Role of Importin-β in the Control of Nuclear Envelope Assembly by Ran
2002; Elsevier BV; Volume: 12; Issue: 6 Linguagem: Inglês
10.1016/s0960-9822(02)00714-5
ISSN1879-0445
AutoresChuanmao Zhang, James R. A. Hutchins, Petra Mühlhäusser, Ulrike Kutay, Paul R. Clarke,
Tópico(s)Genomics and Chromatin Dynamics
ResumoCompartmentalization of the genetic material into a nucleus bounded by a nuclear envelope (NE) is the hallmark of a eukaryotic cell. The control of NE assembly is poorly understood, but in a cell-free system made from Xenopus eggs, NE assembly involves the small GTPase Ran [1Hetzer M. Bilbao-Cortés D. Walter T.C. Gruss O.J. Mattaj I.W. GTP hydrolysis by Ran is required for nuclear envelope assembly.Mol. Cell. 2000; 5: 1013-1024Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 2Zhang C. Clarke P.R. Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.Science. 2000; 288: 1429-1432Crossref PubMed Scopus (158) Google Scholar]. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin [2Zhang C. Clarke P.R. Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.Science. 2000; 288: 1429-1432Crossref PubMed Scopus (158) Google Scholar]. Here, we show that importin-β, an effector of Ran involved in nucleocytoplasmic transport and mitotic spindle assembly, is required for NE assembly induced by Ran. Concentration of importin-β on beads is sufficient to induce NE assembly in Xenopus egg extracts. The function of importin-β in NE assembly is disrupted by a mutation that decreases affinity for nucleoporins containing FxFG repeats. By contrast, a truncated protein that cannot interact with importin-α is functional. Thus, importin-β functions in NE assembly by recruiting FxFG nucleoporins rather than by interaction through importin-α with karyophilic proteins carrying classical nuclear localization signals. Importin-β links NE assembly, mitotic spindle assembly, and nucleocytoplasmic transport to regulation by Ran and may coordinate these processes during cell division. Compartmentalization of the genetic material into a nucleus bounded by a nuclear envelope (NE) is the hallmark of a eukaryotic cell. The control of NE assembly is poorly understood, but in a cell-free system made from Xenopus eggs, NE assembly involves the small GTPase Ran [1Hetzer M. Bilbao-Cortés D. Walter T.C. Gruss O.J. Mattaj I.W. GTP hydrolysis by Ran is required for nuclear envelope assembly.Mol. Cell. 2000; 5: 1013-1024Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 2Zhang C. Clarke P.R. Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.Science. 2000; 288: 1429-1432Crossref PubMed Scopus (158) Google Scholar]. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin [2Zhang C. Clarke P.R. Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.Science. 2000; 288: 1429-1432Crossref PubMed Scopus (158) Google Scholar]. Here, we show that importin-β, an effector of Ran involved in nucleocytoplasmic transport and mitotic spindle assembly, is required for NE assembly induced by Ran. Concentration of importin-β on beads is sufficient to induce NE assembly in Xenopus egg extracts. The function of importin-β in NE assembly is disrupted by a mutation that decreases affinity for nucleoporins containing FxFG repeats. By contrast, a truncated protein that cannot interact with importin-α is functional. Thus, importin-β functions in NE assembly by recruiting FxFG nucleoporins rather than by interaction through importin-α with karyophilic proteins carrying classical nuclear localization signals. Importin-β links NE assembly, mitotic spindle assembly, and nucleocytoplasmic transport to regulation by Ran and may coordinate these processes during cell division. Ran GTPase plays roles in nuclear structure and function throughout the cell division cycle [3Clarke P.R. Zhang C. Ran GTPase: a master regulator of nuclear structure and function during the eukaryotic cell division cycle?.Trends Cell Biol. 2001; 11: 366-371Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. The activity of Ran is determined both by its localization and its guanine nucleotide-bound state. During interphase, Ran is concentrated in the nucleus, mainly in the GTP-bound form, and determines the directionality of nucleocytoplasmic transport by controlling the stability of complexes formed between cargo proteins carrying specific targeting signals and transport proteins. Import complexes formed between proteins with Lys-rich nuclear localization signals (NLS) and importin-β, either directly or through importin-α, are assembled in the cytoplasm and dissociated in the nucleus by the binding of Ran-GTP to importin-β [4Görlich D. Kutay U. Transport between the cell nucleus and the cytoplasm.Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1615) Google Scholar]. During mitosis, Ran, mainly GDP bound, is dispersed throughout the cell, while localized generation of Ran-GTP in the vicinity of chromatin releases proteins required for mitotic spindle assembly from inhibitory complexes with importin-β [5Gruss O.J. Carazo-Salas R.E. Schatz C.A. Guarguaglini G. Kast J. Wilm M. Le Bot N. Vernos I. Karsenti E. Mattaj I.W. Ran induces spindle assembly by reversing the inhibitory effect of importin α on TPX2 activity.Cell. 2001; 104: 83-93Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar, 6Nachury M.V. Maresca T.J. Salmon W.C. Waterman-Storer C.M. Heald R. Weis K. Importin β is a mitotic target of the small GTPase Ran in spindle assembly.Cell. 2001; 104: 95-106Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 7Wiese C. Wilde A. Moore M.S. Adam S.A. Merdes A. Zheng Y. Role of importin-β in coupling Ran to downstream targets in microtubule assembly.Science. 2001; 291: 653-656Crossref PubMed Scopus (267) Google Scholar]. Ran also functions in the assembly of the nuclear envelope (NE) at the end of mitosis when the compartmentalization of the nucleus is reestablished [1Hetzer M. Bilbao-Cortés D. Walter T.C. Gruss O.J. Mattaj I.W. GTP hydrolysis by Ran is required for nuclear envelope assembly.Mol. Cell. 2000; 5: 1013-1024Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 2Zhang C. Clarke P.R. Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.Science. 2000; 288: 1429-1432Crossref PubMed Scopus (158) Google Scholar, 8Zhang C. Clarke P.R. The roles of Ran-GTP and Ran-GDP in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system.Curr. Biol. 2001; 11: 208-212Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar]. In cell-free systems for NE assembly made from Xenopus laevis eggs, NE assembly requires the generation of Ran-GTP from Ran-GDP by the guanine nucleotide exchange factor RCC1, as well as GTP hydrolysis on Ran [1Hetzer M. Bilbao-Cortés D. Walter T.C. Gruss O.J. Mattaj I.W. GTP hydrolysis by Ran is required for nuclear envelope assembly.Mol. Cell. 2000; 5: 1013-1024Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 2Zhang C. Clarke P.R. Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.Science. 2000; 288: 1429-1432Crossref PubMed Scopus (158) Google Scholar]. Localized concentration of Ran is sufficient to induce NE assembly in Xenopus egg extracts in the absence of chromatin, since Sepharose beads coated with Ran will assemble NE-like structures containing nuclear pore complexes (NPCs) around them, forming pseudonuclei that actively import karyophilic proteins [2Zhang C. Clarke P.R. Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.Science. 2000; 288: 1429-1432Crossref PubMed Scopus (158) Google Scholar]. However, the mechanism by which Ran controls NE assembly is unknown. To investigate the possible role of Ran-interacting proteins such as importin-β in NE assembly, we depleted Xenopus egg extracts using RanQ69L, a mutant defective in GTPase activity and therefore locked in the GTP-bound form [9Klebe C. Bischoff F.R. Ponstingl H. Wittinghofer A. Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1.Biochemistry. 1995; 34: 639-647Crossref PubMed Scopus (257) Google Scholar]. This procedure removed more than 90% of importin-β from the extracts (Figure 1A), as well as other Ran-GTP binding proteins [6Nachury M.V. Maresca T.J. Salmon W.C. Waterman-Storer C.M. Heald R. Weis K. Importin β is a mitotic target of the small GTPase Ran in spindle assembly.Cell. 2001; 104: 95-106Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar]. Extracts depleted of Ran binding proteins (ΔRanBP extracts) were deficient in the ability to promote membrane vesicle recruitment and fusion to form continuous membranes around Sepharose beads coated with Ran (Figure 1B). NE assembly activity in ΔRanBP extracts was restored by the addition of 5 μM importin-β, a concentration similar to that of the endogenous protein in nondepleted extracts [6Nachury M.V. Maresca T.J. Salmon W.C. Waterman-Storer C.M. Heald R. Weis K. Importin β is a mitotic target of the small GTPase Ran in spindle assembly.Cell. 2001; 104: 95-106Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar], but not by a 10-fold excess (50 μM) of importin-β (Figure 1B), which had a dominant inhibitory effect in nondepleted extracts (Figure 2B). By contrast, the related import factors importin-5 and transportin (Trn) were unable to recover NE assembly activity in ΔRanBP extracts, although importin-α and importin-7 weakly promoted vesicle recruitment (Figure 1C).Figure 2Analysis of the Structural Requirements for Importin-β to Permit NE Assembly around Ran BeadsShow full caption(A) Full-length importin-β (Impβ-FL) or truncated proteins (Impβ45–462, Impβ1–409) were added to ΔRanBP extracts.(B) Dominant inhibitory effects of importin-β proteins on NE assembly in nondepleted extracts.(C) A mutation that decreases the affinity for nucleoporins (I178D) prevents importin-β from rescuing NE assembly around Ran beads in ΔRanBP extracts. NE assembly was monitored by DHCC staining of membranes and the exclusion of FITC-IgG. Proteins were added at 5 μM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Full-length importin-β (Impβ-FL) or truncated proteins (Impβ45–462, Impβ1–409) were added to ΔRanBP extracts. (B) Dominant inhibitory effects of importin-β proteins on NE assembly in nondepleted extracts. (C) A mutation that decreases the affinity for nucleoporins (I178D) prevents importin-β from rescuing NE assembly around Ran beads in ΔRanBP extracts. NE assembly was monitored by DHCC staining of membranes and the exclusion of FITC-IgG. Proteins were added at 5 μM. Similar to full-length importin-β, a truncated protein (importin-β1–409) that lacks importin-α binding activity [10Kutay U. Izaurralde E. Bischoff F.R. Mattaj I.W. Gorlich D. Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex.EMBO J. 1997; 16: 1153-1163Crossref PubMed Scopus (305) Google Scholar] restored NE assembly around Ran beads in ΔRanBP extracts (Figure 2A), demonstrating that importin-β does not function in NE assembly by interaction through importin-α with karyophilic proteins carrying Lys-rich NLS motifs. By contrast, importin-β45–462, which lacks the Ran binding region [10Kutay U. Izaurralde E. Bischoff F.R. Mattaj I.W. Gorlich D. Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex.EMBO J. 1997; 16: 1153-1163Crossref PubMed Scopus (305) Google Scholar], was defective in restoring NE assembly activity to ΔRanBP extracts (Figure 2A). Indeed, in nondepleted extracts, 5 μM importin-β45–462 (but not 5 μM full-length importin-β or importin-β1–409) had a dominant inhibitory effect on NE assembly around Ran beads (Figure 2B). Thus, importin-β is required for NE assembly induced by Ran. These results suggest that importin-β functions as an adaptor that recruits target proteins to Ran during this process. In addition to transported cargoes and Ran, importin-β interacts directly with protein components of the nuclear pore (nucleoporins) containing FxFG (Phe-x-Phe-Gly, where x is usually Ser, Gly, or Ala) repeats [11Bayliss R. Littlewood T. Stewart M. Structural basis for the interaction between FxFG nucleoporin repeats and importin-beta in nuclear trafficking.Cell. 2000; 102: 99-108Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar]. Both importin-β and nucleoporins were recruited to Ran beads during NE assembly, and this binding was not prevented by the disruption of membrane assembly by the detergent Triton X-100 (see the Supplementary Material available with this article online), suggesting a direct interaction. To test the possible role of importin-β in nucleoporin recruitment, we used proteins in which Ile178 is changed to Asp (I178D), which decreases the affinity of importin-β for FxFG nucleoporins but not nucleoporins containing GLFG (Gly-Leu-Phe-Gly) repeats or transport cargoes [11Bayliss R. Littlewood T. Stewart M. Structural basis for the interaction between FxFG nucleoporin repeats and importin-beta in nuclear trafficking.Cell. 2000; 102: 99-108Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar]. This mutation inhibited the ability of full-length importin-β to restore NE assembly activity to ΔRanBP extracts (Figure 2C). If importin-β serves to recruit FxFG nucleoporins to Ran, then the immobilization and concentration of importin-β might be sufficient to induce NE formation. To test this possibility, we coated Sepharose beads with importin-β proteins and analyzed their ability to induce NE formation in nondepleted Xenopus egg extracts. Beads coated with full-length importin-β recruited nucleoporins and membrane vesicles that fused to form a continuous envelope, whereas pretreatment of importin-β with N-ethylmaleimide (NEM), which reacts with cysteine residues on importin-β and inactivates it [12Chi N.C. Adam S.A. Functional domains in nuclear import factor p97 for binding the nuclear localization sequence receptor and the nuclear pore.Mol. Biol. Cell. 1997; 8: 945-956Crossref PubMed Scopus (63) Google Scholar], abolished the ability to induce NE formation (Figure 3A). Importin-β may therefore account, at least in part, for the NEM sensitivity of NE assembly [13Macaulay C. Forbes D.J. Assembly of the nuclear pore: biochemically distinct steps revealed with NEM, GTP gamma S, and BAPTA.J. Cell Biol. 1996; 132: 5-20Crossref PubMed Scopus (147) Google Scholar]. Beads coated with importin-β proteins containing the I178D mutation also did not induce NE formation (Figure 3A). Thus, immobilized importin-β, acting through recruitment of FxFG nucleoporins, induces NE assembly. When directly bound to beads, importin-β45–462 efficiently induced NE formation (Figure 3A), demonstrating that the interaction of Ran with the immobilized importin-β is not required. Beads coated with importin-α, importin-5, importin-7, or transportin did not induce NE assembly in ΔRanBP extracts (Figure 3B); although, beads coated with importin-7 (Figure 3C) or importin-α (data not shown) were able to form NE when ΔRanBP extracts were supplemented with 5 μM importin-β, but not proteins carrying the I178D mutation (Figure 3C). Both importin-α and importin-7 form complexes with importin-β [14Görlich D. Vogel F. Mills A.D. Hartmann E. Laskey R.A. Distinct functions for the two importin subunits in nuclear protein import.Nature. 1995; 377: 246-248Crossref PubMed Scopus (401) Google Scholar, 15Jäkel S. Albig W. Kutay U. Bischoff F.R. Schwamborn K. Doenecke D. Görlich D. The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1.EMBO J. 1999; 18: 2411-2423Crossref PubMed Scopus (201) Google Scholar], suggesting that they are able to concentrate importin-β and thereby induce NE assembly. These results indicate that importin-β plays a role during NE assembly induced by Ran through interaction with FxFG domains on nucleoporins. Considering the complexity of protein components of the NE, multiple FxFG nucleoporins present in either the soluble or vesicle fractions of Xenopus extracts may be involved. Binding of Ran-GTP to importin-β reduces the affinity of importin-β for many FxFG nucleoporins [10Kutay U. Izaurralde E. Bischoff F.R. Mattaj I.W. Gorlich D. Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex.EMBO J. 1997; 16: 1153-1163Crossref PubMed Scopus (305) Google Scholar, 16Stewart M. Baker R.P. Bayliss R. Clayton L. Grant R.P. Littlewood T. Matsuura Y. Molecular mechanism of translocation through nuclear pore complexes during nuclear protein import.FEBS Lett. 2001; 498: 145-149Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 17Rexach R. Blobel G. Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins.Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (649) Google Scholar, 18Görlich 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; 15: 5584-5594Crossref PubMed Scopus (519) Google Scholar]. Thus, the generation of Ran-GTP by RCC1 concentrated on Ran beads [2Zhang C. Clarke P.R. Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.Science. 2000; 288: 1429-1432Crossref PubMed Scopus (158) Google Scholar, 8Zhang C. Clarke P.R. The roles of Ran-GTP and Ran-GDP in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system.Curr. Biol. 2001; 11: 208-212Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar] or chromatin [1Hetzer M. Bilbao-Cortés D. Walter T.C. Gruss O.J. Mattaj I.W. GTP hydrolysis by Ran is required for nuclear envelope assembly.Mol. Cell. 2000; 5: 1013-1024Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar] may recruit importin-β and release FxFG nucleoporins locally, allowing for the formation of multiprotein complexes that facilitate the recruitment of membrane vesicles. GTP hydrolysis by Ran would permit the recycling of importin-β and the recruitment of further nucleoporins. Ran-GTP may also interact with some nucleoporins directly [19Yokoyama N. Hayashi N. Seki T. Pante N. Ohba T. Nishii K. Kuma K. Hayashida T. Miyata T. Aebi U. et al.A giant nucleopore protein that binds Ran/TC4.Nature. 1995; 376: 184-188Crossref PubMed Scopus (398) Google Scholar] or through importin-β [10Kutay U. Izaurralde E. Bischoff F.R. Mattaj I.W. Gorlich D. Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex.EMBO J. 1997; 16: 1153-1163Crossref PubMed Scopus (305) Google Scholar, 20Delphin C. Guan T. Melchior F. Gerace L. RanGTP targets p97 to RanBP2, a filamentous protein localised at the cytoplasmic perifery of the nuclear pore complex.Mol. Biol. Cell. 1997; 8: 2379-2390Crossref PubMed Scopus (112) Google Scholar], whereas Ran-GDP can interact with FxFG nucleoporins via NTF2 [21Bayliss R. Ribbeck K. Akin D. Kent H.M. Feldherr C.M. Gorlich D. Stewart M. Interaction between NTF2 and xFxFG-containing nucleoporins is required to mediate nuclear import of RanGDP.J. Mol. Biol. 1999; 293: 579-593Crossref PubMed Scopus (138) Google Scholar]. Further interactions between Ran, importin-β, and nucleoporins may occur via zinc-finger domains [22Nakielny S. Shaikh S. Burke B. Dreyfuss G. Nup153 is an M9-containing mobile nucleoporin with a novel Ran-binding domain.EMBO J. 1999; 18: 1982-1995Crossref PubMed Scopus (178) Google Scholar, 23Yaseen N.R. Blobel G. Two distinct classes of Ran-binding sites on the nucleoporin Nup-358.Proc. Natl. Acad. Sci. USA. 1999; 96: 5516-5521Crossref PubMed Scopus (76) Google Scholar] or GLFG repeats [24Radu A. Moore M.S. Blobel G. The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex.Cell. 1995; 81: 215-222Abstract Full Text PDF PubMed Scopus (378) Google Scholar, 25Iovine M.K. Wente S.R. A nuclear export signal in Kap95p is required for both recycling the import factor and interaction with the nucleoporin GLFG repeat regions of Nup116p and Nup100p.J. Cell Biol. 1997; 137: 797-811Crossref PubMed Scopus (72) Google Scholar] on specific nucleoporins. Thus, the complete GDP/GTP cycle on Ran and more than one type of interaction might be required to recruit the full compliment of nucleoporins for nuclear pore complex (NPC) assembly. Ran controls NE formation through the same primary effector molecule, importin-β, that controls mitotic spindle assembly during mitosis [5Gruss O.J. Carazo-Salas R.E. Schatz C.A. Guarguaglini G. Kast J. Wilm M. Le Bot N. Vernos I. Karsenti E. Mattaj I.W. Ran induces spindle assembly by reversing the inhibitory effect of importin α on TPX2 activity.Cell. 2001; 104: 83-93Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar, 6Nachury M.V. Maresca T.J. Salmon W.C. Waterman-Storer C.M. Heald R. Weis K. Importin β is a mitotic target of the small GTPase Ran in spindle assembly.Cell. 2001; 104: 95-106Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 7Wiese C. Wilde A. Moore M.S. Adam S.A. Merdes A. Zheng Y. Role of importin-β in coupling Ran to downstream targets in microtubule assembly.Science. 2001; 291: 653-656Crossref PubMed Scopus (267) Google Scholar] and nuclear import during interphase [4Görlich D. Kutay U. Transport between the cell nucleus and the cytoplasm.Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1615) Google Scholar]. At the end of mitosis, relocalization of Ran to chromatin [26Zhang C. Hughes M. Clarke P.R. Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts.J. Cell Sci. 1999; 112: 2453-2461Crossref PubMed Google Scholar] and the concentration of importin-β could recruit nucleoporins while inhibiting microtubule-stabilizing factors in the vicinity of chromatin. Thus, changes in the localization, concentration, and molecular interactions of Ran and importin-β may help coordinate disassembly of the mitotic spindle with reassembly of the NE and the restarting of nucleocytoplasmic transport. Recombinant human Ran proteins were prepared as glutathione-S-transferase (GST) fusions in Escherichia coli, purified, and loaded with nucleotides as described previously [27Hughes M. Zhang C. Avis J.M. Hutchison C.J. Clarke P.R. The role of Ran GTPase in nuclear assembly and DNA replication: characterisation of the effects of Ran mutants.J. Cell Sci. 1998; 111: 3017-3026Crossref PubMed Google Scholar]. Wild-type Ran was loaded with GDP, and RanQ69L was loaded with GTP. Importin-β, including mutants, importin-α, importin-5, importin-7, and transportin were prepared as described previously [10Kutay U. Izaurralde E. Bischoff F.R. Mattaj I.W. Gorlich D. Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex.EMBO J. 1997; 16: 1153-1163Crossref PubMed Scopus (305) Google Scholar] in 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 2 mM MgCl2, and 250 mM sucrose. Xenopus laevis egg extracts were prepared by the method of Hutchison [28Hutchison C.J. The use of cell-free extracts of Xenopus eggs for studying DNA replication in vitro.in: Fantes P. Brooks R. The Cell Cycle: A Practical Approach. IRL Press, Oxford1994: 177-195Google Scholar], frozen, and stored in aliquots in liquid nitrogen. To deplete extracts of Ran binding proteins, 100 μl extract was mixed with an equal volume of packed glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) prebound with GST-RanQ69L loaded with GTP. The extract was incubated with mixing at 4°C for 60 min, and the beads were pelleted by centrifugation. The supernatant was subjected to this procedure three times to produce Ran binding protein-depleted (ΔRanBP) extract. Mock-depleted extracts were treated in the same way using glutathione-Sepaharose 4B beads with bound GST. Nuclear envelope assembly around glutathione-Sepharose 4B beads coated with GST-Ran was carried out as described previously [2Zhang C. Clarke P.R. Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.Science. 2000; 288: 1429-1432Crossref PubMed Scopus (158) Google Scholar], except that beads were washed in KHM buffer (78 mM KCl, 50 mM HEPES (pH 7.0), 4 mM MgCl2, 2 mM EGTA, and 1 mM dithiothreitol) before the addition to extracts. The beads have a diameter of approximately 100 μm. Importins were coupled directly to NHS-Sepharose (Amersham Pharmacia Biotech). After incubation at 23°C for 30 min in Xenopus egg extracts supplemented with an ATP-regenerating system and cycloheximide [28Hutchison C.J. The use of cell-free extracts of Xenopus eggs for studying DNA replication in vitro.in: Fantes P. Brooks R. The Cell Cycle: A Practical Approach. IRL Press, Oxford1994: 177-195Google Scholar], samples were removed and stained on a slide with 3,3′-dihexyloxacarbocyanine (DHCC) without fixation. In some instances, NE integrity was assessed after incubation for 60 min with the addition of FITC-IgG, which is too large to diffuse across nuclear pores and is excluded by an intact NE. In each case, phase optics were used to focus before capturing fluorescence images. For immunofluorescence, samples were fixed for 30 min in 4% formaldehyde (in 10 mM PIPES (pH 7.2), 80 mM KCl, 5 mM EDTA, and 15 mM NaCl). Beads were then recovered onto coverslips, permeabilized with 0.1% Tritron X-100 for 5 min, and prepared for immunofluorescence microscopy using specific primary antibodies against nucleoporins (mAb414, 1:500) or importin-β (Transduction Laboratories, 1:100). Images were captured immediately on a Zeiss Axioskop microscope using a cooled charged-coupled device camera (Hamamatsu Photonics) and were processed with Improvision Openlab and Adobe PhotoShop software. For Western blotting, beads were recovered from Xenopus egg extracts, and bound proteins were separated on polyacrylamide gels, transferred to nitrocellulose, and developed by chemiluminecence as described previously [2Zhang C. Clarke P.R. Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.Science. 2000; 288: 1429-1432Crossref PubMed Scopus (158) Google Scholar] using the same primary antibodies used for immunofluorescence. Supplementary Material including figures showing that Sepharose beads coated with Ran recruit FxFG nucleoporins and importin-β when incubated with Xenopus egg extracts and beads coated with Ran or importin-β form intact nuclear envelopes that exclude a fluorescent IgG is available at http://images.cellpress.com/supmat/supmatin.htm. This work was supported by grants from The Cancer Research Campaign (now Cancer Research UK), the Biotechnology and Biological Sciences Research Council, the Medical Research Council (PRC), the Special Fund for Major Basic Research of China (C.Z.; G19990539), and the Swiss National Science Foundation (U.K.). Download .pdf (.1 MB) Help with pdf files Supplementary material
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