for transport is supplied by the cytosol. This activity, called nuclear import factor-1, or NIF-1, is ammonium-sulfate-precipitable, protease- sensitive, and heat-labile; it is therefore at least partly proteinaceous. NIF-1 stimulates, in a concentration-dependent manner, the rate at which individual nuclei accumulate protein. The effect of NIF-1 is enhanced by a second cytosolic NEM-sensitive factor, NIF-2. Earlier we identified two steps in the nuclear import reaction: (a) ATP- independent binding of a signal-sequence-bearing protein to the nuclear pore; and (b) ATP-dependent translocation of that protein through the pore. We now show that NEM inhibits signal-mediated binding, and that readdition of NIF-1 restores binding. Thus, NIF-1 is required for at least the binding step and does not require ATP for its activity. NIF-1 may act as a cytoplasmic signal receptor that escorts signal-bearing proteins to the pore, or may instead promote signal-mediated binding to the pore in another manner, as discussed.

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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, 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]
  • Baeuerle PA, Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell. 1988 Apr 22;53(2):211–217. [PubMed]
  • Baeuerle PA, Baltimore D. I kappa B: a specific inhibitor of the NF-kappa B transcription factor. Science. 1988 Oct 28;242(4878):540–546. [PubMed]
  • Bankaitis VA, Malehorn DE, Emr SD, Greene R. The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J Cell Biol. 1989 Apr;108(4):1271–1281. [PMC free article] [PubMed]
  • Block MR, Glick BS, Wilcox CA, Wieland FT, Rothman JE. Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7852–7856. [PubMed]
  • Blow JJ, Laskey RA. Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell. 1986 Nov 21;47(4):577–587. [PubMed]
  • Blow JJ, Watson JV. Nuclei act as independent and integrated units of replication in a Xenopus cell-free DNA replication system. EMBO J. 1987 Jul;6(7):1997–2002. [PubMed]
  • Blow JJ, Laskey RA. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature. 1988 Apr 7;332(6164):546–548. [PubMed]
  • Bürglin TR, De Robertis EM. The nuclear migration signal of Xenopus laevis nucleoplasmin. EMBO J. 1987 Sep;6(9):2617–2625. [PubMed]
  • Cambier JC, Newell MK, Justement LB, McGuire JC, Leach KL, Chen ZZ. Ia binding ligands and cAMP stimulate nuclear translocation of PKC in B lymphocytes. Nature. 1987 Jun 18;327(6123):629–632. [PubMed]
  • Dabauvalle MC, Schulz B, Scheer U, Peters R. Inhibition of nuclear accumulation of karyophilic proteins in living cells by microinjection of the lectin wheat germ agglutinin. Exp Cell Res. 1988 Jan;174(1):291–296. [PubMed]
  • Dang CV, Lee WM. Identification of the human c-myc protein nuclear translocation signal. Mol Cell Biol. 1988 Oct;8(10):4048–4054. [PMC free article] [PubMed]
  • Davey J, Dimmock NJ, Colman A. Identification of the sequence responsible for the nuclear accumulation of the influenza virus nucleoprotein in Xenopus oocytes. Cell. 1985 Mar;40(3):667–675. [PubMed]
  • Deshaies RJ, Koch BD, Werner-Washburne M, Craig EA, Schekman R. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature. 1988 Apr 28;332(6167):800–805. [PubMed]
  • Dingwall C, Dilworth SM, Black SJ, Kearsey SE, Cox LS, Laskey RA. Nucleoplasmin cDNA sequence reveals polyglutamic acid tracts and a cluster of sequences homologous to putative nuclear localization signals. EMBO J. 1987 Jan;6(1):69–74. [PubMed]
  • Dingwall C, Robbins J, Dilworth SM, Roberts B, Richardson WD. The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen. J Cell Biol. 1988 Sep;107(3):841–849. [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]
  • Finlay DR, Newmeyer DD, Price TM, Forbes DJ. Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores. J Cell Biol. 1987 Feb;104(2):189–200. [PMC free article] [PubMed]
  • Franke WW. On the universality of nuclear pore complex structure. Z Zellforsch Mikrosk Anat. 1970;105(3):405–429. [PubMed]
  • Franke WW. Structure, biochemistry, and functions of the nuclear envelope. Int Rev Cytol. 1974;Suppl 4:71–236. [PubMed]
  • Franke WW, Scheer U. The ultrastructure of the nuclear envelope of amphibian oocytes: a reinvestigation. I. The mature oocyte. J Ultrastruct Res. 1970 Feb;30(3):288–316. [PubMed]
  • Franke WW, Scheer U, Krohne G, Jarasch ED. The nuclear envelope and the architecture of the nuclear periphery. J Cell Biol. 1981 Dec;91(3 Pt 2):39s–50s. [PMC free article] [PubMed]
  • Fujisawa-Sehara A, Yamane M, Fujii-Kuriyama Y. A DNA-binding factor specific for xenobiotic responsive elements of P-450c gene exists as a cryptic form in cytoplasm: its possible translocation to nucleus. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5859–5863. [PubMed]
  • Glick BS, Rothman JE. Possible role for fatty acyl-coenzyme A in intracellular protein transport. Nature. 1987 Mar 19;326(6110):309–312. [PubMed]
  • Goldfarb DS, Gariépy J, Schoolnik G, Kornberg RD. Synthetic peptides as nuclear localization signals. Nature. 1986 Aug 14;322(6080):641–644. [PubMed]
  • Gritz L, Abovich N, Teem JL, Rosbash M. Posttranscriptional regulation and assembly into ribosomes of a Saccharomyces cerevisiae ribosomal protein-beta-galactosidase fusion. Mol Cell Biol. 1985 Dec;5(12):3436–3442. [PMC free article] [PubMed]
  • Hutchison CJ, Cox R, Ford CC. The control of DNA replication in a cell-free extract that recapitulates a basic cell cycle in vitro. Development. 1988 Jul;103(3):553–566. [PubMed]
  • Imamoto-Sonobe N, Yoneda Y, Iwamoto R, Sugawa H, Uchida T. ATP-dependent association of nuclear proteins with isolated rat liver nuclei. Proc Natl Acad Sci U S A. 1988 May;85(10):3426–3430. [PubMed]
  • Kalderon D, Roberts BL, Richardson WD, Smith AE. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. [PubMed]
  • Kleinschmidt JA, Seiter A. Identification of domains involved in nuclear uptake and histone binding of protein N1 of Xenopus laevis. EMBO J. 1988 Jun;7(6):1605–1614. [PubMed]
  • Krohne G, Benavente R. The nuclear lamins. A multigene family of proteins in evolution and differentiation. Exp Cell Res. 1986 Jan;162(1):1–10. [PubMed]
  • Lanford RE, Butel JS. Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell. 1984 Jul;37(3):801–813. [PubMed]
  • Lanford RE, Kanda P, Kennedy RC. Induction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal. Cell. 1986 Aug 15;46(4):575–582. [PubMed]
  • Lanford RE, White RG, Dunham RG, Kanda P. Effect of basic and nonbasic amino acid substitutions on transport induced by simian virus 40 T-antigen synthetic peptide nuclear transport signals. Mol Cell Biol. 1988 Jul;8(7):2722–2729. [PMC free article] [PubMed]
  • Loewinger L, McKeon F. Mutations in the nuclear lamin proteins resulting in their aberrant assembly in the cytoplasm. EMBO J. 1988 Aug;7(8):2301–2309. [PubMed]
  • Lohka MJ, Masui Y. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science. 1983 May 13;220(4598):719–721. [PubMed]
  • Lohka MJ, Masui Y. Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs. J Cell Biol. 1984 Apr;98(4):1222–1230. [PMC free article] [PubMed]
  • Lyons RH, Ferguson BQ, Rosenberg M. Pentapeptide nuclear localization signal in adenovirus E1a. Mol Cell Biol. 1987 Jul;7(7):2451–2456. [PMC free article] [PubMed]
  • Malhotra V, Orci L, Glick BS, Block MR, Rothman JE. Role of an N-ethylmaleimide-sensitive transport component in promoting fusion of transport vesicles with cisternae of the Golgi stack. Cell. 1988 Jul 15;54(2):221–227. [PubMed]
  • Markland W, Smith AE, Roberts BL. Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system. Mol Cell Biol. 1987 Dec;7(12):4255–4265. [PMC free article] [PubMed]
  • Moreland RB, Nam HG, Hereford LM, Fried HM. Identification of a nuclear localization signal of a yeast ribosomal protein. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6561–6565. [PubMed]
  • Moreland RB, Langevin GL, Singer RH, Garcea RL, Hereford LM. Amino acid sequences that determine the nuclear localization of yeast histone 2B. Mol Cell Biol. 1987 Nov;7(11):4048–4057. [PMC free article] [PubMed]
  • Munro S, Pelham HR. Use of peptide tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila hsp 70. EMBO J. 1984 Dec 20;3(13):3087–3093. [PubMed]
  • Murakami H, Pain D, Blobel G. 70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria. J Cell Biol. 1988 Dec;107(6 Pt 1):2051–2057. [PMC free article] [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, Lucocq JM, Bürglin TR, De Robertis EM. Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation. EMBO J. 1986 Mar;5(3):501–510. [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]
  • Newport J. Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. Cell. 1987 Jan 30;48(2):205–217. [PubMed]
  • Newport JW, Forbes DJ. The nucleus: structure, function, and dynamics. Annu Rev Biochem. 1987;56:535–565. [PubMed]
  • Newport J, Spann T. Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes. Cell. 1987 Jan 30;48(2):219–230. [PubMed]
  • Picard D, Yamamoto KR. Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J. 1987 Nov;6(11):3333–3340. [PubMed]
  • Richardson WD, Roberts BL, Smith AE. Nuclear location signals in polyoma virus large-T. Cell. 1986 Jan 17;44(1):77–85. [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]
  • Richter JD, Young P, Jones NC, Krippl B, Rosenberg M, Ferguson B. A first exon-encoded domain of E1A sufficient for posttranslational modification, nuclear-localization, and induction of adenovirus E3 promoter expression in Xenopus oocytes. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8434–8438. [PubMed]
  • Rihs HP, Peters R. Nuclear transport kinetics depend on phosphorylation-site-containing sequences flanking the karyophilic signal of the Simian virus 40 T-antigen. EMBO J. 1989 May;8(5):1479–1484. [PubMed]
  • Siegel V, Walter P. Each of the activities of signal recognition particle (SRP) is contained within a distinct domain: analysis of biochemical mutants of SRP. Cell. 1988 Jan 15;52(1):39–49. [PubMed]
  • Silver PA, Keegan LP, Ptashne M. Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5951–5955. [PubMed]
  • Smith AE, Kalderon D, Roberts BL, Colledge WH, Edge M, Gillett P, Markham A, Paucha E, Richardson WD. The nuclear location signal. Proc R Soc Lond B Biol Sci. 1985 Oct 22;226(1242):43–58. [PubMed]
  • Stone J, de Lange T, Ramsay G, Jakobovits E, Bishop JM, Varmus H, Lee W. Definition of regions in human c-myc that are involved in transformation and nuclear localization. Mol Cell Biol. 1987 May;7(5):1697–1709. [PMC free article] [PubMed]
  • Vale RD. Intracellular transport using microtubule-based motors. Annu Rev Cell Biol. 1987;3:347–378. [PubMed]
  • Walter P, Blobel G. Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7112–7116. [PubMed]
  • Walter P, Gilmore R, Blobel G. Protein translocation across the endoplasmic reticulum. Cell. 1984 Aug;38(1):5–8. [PubMed]
  • Waters MG, Chirico WJ, Blobel G. Protein translocation across the yeast microsomal membrane is stimulated by a soluble factor. J Cell Biol. 1986 Dec;103(6 Pt 2):2629–2636. [PMC free article] [PubMed]
  • Warrick HM, Spudich JA. Myosin structure and function in cell motility. Annu Rev Cell Biol. 1987;3:379–421. [PubMed]
  • Wychowski C, Benichou D, Girard M. A domain of SV40 capsid polypeptide VP1 that specifies migration into the cell nucleus. EMBO J. 1986 Oct;5(10):2569–2576. [PubMed]
  • Yamasaki L, Kanda P, Lanford RE. Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals. Mol Cell Biol. 1989 Jul;9(7):3028–3036. [PMC free article] [PubMed]
  • Yoneda Y, Imamoto-Sonobe N, Yamaizumi M, Uchida T. Reversible inhibition of protein import into the nucleus by wheat germ agglutinin injected into cultured cells. Exp Cell Res. 1987 Dec;173(2):586–595. [PubMed]
  • Yoneda Y, Imamoto-Sonobe N, Matsuoka Y, Iwamoto R, Kiho Y, Uchida T. Antibodies to Asp-Asp-Glu-Asp can inhibit transport of nuclear proteins into the nucleus. Science. 1988 Oct 14;242(4876):275–278. [PubMed]

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