Roles of Ran–GTP and Ran–GDP in Precursor Vesicle Recruitment and Fusion During Nuclear Envelope Assembly in a Human Cell-Free System Chuanmao Zhang†‡ and Paul R

208 Brief Communication

Roles of Ran–GTP and Ran–GDP in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system Chuanmao Zhang†‡ and Paul R. Clarke†

The molecular mechanism of nuclear envelope (NE) and facilitates organization of the mitotic spindle during assembly is poorly understood, but in a cell-free mitosis [4–8]. At the transition between these states, Ran system made from Xenopus eggs NE assembly is also has a role in reassembly of the interphase nucleus controlled by the small GTPase Ran [1,2]. In this [9,10]. In a cell-free system made from Xenopus laevis eggs, system, Sepharose beads coated with Ran induce formation of the NE requires generation of Ran–GTP the formation of functional NEs in the absence of from Ran–GDP by the guanine nucleotide exchange fac- chromatin [1]. Both generation of Ran–GTP by the tor RCC1, as well as GTP hydrolysis on Ran, indicating guanine nucleotide exchange factor RCC1 and GTP that the complete GTP–GDP cycle is involved [1,2]. Re- hydrolysis by Ran are required for NE assembly, markably, Sepharose beads coated with Ran assemble NE- although the roles of the GDP- and GTP-bound like structures containing nuclear pore complexes (NPCs) forms of Ran in the recruitment of precursor vesicles in Xenopus egg extracts in the absence of chromatin, form- and their fusion have been unclear. We now show ing pseudo-nuclei that actively import karyophilic proteins that beads coated with either Ran–GDP or Ran–GTP [1]. Ran is therefore likely to promote both the concentra- assemble functional nuclear envelopes in a cell- tion of vesicles and their fusion during NE assembly. free system derived from mitotic human cells, However, the roles of the GTP- and GDP-bound forms forming pseudo-nuclei that actively transport of Ran in the control of these steps have been unclear. proteins across the NE. Both RCC1 and the GTPase- activating protein RanGAP1 are recruited to the To investigate the function of Ran in NE assembly and to beads, allowing interconversion between Ran–GDP test its conservation between vertebrates, we developed a and Ran–GTP. However, addition of antibodies to cell-free system made from cultured human somatic cells. RCC1 and RanGAP1 shows that Ran–GDP must be An extract containing vesicles required for NE assembly converted to Ran–GTP by RCC1 before precursor was prepared from HeLa cells synchronized in mitosis with vesicles are recruited, whereas GTP hydrolysis by nocodazole. When glutathione–Sepharose beads coated Ran stimulated by RanGAP1 promotes vesicle with Ran (produced as a recombinant fusion protein with recruitment and is necessary for vesicle fusion to glutathione-S-transferase (GST) and loaded with GDP) form an intact envelope. Thus, the GTP–GDP cycle were incubated in the HeLa extract, they became rapidly of Ran controls both the recruitment of vesicles and surrounded by a continuous membrane identified by lipo- their fusion to form NEs. philic dye 3,3-dihexyloxacarbocyanine (DHCC). In contrast, control beads coated with GST did not attract any lipid vesicles (Figure 1a,b). To test whether the membrane Addresses: †Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, that formed around Ran beads was complete, we intro- Scotland, UK. ‡Department of Cell Biology and Genetics, College of duced an immunoglobulin conjugated with a fluorescent Life Sciences, Peking University, Beijing 100871, China. dye (IgG–FITC) after incubation of the beads in the extract. IgG–FITC is not actively imported into nuclei and Correspondence: Paul R. Clarke E-mail: [email protected] is excluded by an intact NE. We found that, whereas IgG–FITC diffused into GST beads, it was completely Received: 25 October 2000 excluded by Ran beads (Figure 1b). The membrane con- Revised: 7 December 2000 tained nucleoporins, detected by the monoclonal antibody Accepted: 21 December 2000 mAb414 [11], suggesting that NPCs were formed (Figures Published: 6 February 2001 1c,2b). Thus, Sepharose beads coated with Ran assemble NE-like structures in HeLa extract, as in Xenopus egg Current Biology 2001, 11:208–212 extract [1].

0960-9822/01/$ – see front matter To determine whether the NE that assembled around  2001 Elsevier Science Ltd. All rights reserved. the Ran beads was functional, we confirmed that pseudo- nuclei formed around Ran beads concentrated FITC-conjugated nucleoplasmin (Figure 1d), a protein containing Results and discussion a nuclear localization signal (NLS) that requires active The multifunctional GTPase Ran has a central role in import for concentration in the nucleus. In contrast, no directing nucleocytoplasmic transport during interphase [3] import was observed using beads coated with GST or Brief Communication 209

Figure 2 Figure 1

Functional NE assembly around Ran beads in human cell extracts. Glutathione–Sepharose beads loaded with either recombinant Ran–GDP produced as a fusion protein with GST [10] or GST alone were incubated with S100 extracts prepared from nocadazole- arrested HeLa cells. After 120 min, samples were removed and Ran beads preloaded with either GDP or GTP induce NE assembly examined by fluorescence microscopy (phase contrast images are in human cell extracts, and recruit nucleoporins, RCC1 and SUMO- also shown). (a) Staining with the fluorescent lipophilic dye DHCC. modified RanGAP1. (a) Beads coated with Ran–GDP or Ran–GTP (b) Ability to exclude a fluorescent dye-conjugated immunoglobulin were incubated with HeLa extracts for 120 min and stained with G (IgG–FITC). IgG–FITC was added after 120 min and fluorescence DHCC. (b,c) 1.6 ϫ 103 beads were isolated and subjected to Western was examined after a further 60 min incubation. (c) Detection of blotting for the detection of (b) nucleoporins (mAb414; BABCO, nucleoporins (‘porins’) by the monoclonal antibody mAb414 and 1:5,000), (c, upper panel) RCC1 (Transduction Laboratories, 1:250) indirect immunofluorescence. (d) Import of fluorescent dye- or (c, lower panel) RanGAP1 (Santa Cruz, 1:500): 2 ␮l total HeLa conjugated nucleoplasmin (NP–FITC). NP–FITC was added after 120 extract was also loaded for comparison. The results shown in (b,c) min and fluorescence was examined after a further 60 min incubation. were derived from the same filter stripped and reprobed with the (e) Accumulation of RanBP1 after treatment with 20 ng/ml leptomycin different antibodies. When RanGAP1 is recruited to the beads, a B (LMB) for 60 min; control, untreated. higher molecular mass form is detected, consistent with the modification of RanGAP1 by covalent attachment of the SUMO-1 polypeptide [16,17]. (d–f) Indirect immunofluorescence [1] of (d) RCC1 (Transduction Laboratories, 1:100), (e) RanGAP1 (Santa Cruz, 1:100) and (f) SUMO-1 (Zymed Laboratoies, 1:100) recruited to RanQ69L (Figure 1d), a Ran mutant defective in GTPase Ran–GDP beads. activity [12]. To examine the ability of pseudo-nuclei to carry out active export of proteins, we used Ran-binding protein 1 (RanBP1), a shuttling protein containing a leu- cine-rich nuclear export signal (NES) [13]. RanBP1 was inhibits the export factor Crm1 [14], caused accumulation taken up only very weakly into the pseudo-nuclei formed of RanBP1 in pseudo-nuclei (Figure 1e). Thus, the NE- around Ran beads, but addition of leptomycin B, which like structures that formed around Ran beads in HeLa 210 Current Biology Vol 11 No 3

Figure 3

Generation of Ran–GTP by RCC1 is required for vesicle binding and NE assembly around the Ran–GDP beads. (a) Control incubation with addition of buffer only. (b) Depletion of Ran–GTP by RanBP1 inhibits vesicle binding and NE assembly. HeLa extracts were incubated with addition of 10 ␮M RanBP1 for 30 min, then Ran–GDP beads were introduced and the incubation continued for a further 60, 120 or 180 min. (c) Addition of a high concentration (10 ␮M) of RCC1 to promote Ran-GTP formation does not inhibit vesicle binding. (d–g) Effect of pre-incubation of extracts with (d) an irrelevant antibody (anti-Bad, 20 ␮g/ml, Transduction Laboratories) or (e–g) an antibody directed against RCC1 (20 ␮g/ml) on vesicle recruitment to (d–f) Ran–GDP beads or (g) Ran–GTP beads. In (f), 2 ␮M exogenous RCC1 protein was added to neutralize the effects of the anti-RCC1 antibody. Numbers indicate time in min.

extracts are functional in both active nuclear import and with similar incorporation of nucleoporins (Figure 2b). export, showing that they contain functional NPCs. Similar results were also obtained in Xenopus egg extracts (data not shown). However, Ran may be rapidly intercon- Beads coated with Ran preloaded with either GDP or verted between the GDP- and GTP-bound states upon GTP induced NE assembly in HeLa extracts (Figure 2a), addition to the extracts. Indeed, both Ran–GDP and Ran– Brief Communication 211

Figure 4 promotes conversion of Ran to the GTP-bound form, had no inhibitory effect on vesicle binding to Ran–GTP beads (Figure 3c). Together, these results suggest that it is Ran– GTP that recruits vesicles on the beads, and if beads coated with Ran–GDP are introduced, Ran–GTP should be generated first to attract the binding of vesicles.

To determine whether RCC1 is required for vesicle binding to Ran beads, we introduced an antibody directed against RCC1 into HeLa extract to neutralize the endoge- nous RCC1, then added Ran beads to assemble NE. As a control, an irrelevant antibody was added which did not affect NE assembly around Ran beads (Figure 3d). By contrast, the anti-RCC1 antibody strongly inhibited vesicle binding to beads coated with Ran-GDP (Figure 3e), an effect that was overcome by 2 ␮M exogenous RCC1 (Figure 3f). When the anti-RCC1 antibody was added and Ran–GTP beads were used, NE assembly also occurred efficiently (Figure 3g). We conclude that, when using Ran–GDP beads to induce NE assembly, Ran–GDP must be converted to Ran–GTP by RCC1 before vesicles are recruited onto the beads.

Beads coated with either RanQ69L–GTP or Ran loaded with the non-hydrolyzable GTP analog GTP␥S fail to assemble complete nuclear envelopes in Xenopus egg extracts [1] and in HeLa extracts (data not shown), indicating that GTP hydrolysis by Ran is required. Ran has a very low intrinsic GTPase activity, but is stimulated 10,000- RanGAP1 is required for vesicle binding and fusion. Effects of pre- incubation of extracts with (a) an irrelevant antibody (anti-Hsp60, fold by RanGAP1 [18]. To test whether RanGAP1 is 20 ␮g/ml, Santa Cruz) or (b–d) an antibody directed against RanGAP1 required for NE assembly, we introduced an antibody (20 ␮g/ml; Santa Cruz). In (c), 0.2 ␮M RanGAP (Rna1p) was added. directed against RanGAP1 into the extract. This antibody In (d), 2 ␮M RanBP1 was added to stimulate GTP hydrolysis by Ran. inhibited nuclear envelope assembly around beads coated In (b) the dot-like staining of unfused vesicles bound to the surface of the bead is shown in the inset. with Ran–GTP (Figure 4). Distinct vesicles that failed to fuse accumulated on the surface of the beads even after prolonged incubation (180 min). Addition of fission yeast RanGAP, which stimulates GTP hydrolysis by Ran [18], GTP beads recruited the exchange factor RCC1 [15] and recovered vesicle fusion activity and caused complete NE the form of RanGAP1 modified by SUMO-1 [16,17], con- assembly (Figure 4c). Vesicle fusion activity was also par- centrating these regulators on the beads (Figure 2c–f). tially recovered by addition of RanBP1, which stimulates GTP hydrolysis by Ran in co-operation with RanGAP1 To examine the respective roles of Ran–GDP and Ran– (Figure 4d). Thus, GTP hydrolysis by Ran, stimulated GTP in NE assembly, we added regulators of Ran to the by RanGAP1 that is localized to the beads surface in its extract at high concentration, sufficient to cause Ran to SUMO-1-modified form, is required for intact NE assem- be predominantly loaded with either GDP or GTP. HeLa bly, acting mainly at the stage of vesicle fusion. The cell extracts were incubated with 10 ␮M RanBP1, which reduction in vesicle binding when GTP hydrolysis is inhibits nucleotide exchange on Ran by RCC1 and, to- blocked (compare Figure 4b with 4b at 60 min) indicates gether with RanGAP1, stimulates the hydrolysis of GTP that GTP hydrolysis by Ran also plays a role in recruit- to GDP, thereby favoring the GDP-bound state of Ran ment of vesicles. [18]. Ran–GDP beads were then introduced and the extracts incubated further. As shown in Figure 3a, vesicle Together with previous results using Xenopus egg extracts binding to Ran–GDP beads was abolished by RanBP1 [1,2], these experiments indicate that the role of Ran until at least 60 min, in contrast to incubations in the in the control of NE assembly is conserved, at least in absence of RanBP1 (Figure 3b), although lipid binding vertebrates. We show that the GTPase cycle of Ran plays did occur with prolonged incubation (120–180 min). Con- roles in both vesicle recruitment and fusion to form com- versely, a high concentration of RCC1 (10 ␮M), which plete envelopes. We propose that concentration of Ran 212 Current Biology Vol 11 No 3 on chromatin at the end of mitosis [8] recruits RCC1, 17. Mahajan R, Delphin C, Guan T, Gerace L, Melchior F: A small ubiquitin-related polypeptide involved in targetting resulting in the local generation of Ran–GTP required RanGAP1 to nuclear pore complex protein RanBP2. Cell 1997, for membrane vesicle binding. Hydrolysis of GTP by Ran 88:97-107. 18. Bischoff FR, Krebber H, Smirnova E, Dong W, Ponstingl H: stimulated by RanGAP1 is necessary for vesicle fusion to Co-activation of RanGTPase and inhibition of GTP form an intact envelope, but may also play a role in vesicle dissociation by Ran-GTP binding protein RanBP1. recruitment, possibly by promoting the fusion of vesicles EMBO J 1995, 14:705-715. to those already bound. The mechanism by which Ran controls these processes is currently unknown, but is likely to involve interactions with specific proteins deter- mined by the guanine-nucleotide bound state of Ran.

Supplementary material Full methodological details are available at http://current-biology.com/ supmat/supmatin.htm.

Acknowledgements We are grateful to F.R. Bischoff, R.A. Laskey, A. Wittinghofer and M. Yoshida for providing reagents. This work was supported by grants to P.R.C. from The Cancer Research Campaign and the Biotechnology and Biological Sciences Research Council. C.Z. is supported by the Special Fund for Major State Basic Research of China (G19990539).

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