Review. The Ran GTPase: Theme and Variations. Mary Dasso

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1 , Vol. 12, R502 R508, July 23, 2002, 2002 Elsevier Science Ltd. All rights reserved. PII S (02) The Ran ase: Theme and Variations Review Mary Dasso The small ase Ran has roles in multiple cellular processes, including nuclear transport, mitotic spindle assembly, the regulation of cell cycle progression and nuclear assembly. The past year has seen a remarkable unification of these different roles with respect to the effectors and mechanisms through which they function. Our emergent understanding of Ran suggests that it plays a central role in spatial and temporal organization of the vertebrate cell. Theme: Ran Gradients Ran is a Ras-related ase that acts in diverse cellular processes, including nuclear transport, mitotic spindle assembly, and post-mitotic nuclear assembly. As I will discuss here, two themes unify Ran s action against these targets: first, the distribution of Ran is used to direct spatially regulated processes with respect to chromosomal localization. Second, gradients of Ran are interpreted through a common set of Ran binding effectors that were first characterized as receptors for nuclear transport. Gradients of Ran are established through the localization of its regulators. Ran s intrinsic rates of nucleotide exchange and are slow. These reactions are respectively accelerated several orders of magnitude in vivo by RCC1, a guanine nucleotide exchange factor [1], and by RanGAP, a ase activating protein [2]. During interphase, RCC1 is nuclear [3], while RanGAP is cytosolic [4,5]. The asymmetric distribution of nucleotide exchange and causes nuclear Ran to be predominantly -bound, and cytosolic Ran to be predominantly -bound [6]. RCC1 binds to chromatin through histones H2A and H2B [7]. Binding of RCC1 to H2A or H2B causes a modest stimulation of RCC1 s exchange activity, increasing the dissociation rate constant for nucleotide exchange around twofold. Such stimulation may help to enhance the formation of Ran gradients by causing differential activation of chromosome-associated RCC1. A fraction of Ran is also associated with chromatin [8]. The majority of chromatin-associated Ran binds through RCC1, but Ran also binds to chromatin in an RCC1- independent manner as well as through histones H3 and H4 [9]. The targeting of RCC1 and Ran to chromatin may facilitate rebuilding of the nuclear envelope (NE) and reestablishment of interphase Ran- gradients at the end of mitosis (see below). Live imaging in Drosophila embryos shows that RCC1 remains largely chromatin-associated during Laboratory of Gene Regulation and Development, NICHD/NIH, Building 18T, Room 106, 18 Library Drive, Bethesda, Maryland, USA. mdasso@helix.nih.gov mitosis [10]. Consistent with this finding, fluorescence resonance energy transfer (FRET)-based measurements in mitotic Xenopus egg extracts show elevated levels of Ran in the immediate vicinity of chromosomes [6]. Interestingly, the bulk of Ran remains tightly associated with non-chromosomal spindle structures during mitosis in Drosophila [10], suggesting that Ran does not diffuse freely after it is generated by chromatin-associated RCC1. RanGAP associates with the mitotic spindle in Drosophila and mammals [10,11], and is strongly associated with kinetochores in mammalian cells [11]. Ran is regulated by a family of proteins that share a homologous Ran binding domain (RanBD) [12] (Figure 1). A well-characterized member of this family in vertebrates is RanBP1. RanBP1 is a cytosolic protein that binds to Ran with high affinity, and moderately accelerates RanGAP-mediated nucleotide [13]. RanBP1 is conserved between budding yeast and humans, and yeast RanBP1 is encoded by an essential gene [14]. Another RanBD family member is RanBP3, which has a lower affinity for Ran and acts as a loading accessory protein for the Crm1 export receptor (see below) [15 17]. Additional RanBDcontaining proteins are found in genomic sequences searches. These sequences encode putative FxFGcontaining nucleoporins (Figure 1) whose localization to the nuclear pore has been confirmed in budding yeast (Nup2) and vertebrates (RanBP2) [18,19]. These sequences vary widely in size, in the number of RanBDs they encode and in their complement of other functional domains. In vitro analysis has suggested that RanBP1 promotes dissociation of Ran from transport receptors, whose binding would otherwise block RanGAPmediated [20,21]. Surprisingly, however, the sequences of the Caenorhabditis elegans and Drosophila melanogaster genomes do not reveal obvious homologs of RanBP1 in those organisms. Moreover, the concentration of RanBP1 varies during the cell cycle, and there is essentially no RanBP1 protein in mammalian tissue culture cells that have been synchronized in G 0 phase [22]. These observations suggest that receptor dissociation from Ran must occur through a RanBP1-independent pathway under some circumstances. Although the heterogeneous class of large RanBD-containing proteins are good candidates to act in lieu of RanBP1 under these circumstances, these proteins are poorly understood and this idea remains to be experimentally tested. Variation I: Ran in transport An accepted paradigm for Ran s function in nuclear trafficking has emerged over the past decade (reviewed in [23]; Figure 2). A family of Ran binding proteins related to importin β act as receptors for nuclear import and export. These proteins translocate across the nuclear pore complex (NPC) freely in a Ran-independent

2 R503 Species RanBP1 -like RanBP3 -like S. cerevisiae Yrb1p Yrb2p Nup2p Heterogeneous RanBD proteins C. elegans n/d R12C12.2 F59A D. melanogaster n/d CG10225 CG H. sapiens RanBP1 RanBP3 RanBP RanBP2L1 Figure 1. RanBD proteins vary between species. RanBD proteins found in budding yeast (Saccharomyces cerevisiae), nematode (Caenorhabditis elegans), fruit fly (Drosophila melanogaster) and mammals (Homo sapiens) are shown. For RanBP1- and RanBP3-related proteins, the gene encoding the closest homolog by sequence similarity in each species is listed. n/d indicates no obvious RanBP1 homolog was found in the genome of the organism. Genes encoding other RanBD proteins are listed under Heterogenous RanBD proteins. The domain structure of each of these proteins is illustrated as follows: RanBDs (orange); zinc fingers (yellow). The zinc finger region of RanBP2 binds to Ran [45], although the functional consequence of this binding has not been elucidated. Leucine rich regions (light blue), with potential TPR repeats within these domains shown by hatching. The internal repeat (IR) domain of RanBP2 (green). The IR domain interacts with RanGAP and Ubc9, the conjugating enzyme of the small ubiquitin-like proteins SUMO-1 [46,47]. This domain has recently been shown to have SUMO-1 ligase activity in vitro [48]. Cyclophilin homology domain of RanBP2 (purple); GRIP domain within RanBP2L1 (pink). Short blue lines below each protein show FG motifs, longer lines show FxFG motifs. fashion [23]. Ran binding regulates the loading and unloading of cargo from these receptors on different sides of the NE: import receptors acquire their cargo in the cytosol, where Ran is scarce, and release it after translocation through the NPC and binding to nuclear Ran. By contrast, export receptors acquire their cargo inside the nucleus within complexes that also contain Ran. After translocation through the NPC, RanGAP-mediated Ran- causes dissociation of export complexes. As both import and export receptors exit the nucleus in association with Ran, nuclear transport results in the cytosolic accumulation of Ran-. Regeneration of Ran requires NTF2 [23], a protein that promotes the nuclear uptake of Ran from the cytosol after each round of transport, thereby allowing Ran to undergo RCC1-mediated nucleotide exchange. Beyond this simple paradigm, however, there are many variations upon the basic mechanism by which transport receptors use the Ran gradient. First, there is no a priori reason that transport receptors are limited to carrying cargo in only one direction. In fact, bi-directional transporters have recently been reported in budding yeast [24] and mammalian cells [25] (Figure 2). For instance, Importin 13 promotes the nuclear import of the SUMO-1 conjugating enzyme Ubc9 and of the RNA binding motif protein RBM8, as well as the export of the translation initiation factor eif1a [25]. Interestingly, the export of eif1a by importin 13 is distinguished from other export pathways by the fact that Ran alone is insufficient to dissociate eif1a from importin 13. Rather, this dissociation comes about through displacement of eif1a by higher affinity interactions between importin 13 and its import substrates in the cytosol after Ran. Ran thus only regulates unloading of export cargo from this receptor indirectly. Second, adaptor proteins are required to mediate the binding of many substrates to their receptors. The first nuclear import pathway elucidated, import of substrates bearing classical nuclear localization signals (NLS), requires an adaptor called importin for substrate recognition [23]. Importin binds to the NLS directly, and the importin β receptor binds the importin NLS complex to mediate its uptake through the NPC. It is possible that this adaptor system evolved to steepen the concentration gradient of NLS-bearing substrates between the nucleus and the cytosol: the exit of importin from the nucleus after each round of import requires its association with Ran and a separate export receptor, CAS. Two rounds of Ran are thus expended for import of each NLS-bearing substrate, increasing the energy that can be used to drive the accumulation of the substrates against a gradient. Even more complex adaptor systems have been described, with notable examples requiring as many as three adaptors for substrate receptor binding [23,26]. Third, accessory proteins regulate cargo loading of receptors. For the purposes of this discussion, accessory proteins are distinguished from adaptors by their lack of direct interactions with the transport cargo. A well-characterized example of such a protein is RanBP3, a RanBD-containing nuclear protein [16,17].

3 Review R504 Cytosol Nucleus Importin cycle Exportin cycle Bi-directional cycle Ran Importin Import cargos Ran Ran Importin 13 Exportin Ran Msn5p Export cargos Ran Figure 2. The distribution of RanGAP, RanBP1 and RCC1 leads to asymmetric distribution of Ran across the nuclear envelope. This asymmetry controls the direction of nuclear transport by regulating transport receptor loading and unloading. Import receptors (importins, left) form complexes with their cargo in the cytosol and transit across the NPC. In the nucleus, Ran binds to the importins and causes cargo release. Importins return to the cytosol in association with Ran. RanGAP and RanBP1 hydrolyze Ran to Ran, promoting receptor recycling. Export receptors (exportins, centre) bind to Ran and their export cargo in the nucleus. These complexes dissociate after transit through the NPC and RanGAPmediated Ran. Some receptors (bi-directional cycle, right) can carry different cargos depending upon their state of association with Ran. This allows them to import one set of proteins while exporting another. While RanBP3 does not bind to export substrates like an adaptor protein, it binds to Crm1 and increases the affinity of Crm1 for both Ran and nuclear export sequences (NESs). Remarkably, RanBP3 inhibits the binding of unloaded Crm1 to the NPC, possibly through direct interaction with the pore-targeting domains of Crm1 [16]. This inhibition is relieved by Ran. RanBP3 thus appears to coordinate efficient cargo loading, Ran binding and nuclear translocation of Crm1. Fourth, cargo unloading can be regulated by events in addition to Ran binding. Two different cases have been reported wherein Ran binding and import cargo release require the assembly of the cargo into macromolecular complexes within the nucleus. Mtr10 is an import receptor for Npl3, a shuttling RNA binding protein. Mtr10 binds Ran with relatively low affinity in vitro, and incubation with both Ran and RNA are required for displacement of Npl3 from Mtr10 [27]. In vivo, this may mean that Npl3 must be correctly deposited on RNA prior to receptor release and attaining functional competence. Similarly, Kap114 is an import receptor for the TATA binding protein (TBP) [28]. DNA containing TBP binding sites stimulates Ran -mediated dissociation of TBP from Kap114, suggesting that TBP may be released from Kap114 as it binds to the promoters of genes that it regulates. These observations suggest that import receptors may control intranuclear targeting or the function of cargo proteins prior to association with appropriate intranuclear partners. Of course, knowing how the loading and unloading of each individual receptor are regulated is very different from understanding the dynamics of the entire system within the intact cell. In order to estimate the true parameters of nuclear trafficking, computational modeling has recently been combined with live image analysis of fluorescent Ran within intact BHK cells [29]. Notably, this combined approach indicates that free Ran levels within the nucleus may be orders of magnitude lower than the dissociation constant for Ran binding to several import receptors [23,29], suggesting that complex loading pathways using adaptors and accessory subunits may be the norm for many transport receptors in vivo. Variation II: Ran and the Spindle Ran regulates spindle assembly in a manner that is independent of nuclear transport (reviewed in [30]). M- phase arrested Xenopus egg extracts (CSF extracts) assemble spindles directly from added chromatin templates. Spindle assembly is severely defective when Ran levels are lowered in CSF extracts, and the resultant spindles are disorganized with low densities of microtubules. Conversely, elevated levels of Ran in CSF extracts cause massive microtubule polymerization, even in the absence of chromosomes or centrosomes. Earlier analysis suggested that microtubules are stabilized in the vicinity of mitotic chromosomes through the localized production of a diffusible microtubule-stabilizing factor by a chromatin-associated enzyme. As RCC1 associates with chromatin [3], it was natural to speculate that Ran could be involved in the stabilization of mitotic microtubules by chromosomes. Recent FRET experiments showing that Ran concentrations are elevated near mitotic chromosomes in CSF extracts are entirely consistent with this idea [6]. Importin and β were subsequently implicated in spindle assembly through their capacity to specifically inhibit spindle formation in a manner that could be relieved by Ran [31 33]. A bacterially expressed fragment of the microtubule motor accessory protein NuMA (NuMA-tail II) induces formation of microtubule asters in CSF extracts that are morphologically similar to asters induced by Ran- [34]. Nachury et al. [32] and Wiese et al. [33] examined whether NuMA might be a mitotic target of Ran. Consistent with the previous finding that NuMA s NLS lies within the tail II domain, both groups showed

4 R505 Figure 3. Elevated Ran levels near chromosomes promote mitotic spindle assembly. Importin /β bind and inhibit spindle assembly factors (SAF) at low Ran concentrations throughout much of the mitotic cell. Near chromosomes, RCC1 generates an elevated concentration of Ran [6], locally disrupting inhibitory complexes and allowing full SAF activity. SAF RanGAP/RanBP1 mediated β SAF β β Nucleotide exchange at chromosome SAF RCC1 Localized activation of spindle assembly Ran β Importin β SAF SAF, active Ran Importin SAF SAF, inactive Microtubule that NuMA-tail II binds to importin β and that this binding can be released by Ran-. Gruss et al. [31] established an assay for purification of Ran-dependent spindle factors from HeLa cells using Xenopus CSF extracts that had been depleted of proteins with affinity for importin. The major component that was purified in this assay was another microtubule motor accessory protein, TPX2. All three groups proposed that the mechanism whereby Ran and Importin /β act in spindle assembly is closely related to their roles in nuclear transport (Figure 3) [31 33]: Importin /β bind and inhibit TPX2, NuMA and other spindle assembly factors. Ran near chromosomes destabilizes the inhibitory complexes, and allows localized spindle assembly factor activity. Far from chromosomes, Ran undergoes nucleotide, recycling importin β and allowing it to re-establish spindle assembly factor inhibition. A central aspect of this model is the prediction that importin and β should import spindle assembly factors into interphase nuclei, which could also ensure that these factors are not inappropriately active on cytosolic microtubules after nuclear envelope assembly. Indeed, NuMA and TPX2 are nuclear throughout interphase. As many other spindle proteins are nuclear during interphase, they could also be considered as potential targets for similar regulation [30]. Consistent with the notion that Ran acts on many targets in spindle assembly, Ran has been reported to direct other aspects of spindle assembly whose molecular basis is not fully established. These aspects include frequencies of microtubule transitions between shrinkage and growth [35,36], centrosomal microtubule nucleation capacity [35], and the activity of additional motor proteins or their accessory subunits [36]. While this model is attractive, several points remain to be addressed. First, quantitative association between importin β and the proposed targets of inhibition has not been well demonstrated. This is a critical point because it would be difficult to imagine effective inhibition without sequestration of a substantial fraction of the target protein. In particular, it is notable that earlier examination of NuMA complexes in Xenopus egg extracts did not reveal importin β as a major NuMA binding partner [34]. Moreover, a human Pinsrelated protein called LGN is essential for correct mitotic spindle assembly and binds to NuMA in the tail II domain [37]. Immunodepletion of LGN from mitotic Xenopus extracts or a recombinant NuMA fragment containing the LGN binding region (but not the NuMA NLS) are sufficient to cause aster assembly in CSF extracts [37], arguing that release from LGN rather than importin β is a critical event in the activation of mitotic NuMA. Second, inhibition of NuMA or TPX2 has not been reconstituted in any in vitro assay, so the biochemistry of inhibition by importin β binding has not been firmly established. Finally, this model focuses only on the localized generation of Ran, but it is important to remember that Ran nucleotide is also closely regulated during mitosis. RanGAP is localized to mitotic spindles [10,11]. In vertebrates, this association is carefully regulated through RanGAP modification by a small ubiquitin-like protein called SUMO-1 [11]. The role of regulated Ran will be an important topic for future consideration. Experiments in tissue culture cells have given contradictory results regarding the requirement for the Ran pathway during mammalian spindle assembly [22,32,38]. Bamba et al. [39] reported in vivo analysis of the Ran pathway in C. elegans by RNA interference (RNAi). RNAi disruption of Ran, RCC1 or RanGAP expression caused chromosome misalignment and missegregation in embryos, with the chromatin becoming detached from spindle microtubules. These treatments had much less impact on the astral microtubules, indicating that

5 Review R506 Ran undergoes / exchange RCC1 Ran binds to chromatin Ran recruits Importin β Chromatin NPC Vesicle RanGAP Ran- and Importin β binding to nucleoporins promote NE formation NPC NPC Nuclear envelope Figure 4. Ran- promotes nuclear envelope assembly. One possible model that would be largely consistent with experimental evidence is shown: a high concentration of Ran is generated near the chromosome by RCC1, allowing the binding of Ran to histones on the surface of the chromatin. This chromatin-associated Ran can recruit importin β and membrane vesicles with NPC proteins. RanGAP-mediated Ran is required for fusion of the vesicles and the formation of the complete nuclear envelope [40,42]. their dynamics are controlled in a different manner that is less dependent upon Ran. Two aspects of these studies were particularly remarkable: first, it was notable that RCC1 or RanGAP caused similar phenotypes, although the penetrance of the effects differed. This was surprising because the results in CSF extracts would suggest that these two conditions should have had opposite effects on microtubule dynamics. Second, there appears to be a significant concentration of Ran on the kinetochores of mitotic C. elegans chromosomes. It will be of considerable interest to investigate the possibility of a previously unsuspected role of Ran in kinetochore function. Variation III: Ran in the Nuclear Envelope During open mitosis, the NE is broken down and its membrane constituents are re-distributed within the cell. At the end of mitosis, these must be re-targeted to the decondensing chromosomes to re-build the nucleus. In interphase Xenopus egg extracts, demembranated sperm chromatin decondenses and recapitulates nuclear assembly in vitro. Experiments in egg extracts have suggested a direct role for Ran at early stages of NE re-assembly. Fusion of NE membranes is inhibited by depletion of RCC1 or Ran, as well as by Ran bound to non-hydrolyzable analogs or constitutively -bound Ran mutants [40]. Even more strikingly, membranes from Xenopus egg or HeLa cell extracts will bind to beads coated with GST Ran and assemble structures that resemble NE, with nuclear pores and a nuclear lamina [41]. Both nucleotide exchange by RCC1 and by RanGAP are required for NE assembly in this bead-based assay, but the requirement for RCC1 can be bypassed by prior loading of Ran with [42]. Notably, RCC1-bound beads do not work in this assay [41]. Together, these observations suggest that concentration of Ran near postmitotic chromatin is necessary and sufficient to direct NE assembly in in vitro systems. Two recent breakthroughs have changed our understanding of how Ran works in NE assembly. First, it has been reported that Ran can bind to chromatin through a relatively low affinity (2 µm) association with histones H3 and H4 [9]. This observation provides a plausible explanation for the observation that Ran precedes RCC1 in binding to sperm chromatin during early stages of nuclear assembly in egg extracts [43], More importantly, as RCC1-bound beads cannot nucleate NE assembly [41], Ran bound in this mode may be uniquely responsible for recruitment of NE membranes. Second, it has recently been shown that importin β is required for NE assembly in bead-based assays [44]. The function of importin β in NE assembly is disrupted by a mutation that decreases importin β s affinity for nucleoporins or Ran, but not by a mutation that disrupts importin β s interactions with importin. It thus appears that importin β functions in NE assembly through recruitment NPC components rather than through importin -dependent interactions with cargo proteins. Furthermore, importin β-coated beads induce NE assembly in a manner similar to Ran-coated beads [44], whereas beads coated with other transport receptors did not assemble NE. Beads coated with importin β mutants that do not bind Ran assembled NE but beads coated with importin β mutants that were deficient in NPC binding did not [44], suggesting the primary role of Ran- during NE assembly may be the recruitment of NPC components via importin β. A possible model for Ran in NE assembly is shown in Figure 4: localized generation of Ran by RCC1 allows the association of Ran to chromatin. Chromatin-associated Ran recruits importin β and membrane vesicles with NPC components. RanGAP-mediated Ran is required for fusion of the vesicles [40,42], although the mechanistic reason for this requirement remains to be elucidated. This model is distinct from Ran s role in nuclear transport because it does not involve the binding or sequestration of cargo proteins, but rather works through the affinity of importin β for the NPC. As in the nuclear transport and spindle assembly models discussed above, however, this scheme still uses Ran to direct nuclear assembly with respect to chromosome localization. Coda: The regulation of Ran It seems reasonable to suggest that different aspects of Ran function are probably regulated with respect to each other. Not only would this allow Ran to act effectively as a beacon for signaling the localization of chromosomes throughout the cell cycle, but it may also enforce distinctions between different parts of the cell cycle, for example that spindle assembly and NE assembly are mutually exclusive events.

6 R507 In addition to its important role in trafficking of NLSbearing proteins, importin β is the principal effector for Ran in both spindle formation [32] and NE assembly [44]. While it is not obligatory that all of these functions should be vested in a single receptor, as other transport receptors should be equally capable of detecting and responding to gradients of Ran, use of a common effector may facilitate their coordination. Importin β may be particularly suited for this role because of its capacity to carry a diverse array of cargo or its relatively avid binding of Ran [23]. Notably, the dissociation from Ran is regulated by both RanBP1 and importin [21], and it is possible that this unusual characteristic may be important in importin β s central role. Two other aspects of this pathway are striking and may become increasingly important in the future. First, we have only just begun to understand the targeting of Ran and its regulators. In some instances, such as the binding of Ran and RCC1 to histones [7,9], it will be of considerable interest to examine how protein protein interactions are controlled. In other instances, such as the localization of Ran to non-chromosomal spindle components in mitosis [10], even the identities of the players remain to be discovered. Second, evolution has clearly been at play in diversifying this pathway in different organisms (Figure 1). The resultant diversification may be extremely informative in telling us what is an essential component of this tune and what is a biological grace note. Eventually, this will lead us to understand not only how a single motif has been expanded to many different processes, but also how it has been adapted to the requirements of different eukaryotic cells. References 1. Bischoff, F.R. and Ponstingl, H. (1991). Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1. 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7 Review R Du, Q., Stukenberg, P.T. and Macara, I.G. (2001). A mammalian Partner of inscuteable binds NuMA and regulates mitotic spindle organization. Nat. Cell Biol. 3, Nishitani, H., Ohtsubo, M., Yamashita, K., Iida, H., Pines, J., Yasudo, H., Shibata, Y., Hunter, T. and Nishimoto, T. (1991). 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. 10, Bamba, C., Bobinnec, Y., Fukuda, M. and Nishida, E. (2002). The ase Ran regulates chromosome positioning and nuclear envelope assembly in vivo. Curr. Biol. 12, Hetzer, M., Bilbao-Cortes, D., Walther, T.C., Gruss, O.J. and Mattaj, I.W. (2000). by Ran is required for nuclear envelope assembly. Mol. Cell 5, Zhang, C. and Clarke, P.R. (2000). Chromatin-independent nuclear envelope assembly induced by Ran ase in Xenopus egg extracts. Science 288, Zhang, C. and Clarke, P.R. (2001). Roles of Ran- and Ran- in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system. Curr. Biol. 11, Clarke, P.R. and Zhang, C. (2001). Ran ase: a master regulator of nuclear structure and function during the eukaryotic cell division cycle? Trends Cell Biol. 11, Zhang, C., Hutchins, J.R., Muhlhausser, P., Kutay, U. and Clarke, P.R. (2002). Role of Importin-beta in the control of nuclear envelope assembly by Ran. Curr. Biol. 12, Yaseen, N.R. and Blobel, G. (1999). Two distinct classes of ranbinding sites on the nucleoporin nup-358. Proc. Natl. Acad. Sci. U.S.A. 96, Saitoh, H., Sparrow, D.B., Shiomi, T., Pu, R.T., Nishimoto, T., Mohun, T.J. and Dasso, M. (1998). Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2. Curr. Biol. 8, Matunis, M.J., Wu, J. and Blobel, G. (1998). SUMO-1 modification and its role in targeting the Ran ase-activating protein, RanGAP1, to the nuclear pore complex. J. Cell Biol. 140, Pichler, A., Gast, A., Seeler, J.S., Dejean, A. and Melchior, F. (2002). The nucleoporin RanBP2 has SUMO1 E3 ligase activity. Cell 108,