Importin- Is a GDP-to-GTP Exchange Factor of Ran IMPLICATIONS FOR THE MECHANISM OF NUCLEAR IMPORT

Thierry G. Lonhienne, Jade K. Forwood, Mary Marfori , Gautier Robin , Bostjan Kobe , and Bernard J. Carroll From the Australian Research Council Centre of Excellence for Integrative Legume Research and School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia QLD 4072, School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, New South Wales 2650, and Institute for Molecular Bioscience and Special Research Centre for Functional and Applied Genomics, University of Queensland, St. Lucia QLD 4072, Australia

Ran (Gsp1p in yeast) is a Ras-like GTPase that regulates diverse cellular processes, including nuclear transport, mitotic spindle assembly, and post-mitotic nuclear assembly (1,2). Like other GTPases, Ran can bind GTP and GDP. Ran-GTP is generated in the nucleus by the guanine exchange factor RCC1 (regulator of chromosome condensation 1), which is associated with the chromatin (3). Ran-GDP is produced in the cytoplasm by the activation of the intrinsic GTPase activity of Ran by Ran-GAP1 (GTPase-activating protein) (4) and RanBP1 (Ran-bind-ing protein-1, Yrb1p in yeast). The compartmentalization of RanGAP1 (cytoplasm) and RCC1 (nucleus) gives rise to the asymmetric distribution of Ran-GDP (cytoplasm) and Ran-GTP (nucleus) across the nuclear envelope. This asymmetric distribution of Ran-GDP and Ran-GTP plays a central role in nucleocytoplasmic transport by mediating assembly and disassembly of import and export complexes through interaction with the nuclear import machinery (for reviews, see Refs. [5][6][7][8][9]. The passage of molecules into the nucleus occurs through the nuclear pore complexes (NPCs) 6 (10). Nucleocytoplasmic transport is driven by a series of protein-protein interactions and involves several soluble carriers named ␤-karyopherins. Import carriers are called importins and export carriers are called exportins. The classical nuclear import pathway involves importin-␤ (Kap95p in yeast) and the adaptor protein importin-␣ (Kap60p in yeast). In the cytoplasm importin-␤ binds to importin-␣. Their interaction triggers a conformational change of importin-␣ that increases its affinity for cargo proteins containing a nuclear localization signal (NLS) (11,12). The translocation of the resulting complex (importin-␤⅐importin-␣⅐NLS) involves interactions with the NPC proteins (nucleoporins), particularly the FXFG-repeat domains (11). The protein cargo is released in the nucleus by the action of Ran-GTP, which induces the dissociation of importin-␣ from importin-␤ by forming a stable complex with importin-␤. The importins are then recycled to the cytoplasm. Importin-␤ transfers to the cytoplasm associated with Ran-GTP, and importin-␣ is exported by CAS (exportin2; Cse1p in yeast) in the form of an importin-␣⅐CAS⅐Ran-GTP complex (13). Importin-␤ and importin-␣ are released from their complexes in the cytoplasm by the combined action of RanBP1 and RanGAP1. Importin-␤ and importin-␣ are then able to function in a new cycle of transport, whereas Ran-GDP is transported into the nucleus by NTF2 (nuclear-transport factor-2, Ntf2p in yeast) (14). In the nucleus Ran-GDP is transformed to Ran-GTP by the action of RCC1 (3).
The complexity of the nuclear import mechanism is highlighted by the fact that it involves the active participation of soluble factors other than Ran-GTP, importin-␤, and importin-␣. Indeed, Ran-GDP, RanBP1, and NTF2 have been shown to be involved in the docking and translocation events of nuclear import. Chi et al. (15) have demonstrated that Ran-GDP forms a stable complex with RanBP1 and importin-␤; they suggested a role for Ran-GDP in the association of the importin-␤⅐importin-␣⅐NLS complex with the nuclear pore and speculated that the importin-␤⅐importin-␣⅐NLS⅐Ran-GDP⅐RanBP1 pentameric complex was the actual translocation complex that moved through the pore. This model has also been adopted by others (16 -18) who have proposed that a stable Ran-GDP-containing complex was created on nucleoporin Nup358 (also called RanBP2) and that upon displacement of the importin-␤⅐importin-␣⅐Ran-GDP complex from the RBH (domain homologous to RanBP1) domains of Nup358 by RanBP1, binding of NTF2 triggered translocation to the nucleus. The role of NTF2 as the factor responsible for the translocation of the transport complex through the nuclear envelope has also been proposed by Paschal et al. (19). The role of Ran-GDP and RanBP1 in nuclear import has been demonstrated by a single mutation of a cysteine residue of importin-␤; the mutation was required for binding Ran-GDP⅐RanBP1, but not Ran-GTP⅐RanBP1, and inhibited the nuclear import in permeabilized cells (20). The active role of RanBP1 in nuclear import has been further demonstrated by Künzler et al. (21), who showed that mutations in the Yrb1 gene encoding the yeast ortholog of RanBP1 impair nucleocytoplasmic transport.
Despite considerable evidence for the involvement of Ran-GDP, RanBP1, and NTF2 in nuclear protein import, the precise mechanism by which these molecules regulate this process has been unknown. Here we characterize the interaction between Kap95p and Gsp1p-GDP. We show that this interaction results in GDP-to-GTP exchange on Gsp1p. Furthermore, we demonstrate that Gsp1p, Kap60p, Kap95p, Yrb1p, and Ntf2p interact to regulate the GDP-to-GTP exchange on Gsp1p. We suggest a mechanism of nuclear import additional to the RCC1-dependent pathway that incorporates our observations.
Expression and Purification of Recombinant Proteins-All constructs were expressed as glutathione S-transferase fusion proteins. The plasmids were introduced into Escherichia coli strain BL21(DE3) (Novagen) carrying the plasmid pGTf2 (Takara Bio Inc.) coding for a set of chaperones (GroES-GroELtig). Cells were grown in 2 liters of terrific broth (TB) medium containing 200 g/ml ampicillin and 35 g/ml chloramphenicol at 37°C to a cell density of 1-2 A 600 (absorption at 600 nm) units. Cells were cooled down on ice, and 1 mM isopropyl-1thio-␤-D-galactopyranoside and 10 g/ml tetracycline were added to induce expression of recombinant proteins and chaperones, respectively. After incubation (160 rpm) for 16 -20 h at 18°C, cells were harvested by centrifugation, and the pellets were frozen at Ϫ20°C.
All purification steps were carried out at 5°C. The pellets were thawed and resuspended in 50 ml of phosphate-buffered saline containing 1ϫ FastBreak Cell Lysis (Promega), 1 mM dithiothreitol, 1 g/ml aprotinin, leupeptin, and pepstatin, 1 mM phenylmethylsulfonyl fluoride, and 100 units of DNase. The homogenates were spun down at 25,000 ϫ g for 1 h, and the cleared supernatants were subjected to glutathione affinity chromatography (glutathione-agarose; Scientifix). Fusion tags were cleaved using thrombin and separated by size exclusion chromatography (Superdex S200; GE Healthcare). Fractions containing the purified recombinant protein were passed once more through glutathione-Sepharose beads to remove residual traces of glutathione S-transferase and uncleaved protein. Purified proteins were equilibrated with 50 mM Hepes, pH 7.5, 100 mM KCl and concentrated in Amicon Ultra 10k (Millipore). Aliquots of purified proteins were snap-frozen and kept at Ϫ80°C. For production of Ran-GDP and Gsp1p-GDP, purified proteins were incubated with a 50-fold excess of GDP and 5 mM EDTA for 40 min at 25°C followed by the addition of 10 mM Mg 2ϩ . Proteins were then equilibrated with 50 mM Hepes, pH 7.5, 100 mM KCl, concentrated, and snap-frozen at Ϫ80°C.
Radiolabeled Nucleotide Assays-All such assays were performed in a total volume of 50 l.   ) or Ntf2p, and MgCl 2 (or EDTA) in the incubating buffer. Exchange reactions were started by the addition of 2 l of 100 mM GDP or GTP. At various time points, 8-l aliquots were filtered through BA85 nitrocellulose and washed with the rinsing buffer (50 mM Hepes, pH 7.5, 100 mM KCl, 20 mM MgCl 2 ). The protein-bound radioactivity retained on the filter was measured by scintillation counting and expressed as cpm units. For Gsp1p-GDP loading assays, 5 l of Gsp1p-GDP (5 or 8 M) was preincubated for 15 min at 25°C with 45 l of the incubating buffer containing Kap95p, Yrb1p, Kap60p, or Ntf2p, and MgCl 2 (or EDTA). Loading reactions were started by the addition of 2 l of the nucleotide mixture (GDP, [ 3 H]GDP, GTP, or [ 3 H]GTP). At various time points protein-bound radioactivity contained in 8-l aliquots was measured as mentioned above.
Purification of Ran⅐Kap95p Complex by Gel Filtration-Recombinant human Ran (1.5 mg (22)) was incubated at 25°C for 40 min in the running buffer (20 mM Tris, pH 7.4, 100 mM NaCl) supplemented with 5 mM EDTA, 0.89 M [ 3 H]GDP, and 1 mM GDP in a total volume of 1 ml. The loading reaction was stopped by the addition of 20 l of 1 M MgCl 2 . The sample was applied to a Superdex S200 column, and gel filtration was performed in the running buffer. Fractions corresponding to purified Ran-[ 3 H]GDP were pooled and concentrated to 0.5 ml. Purified Ran-[ 3 H]GDP was then incubated with 4.2 mg of Kap95p in the running buffer for 30 min at 25°C in a total volume of 1 ml. The sample was applied to a Superdex S200 column. Fractions corresponding to protein peaks were analyzed for their protein content by SDS-PAGE. The radioactivity corresponding to [ 3 H]GDP contained in the fractions was measured by scintillation counting.
HPLC Analysis-The nucleotide-bound state of human Ran and its complex with Kap95p was assessed using isocratic HPLC. In each experiment 0.86 nmol of Ran protein was loaded onto a C18 reverse-phase column (Jupiter, Phenomenex) under ion-pair conditions in 100 mM potassium phosphate, pH 7.0, 10 mM tetrabutylammonium bromide, and 4% (v/v) acetonitrile. The stoichiometry of nucleotide bound was assessed using GDP and GTP nucleotide standards.  (Fig. 1A). In the binding buffer alone containing 1 mM Mg 2ϩ , Gsp1p-[ 3 H]GDP was stable over 60 min, whereas it was strongly destabilized in the presence of EDTA, consistent with previous observations (3,23). In 1 mM Mg 2ϩ , Kap95p promoted Gsp1p-[ 3 H]GDP dissociation. Because the nucleotide binding of Ran depends on Mg 2ϩ (24), we examined its effect on the GDP exchange activity of Kap95p. As expected, the release of [ 3 H]GDP from Gsp1p by Kap95p was inhibited by increasing concentrations of Mg 2ϩ (Fig. 1A).

Kap95p (Importin-␤) Promotes GDP Release and GTP Binding to Gsp1p (Ran)-To
We next examined the effect of Kap95p on the loading of nucleotides on Gsp1p by incubating Gsp1p-GDP with equal amounts of [ 3 H]GTP and GDP (Fig. 1B). In the absence of Kap95p in the binding buffer containing 1 mM Mg 2ϩ , as expected, no loading of [ 3 H]GTP was observed. Kap95p, when compared with EDTA, caused high [ 3 H]GTP loading on Gsp1p in the presence of 1 mM Mg 2ϩ . Ran has a 10-fold higher affinity for GDP compared with GTP (23); therefore, this result suggested that upon formation of the Kap95p⅐Gsp1p complex, Gsp1p adopted a conformational state that favored the binding of GTP over GDP. Jointly, the results demonstrate that Kap95p promotes the conversion of Gsp1p-GDP to Gsp1p-GTP in vitro. Additional experiments demonstrated that Kap95 also induced the nucleotide exchange of human Ran with equal effectiveness (data not shown), consistent with the high degree of conservation between the human and yeast nuclear import pathways (6).

Nucleotide-free Ran and Kap95p (Importin-␤) Form a Stable Complex That Resists Dissociation by Kap60p (Importin-␣)-
During the GDP nucleotide exchange occurring on the Ran⅐Gsp1p⅐Kap95p complex, Ran⅐Gsp1p must pass through an intermediate free of nucleotide. We analyzed the stability of the nucleotide-free human Ran⅐Kap95p complex ( Fig. 2A). Kap95p was incubated with an excess of Ran preloaded with [ 3 H]GDP, and the sample was resolved by gel filtration. Two protein peaks were observed, one corresponding to a 1:1 complex between Ran and Kap95p and another one corresponding to free Ran. The radioactivity profile showed two peaks, one overlapping the free Ran protein peak the other one corresponding to free GDP. The integration of the radioactivity found in the two peaks corresponded to the initial activity associated with Ran-[ 3 H]GDP before the addition to Kap95p. No radioactivity was detected in the protein peak containing the Ran⅐Kap95p complex, demonstrating that the addition of Kap95p to Ran-[ 3 H]GDP generated a stable 1:1 complex between Kap95p and nucleotide-free Ran. To confirm the nucleotide free status of the Ran⅐Kap95p complex, we conducted a similar experiment with Ran preloaded with GDP instead of [ 3 H]GDP. We analyzed the two resultant protein peaks by reverse-phase HPLC (Fig. 2B). The HPLC data confirmed that Ran was associated with GDP in a 1:1 stoichiometry and that GDP was excluded from the Ran⅐Kap95 complex.
To further investigate the stability of the Ran⅐Kap95p complex, we examined the ability of Kap60p to dissociate the complex (Fig. 3). Excess of Kap60p protein was added to Ran⅐Kap95p complex in the binding buffer alone or in the presence of excess of Mg 2ϩ and GDP. Kap60p was unable to dissociate the Ran⅐Kap95p complex in buffer alone, implying that under these conditions the affinity between nucleotide-free Ran and Kap95p was higher than between Kap60p and Kap95p (3 ϫ 10 Ϫ8 M Ϫ1 for importin-␣⅐importin-␤ (25)). However, the addition of excess of Mg 2ϩ and GDP allowed Kap60p to dissociate the Ran⅐Kap95p complex, suggesting that GDP and Mg 2ϩ destabilized the Ran⅐Kap95p interaction. Together, these results demonstrated that upon binding with Ran-GDP, Kap95p is able to release the nucleotide and thereby increase the affinity between Kap95p and nucleotide-free Ran such that they cannot be dissociated by Kap60p.
Yrb1p (RanBP1) Co-activates the GDP Nucleotide Exchange by Kap95p (Importin-␤)-Ran-GDP has the ability to form a trimeric complex with RanBP1 and importin-␤ (15). It was, therefore, of interest to assess the GDP exchange activity of Gsp1p-GDP in the presence of Yrb1p and Kap95p. Yrb1p promoted Kap95p-mediated dissociation of Gsp1p-[ 3 H]GDP, whereas in the absence of Kap95p, no dissociation was observed with or without Yrb1p (Fig. 4A).
We then carried out nucleotide loading assays on Gsp1p in complex with Kap95p and/or Yrb1p and with equal amounts of [ 3 H]GTP and GDP (Fig. 4B) (Fig. 4A), Yrb1p had no effect on the loading of   (Fig. 5A). However, the addition of Yrb1p counteracted this inhibition, nearly restoring the rate of dissociation of Gsp1p-GDP obtained in presence of Kap95p alone.  We carried out nucleotide loading assays on Gsp1p in the Gsp1p⅐Yrb1p⅐Kap95p⅐Kap60p complex upon GDP release. In loading assays with 1.6 M [ 3 H]GTP and GDP, the amount of Gsp1p loaded with [ 3 H]GTP in the presence of Kap95p, Kap60p, and Yrb1p (Fig. 5B) was small compared with the amount that underwent GDP dissociation (Fig. 5A) (26)). We, therefore, instead monitored the loading of [ 3 H]GDP in the presence of a gradient of GTP concentration reaching 470 M at its maximum (Fig. 5C) (Fig. 5D) confirmed our previous results establishing that the conformation of Gsp1p in the Gsp1p⅐Yrb1p⅐Kap95p complex favors the selective binding of GTP over GDP (Fig. 3). Only a slight release of [ 3 H]GTP from Gsp1p was observed when Kap60p was added to Yrb1p and Kap95p. This result clearly indicated that Kap60p did not form a stable complex with Gsp1p-[ 3 H]GTP, Kap95p, and Yrb1p.

Ntf2p (NTF2) Inhibits Gsp1p-GDP (Ran-GDP) Nucleotide Exchange in the Presence of Kap95p (Importin-␤), Yrb1p (RanBP1), and Kap60p (Importin-␣)-NTF2
is a transport factor that imports Ran-GDP to the nucleus (14). NTF2 is an inhibitor of Ran-GDP dissociation (27), and it is involved in the formation of a pentameric complex with Gsp1p-GDP, Kap60p, Kap95p, and Nup36p, a nucleoporin containing an RBH domain (28). We, therefore, investigated the effect of Ntf2p on the GDP nucleotide exchange by Kap95p in presence of Kap60p and Yrb1p. We found that Ntf2p partially inhibited both GDP dissociation and GTP loading on Gsp1p (Fig. 6).
Our results demonstrate that Kap95p (importin-␤) induces the GDP-to-GTP exchange on Gsp1p (Ran), mimicking the activity of the known guanine exchange factor RCC1 (Fig. 1). However, although RCC1 has almost no preference for the nucleotide state of Ran and catalyzes the nucleotide exchange in both directions (23), nucleotide exchange by Kap95p (importin-␤) is unidirectional because of the stable association of Kap95p (importin-␤) with Gsp1p-GTP (Ran-GTP). The stability of the nucleotide-free Ran⅐Kap95p complex, demonstrated by the inability of Kap60p to dissociate it (Fig. 3), is probably the feature that accounts for the high affinity of Ran-GTP for Kap95p (importin-␤) compared with Ran-GDP and, therefore, for the unidirectional path of the nucleotide exchange.
RCC1 and Kap95p share two interesting similarities with regard to their GDP nucleotide exchange activity. First, both RCC1 and Kap95p have a weak affinity for Ran-GDP. The reaction between Ran-GDP and RCC1 has a K m of 1.1 M (23), whereas the interaction between Ran-GDP and importin-␤ has a K D of 2 M (22). Another remarkable similarity between Kap95p and RCC1 is their ability to form a stable complex with nucleotide-free Ran. In the absence of Mg 2ϩ and free nucleotides, RCC1 mixed with Ran-GDP or Ran-GTP forms a stable complex with nucleotide-free Ran (24). We show that under similar conditions Kap95p mixed with Ran-GDP forms a stable complex with nucleotide-free Ran (Fig. 2). The ability to form a stable complex with nucleotide-free Ran is the feature that confers to both RCC1 and Kap95p their nucleotide exchange activity. GDP and Mg 2ϩ , which stabilize the Ran-GDP association, dissociate RCC1⅐Ran complex when present in excess (24). We show similar GDP and Mg 2ϩ dependence of the stability of the Gsp1p⅐Kap95p complex (Fig. 3). Increased concentration of Mg 2ϩ inhibits the nucleotide exchange of Ran by RCC1 (24) and of Gsp1p by Kap95p (Fig. 1).
We do not consider it possible to directly compare the catalytic activities of Kap95p and RCC1 because their mode of action is different. RCC1 works as an enzyme and requires a high turnover to efficiently convert free Ran-GDP to Ran-GTP in the nucleus. The rate of the reaction depends of both Ran-GDP and RCC1 concentrations (23). By contrast, Kap95p does not work as an enzyme because it remains associated with the product. Indeed, in our assays (Fig. 5) Kap95p nucleotide exchange activity occurs in a tetramer complex (Kap95p⅐ Gsp1p-GDP⅐Yrb1p⅐Kap60p), and the reaction gives rise to free Kap60p and Kap95p⅐Gsp1p-GTP⅐Yrb1p trimer. The rate of the reaction does not depend of the concentration of Ran-GDP and Kap95 because it occurs in a pre-established complex that travels from the cytoplasm to the nucleus. It most probably depends of the biochemical environment specific to the interior of nuclear pore (FXFG repeats and ionic strength).
Our previous research demonstrated the formation of a stable Ran-GDP⅐Kap95 complex (22). Here, we show that Kap95 can also form a complex with free Ran that remains stable in the absence of free Mg 2ϩ and GDP (Fig. 3).
Kap60p (Importin-␣), Yrb1p (RanBP1), and Ntf2p (NTF2) Regulate the GDP Nucleotide Exchange Reaction by the Gsp1p- GDP⅐Kap95p (Ran-GDP⅐Importin-␤) Complex-The observation that the interaction of Gsp1p-GDP with Kap95p leads to the generation of the Gsp1p-GTP⅐Kap95p complex in vitro suggests that such a reaction could occur in vivo. Such a reaction would be undesirable in the cytoplasm but is unlikely because of the high local concentration of Kap60p, which inhibits the GDP nucleotide exchange by Kap95p (Figs. 3B and 5A), consistent with the higher affinity between importin-␤ and importin-␣ (1.1 ϫ 10 Ϫ8 M, (25)) than between Ran-GDP and importin-␤ (2 M, (22)).
Yrb1p (RanBP1) has the opposite effect to Kap60p on the GDP nucleotide exchange by Kap95p, strongly enhancing Gsp1p⅐GDP dissociation and selective binding of GTP (Fig. 4). It acts as a co-activator of Kap95p because it has no effect on the GDP nucleotide exchange in the absence of Kap95p.
The human counterparts of Gsp1p-GDP, Yrb1p, Kap95p, and Kap60p have been reported to form a tetramer that may be involved in the translocation of cargo proteins across the NPC (15,17). Our results show that GDP dissociation activity occurs on Gsp1p in the presence of Yrb1p, Kap95p, and Kap60p (  (Fig. 5D), one can predict that the binding of GTP to Gsp1p⅐Yrb1p⅐Kap95p⅐Kap60p induces the dissociation of Kap60p from the complex.
NTF2 has been shown to function as a Ran-GDP dissociation inhibitor (27). Our data (Fig. 6) do not allow one to distinguish if the inhibitory activity of Ntf2p on the GDP nucleotide exchange occurs through the formation of a pentameric complex with Gsp1p, Yrb1p, Kap95p, and Kap60p or if it occurs through the formation of a dimer with Gsp1p-GDP.
Biological Significance of the Nucleotide Exchange Activity of Kap95p (Importin-␤)-Is the GDP nucleotide exchange activity of Kap95p described here of relevance to nucleocytoplasmic transport in vivo? A number of research groups have suggested that a nucleotide exchange reaction can occur locally at the NPCs, independently of RCC1 (11,15,17,33,36,41,42). For example, Chi et al. (15) proposed a model in which importin-␤⅐NLS⅐importin-␣⅐Ran-GDP⅐RanBP1 is the translocation complex that moves through the pore. They suggested that RanBP2 (Nup358), a nucleoporin localized at the cytoplasmic surface of the NPC, is the site of a series of docking, undocking, diffusion, and redocking events of the translocation complex and that at some point after docking, an exchange reaction to replace the GDP with GTP on Ran could occur, catalyzed by an unknown factor. In this model newly formed Ran-GTP releases the receptor complex from the docking site and is rapidly hydrolyzed by the concerted action of RanBP1 and RanGAP to allow redock-ing. Our data fit perfectly with such a model because we show that in the presence of physiological concentrations of GDP and GTP, conversion of Ran-GDP to Ran-GTP occurs in the Gsp1p-GDP⅐Yrb1p⅐Kap95p⅐Kap60p complex.
Another model consistent with our results involving the formation of a translocation complex including Ran-GDP and RanBP1 has been proposed by Melchior and Gerace (17). In this model, upon binding of the Ran-GTP⅐importin-␤ complex to RanBP2 (Nup358), the interaction of importin-␣ with importin-␤ induces GTP hydrolysis by the RanBP2associated RanGAP1, giving rise to a stable Ran-GDP-containing transport complex with RanBP2. RanBP1 dissociates the complex from RanBP2, and the interaction with NTF2 induces translocation of the complex. Once the import complex has reached the nucleoplasm, either conversion of Ran-GDP to Ran-GTP in the complex or its replacement by free Ran-GTP produced by chromatin-bound RCC1 (36) leads to complex disassembly. In addition to the RCC1-dependent pathway being operative, our data are consistent with the former mechanism also being a possibility. In the vicinity of the nuclear pore, it is difficult to conceive how free Ran-GTP would replace Ran-GDP in the Ran-GDP⅐RanBP1⅐importin-␤⅐importin-␣ complex because both Ran-GDP⅐RanBP1⅐importin-␤⅐importin-␣ and Ran-GDP⅐RanBP1⅐importin-␤ complexes are stable and because the affinities of Ran-GDP and Ran-GTP for importin-␤ in presence of RanBP1 are indistinguishable (15). Therefore, in situ formation of Ran-GTP by the nucleotide exchange activity of importin-␤ (Kap95p) in the vicinity of the nuclear pore is a plausible event. NTF2 could then be a factor that protects the import complex from premature dissociation via importin-␤ (Kap95p)-mediated nucleotide exchange. Although the inhibition by Ntf2p of the conversion of Gsp1p-GDP to Gsp1p-GTP in the Gsp1p-GDP⅐Yrb1p⅐Kap95p⅐Kap60p complex is not fully efficient in our assays, one cannot exclude the possibility that the inhibition would be more efficient in presence of nucleoporins, particularly the FXFG repeat motifs that bind both NTF2 and importin-␤ (11,43). Fig. 7 shows a hypothetical model for nuclear protein import adapted from the models proposed previously (17,44), which involves in situ GDP nucleotide exchange. In our model, which uses the more common human nomenclature for the nuclear import proteins, a Ran-GDP⅐RanBP1⅐importin-␤⅐importin-␣⅐NLS complex produced in the cytoplasm would interact with NTF2, leading to two effects (i) to generate the translocation of the complex by interacting with the FXFG repeats at the NPC and (ii) to protect the complex against in situ GDP nucleotide exchange. Arriving in the nucleus, NTF2 would be displaced by an unknown factor, rendering the cargo complex ready for GDP exchange. Subsequent to Ran-GTP formation, importin-␣ and NLS would dissociate from the complex, giving rise to a Ran-GTP⅐RanBP1⅐importin-␤ complex. Such a complex has been detected in vivo in the nucleus (40).
Our model confers a biochemical function to RanBP1 in the nucleus. The requirement for RanBP1 in nuclear import has been demonstrated by many researchers; however, its role in this part of the pathway has remained obscure (15,20,34,35,39,40). In our model the biochemical function of RanBP1 in the nucleus is to co-activate the GDP exchange by importin-␤ in the import complex. This function is accomplished through two biochemical effects; that is, the formation of a tetrameric complex that allows the association of Ran-GDP with the importin-␤⅐importin-␣ complex and the stimulation of GDP dissociation from Ran followed by the formation of Ran-GTP. In combination with its function as RanGAP co-activator, RanBP1 could be seen as an adaptor of Ran, allowing the regulation of its nucleotide-bound status by different factors. Given the high occurrence of RBH domains in nucleoporins and their similar biochemical characteristic compared with RanBP1 (45), it would be interesting to test these domains for their potential to have a role in nucleotide exchange.
Our model of cargo complex disassembly involving the in situ formation of Ran-GTP in the vicinity of the nuclear pore can easily co-occur with the accepted model featuring free Ran-GTP produced by chromatin-bound RCC1 that dissociates the cargo from importin-␤. Interestingly, there is no obvious ortholog of RCC1 in plants (46), and loss of RCC1 function in a temperature-sensitive yeast mutant leads to only a decrease of nuclear import efficiency in vivo (47). These observations suggest the existence of a nuclear import pathway independent of RCC1, which could correspond to the pathway we describe here. This hypothesis is further supported by the fact that RanBP1 mutants are defective in nuclear import (35). A mechanism involving an in situ formation of Ran-GTP in the transport complex would present the advantage of being independent of RCC1 and Ran-GTP, two versatile proteins that are involved in many vital functions in the cell other than nucleocytoplasmic transport, such as chromatin condensation, mitosis regulation, spindle assembly, and post-mitotic nuclear envelope assembly (2,48). In different physiological stages of the cell, their concentrations may vary significantly. For example, RCC1 is absent from frog sperm chromatin (7) and is inactivated at the end of the S phase (49). A mechanism of cargo disassembly independent of RCC1 and free Ran-GTP would ensure the maintenance of a basal nuclear import activity independent of the physiological stage of the cell.
In summary, we have characterized new biochemical interactions between soluble factors involved in nuclear import. Our data obtained in vitro suggest that these interactions may have an important role in the disassembly of cargo complex in the nucleus, although the function remains to be demonstrated in vivo. The determination of the structure of the complex of Ran-GDP, RanBP1, importin-␤, and importin-␣ could allow the design of mutants that would block the nucleotide exchange occurring on the complex and could be used as probes for in vivo experiments.
The model described in this paper answers many questions raised with regard to the factors responsible for the nucleocytoplasmic exchange observed at the NPC and to the function of RanBP1 in the nucleus. Given the high occurrence of RBH domains in nucleoporins and their similar biochemical characteristic compared with RanBP1 (45), it would be interesting to test these RanBP domains for their potential to have a role in nucleotide exchange.