RanBP3 Stimulates Crm1-mediated Nuclear Protein Export
Genetic and biochemical evidence has linked the yeast orthologue of RanBP3, Yrb2p, to Xpo1p-dependent nuclear protein export, but the function of Yrb2p in export remains unknown (Taura et al. 1998
; Noguchi et al. 1999
). To test whether RanBP3 is involved in Crm1-mediated nuclear export, we used an in vitro nuclear export assay that uses the NES of PKI (Holaska and Paschal 1998
). This two-step assay involves an import and an export phase. In the import phase, a fluorescein isothiocyanate–conjugated streptavidin–NLS fusion protein (FITC–STV–NLS) is imported into nuclei of digitonin-permeabilized HeLa cells with lysate. In the export phase, biotinylated PKI (bPKI) is added, which is small enough to diffuse into the nucleus. There it can bind the fluorescent streptavidin and, in the presence of soluble transport factors, mediate export of bPKI/FITC–STV–NLS into the cytoplasm. Residual nuclear fluorescence is quantitated, and export activity is measured as the loss of fluorescence compared with controls that lack the necessary transport factors.
To seek an effect of RanBP3 on nuclear protein export, it was necessary to find levels of Crm1 and Ran that were limiting with respect to export activity. A limiting concentration of Crm1 (90 nM) alone or Crm1 plus Ran (1.0 μM) did not significantly increase the amount of export over buffer alone ( and ). However, when this amount of Crm1 and Ran was supplemented with GST-RanBP3 (1.0 μM), a significant loss of nuclear fluorescence was measured. This activity was fully inhibited by the addition of LMB, which reacts covalently with Crm1 and inhibits its interaction with NES cargo (Kudo et al. 1999
). RanBP3 lacking the GST tag also activated export (data not shown). Therefore, RanBP3 can stimulate Crm1-dependent export activity in a permeabilized cell nuclear export assay.
Figure 1 RanBP3 stimulates Crm1-mediated nuclear protein export. (A) Permeabilized HeLa cells were preloaded with bPKI/FITC–STV–NLS export cargo and then exposed to Crm1 (90 nM), Ran (1.0 μM), ±RanBP3 (1.0 μM), ±LMB (more ...)
RanBP3 Binds Directly and Specifically to Crm1
Next, we asked whether a direct association could be observed between RanBP3 and Crm1. As shown in A, His-tagged Crm1 (Crm1-H6) associated with GST-RanBP3 directly (lane 8). RanQ69L, a GTPase-deficient Ran mutant, was efficiently incorporated into the Crm1–RanBP3 complex without reducing the levels of bound Crm1 (lanes 8 and 9). These data suggest that Crm1, RanBP3, and Ran can form a stable ternary complex.
Figure 2 Crm1 binds directly and specifically to RanBP3. (A) Crm1 binds directly to RanBP3 in the presence of RanQ69L and NES peptide. Recombinant Crm1-H6 (200 nM) was incubated with GST-tagged RanBP3 attached to glutathione–Sepharose beads in the presence (more ...)
Crm1 has been shown to form ternary complexes with Ran:GTP and NES-bearing proteins. We predicted that RanBP3 was not acting as a classic NES, because it could enhance Crm1-mediated export in vitro () and its binding to Crm1 was not enhanced by Ran. Nevertheless, to determine whether binding of RanBP3 to Crm1 was competitive with NES binding, we analyzed the Crm1–RanBP3 interaction in the presence of a large molar excess of a NES peptide. Neither NES peptide alone nor NES peptide plus RanQ69L prevented Crm1 from binding GST-RanBP3 ( A, lanes 10 and 11). Moreover, LMB did not inhibit binding of Crm1 or RanQ69L to GST-RanBP3 (lane 12), although it completely inhibited Crm1 binding to NES ( A). Together, these data demonstrate that the interaction between Crm1 and RanBP3 is distinct from that between Crm1 and NES.
Figure 3 RanBP3 increases the affinity of Crm1 for Ran:GTP. (A) Various combinations of Crm1-H6 (330 nM), GST-RanBP3 (250 nM), and RanQ69L (2.0 μM) were incubated with MVM NS2 NES peptide–conjugated beads (5 μl). Bound and unbound fractions (more ...)
To assess the selectivity of this interaction, the binding of RanBP3 to other karyopherin-β family members was assayed in a yeast dihybrid assay. Only strains harboring Crm1 and RanBP3 were able to support growth in the absence of histidine ( B). Importin-11, CAS, and importin-β were functional in this assay as they were able to interact with VP16-Ran. The poor ability of Crm1 to interact with Ran in this assay has been reported by others (Stade et al. 1997
). These data suggest that RanBP3 may be a specific partner of Crm1.
RanBP3 Increases Crm1 Binding to Ran:GTP and Export Cargo
Can Crm1 within a RanBP3–Crm1–Ran:GTP complex still bind NES cargo? To address this question, we used beads coupled to an MVM-NS2 NES peptide. These beads were exposed to solutions containing combinations of transport factors ( A). Neither GST-RanBP3 nor RanQ69L alone bound significantly to the beads without Crm1 ( A, lane 4), nor did any of these factors bind to control beads generated from an unrelated peptide encoding an NH2
-terminal sequence from the small GTPase TC10 ( A, lanes 1–3). As expected from previous work, the NES beads associated weakly with Crm1 alone, and RanQ69L substantially increased the binding efficiency, reflecting the increased affinity of Crm1 for NES in the presence of Ran:GTP ( A, lane 5 vs. 6) (Fornerod et al. 1997a
). When GST-RanBP3 was added to the assay, a small amount bound to Crm1 on the NES beads in the absence of RanQ69L (lane 7). The amount of bound GST-RanBP3 increased when RanQ69L was included ( A, lane 8), most likely reflecting the increased amount of Crm1 bound to the beads. Addition of LMB to the assay abrogated the ability of this complex to bind the beads ( A, lane 9). Therefore, RanBP3 is able to bind in a quarternary complex containing Crm1, RanBP3, NES, and Ran:GTP.
To more quantitatively determine the interaction of Ran:GTP within this complex, we immobilized Crm1-H6 on Ni2+-NTA beads. The beads were then exposed to a titration of Ran:γ[32P]GTP in the absence or presence of GST-RanBP3 (4:1 molar ratio of RanBP3 to Crm1). The titration was repeated with three levels of NES peptide: a saturating level (40 μM), an intermediate level (100 nM), or no peptide. At the lower levels of NES peptide (100 or 0 nM), RanBP3 significantly increased the amounts of Ran:GTP bound within the complex ( B). These results indicate that, at subsaturating levels of NES, the affinity of the Crm1–RanBP3 complex for Ran:GTP is greater than that of Crm1 alone. At very high NES concentration (40 μM), the binding of Ran:γ[32P]GTP to Crm1 was saturable in both the presence and absence of RanBP3. Interestingly, although RanBP3 and Crm1 both contain a Ran-binding domain (RBD), the amount of Ran associated with Crm1 under saturating conditions was identical in either the presence or absence of RanBP3. This experiment indicates that the stoichiometry of Ran:GTP with respect to Crm1 is not increased by binding to RanBP3 and implies that RanBP3 acts by increasing the affinity of the Crm1–RanBP3 complex for Ran:GTP, rather than by independently binding a second Ran:GTP per complex.
It is important to note that the relative concentrations of RanBP3 and Crm1 are critical to the modulation of Ran's binding to Crm1. At low RanBP3:Crm1 ratios, such as the one used above (4:1), RanBP3 increases the amount of Ran:GTP bound to Crm1. However, when this ratio is increased to 40:1, RanBP3 is unable to increase the amount of Ran:GTP associated with the Crm1 beads ( C). This behavior is predicted from the nature of the Crm1–RanBP3–Ran:GTP complex. A large excess of free RanBP3 is able to compete for available Ran:GTP with the Crm1–RanBP3 complexes present on the beads. The ability to stimulate Ran:GTP binding is dependent on the presence of the RBD of RanBP3, as a mutant that lacks this domain (NF; see below) was unable to mediate the effect ( C).
RanBP3 Increases the Affinity of Crm1 for NESs
To determine the effect of RanBP3 on the binding of cargo to Crm1, we performed RanGAP protection assays. However, in light of the previously reported ability of Yrb2p to weakly coactivate RanGAP (Noguchi et al. 1997
), we first tested whether RanBP3 had a similar biochemical activity. Using a low concentration of RanGAP (150 pM) such that only ~25% of the total Ran-bound γ[32
P]GTP was hydrolyzed within the time frame of the assay, we observed that RanBP3 has a weak ability to coactivate RanGAP in the absence of other factors ( A). However, when Crm1 and NES were added to the assay, Ran was protected from the action of RanGAP (data not shown).
Figure 4 RanBP3 increases the affinity of Crm1 for Ran:GTP and NES. (A) RanBP3 is a weak coactivator of RanGAP. GST-RanGAP (0.15 nM) was added to preassembled complexes of Ranγ[32P]GTP (40 nM) +RanBP3 (1 or 10 μM) for 4 min at 30°C. After (more ...)
The ability of the quaternary complex to protect Ran from the action of RanGAP allowed us to assess quantitatively the interaction of Ran and NES with Crm1. The advantage of the GAP protection assay over the direct binding measurements shown in is that the GAP assay is performed in solution, under pseudoequilibrium conditions, whereas bead binding assays involve repeated washing and are not at equilibrium. RanGAP was increased (to 30 nM) to insure complete hydrolysis of free Ran:GTP during the assay. Crm1 alone has a low affinity for Ran:GTP and as expected was unable to protect Ran from the action of RanGAP ( B). However, when RanBP3 or the NES from MVM-NS2 were added to the solution, ~25 and ~63% of the Ran:GTP were protected, respectively. This result indicates that both RanBP3 and NES are able individually to increase the affinity of Crm1 with Ran:GTP. When both RanBP3 and NES were added, the protection was additive, such that ~85% of the GTP remained. Therefore, the formation of a quaternary complex described above ( A) can also be detected in this assay. The effect was not specific to this particular NES, as the increased protection was also observed with the NES from PKI ( B), however to a lesser extent due to the lower affinity of PKI NES versus the MVM NS2 NES for Crm1 (Askjaer et al. 1999
The fact that NES cargo binds cooperatively with Ran:GTP to Crm1, and that RanBP3 raises the affinity of Crm1 for Ran:GTP ( B), argues that RanBP3 should also increase the affinity of Crm1 for NES cargo. To test this idea, we titrated the RanGAP protection by the MVM-NS2 NES in the absence and presence of RanBP3. Increasing the concentration of NES increased the protection from RanGAP, in a saturable manner ( C), and RanBP3 was able to increase the affinity of Crm1 for the MVM-NS2 NES by 1.6-fold (from Kapp ≈ 540–330 nM).
These data indicate that RanBP3 and NES are both able to facilitate Ran:GTP binding to Crm1 complexes, but by independent mechanisms. RanBP3 also increases the affinity of Crm1 for NES cargo, but this property seems to be dependent on Ran binding because RanBP3 did not alter the efficiency of Crm1 association with NES–peptide beads in the absence of Ran ( A). Therefore, within the nucleus, RanBP3 acts to increase the efficiency of Crm1-mediated export by increasing the affinity of the Crm1–RanBP3 complex for Ran:GTP, which cooperatively promotes cargo binding.
The FxFG Domain and RBD of RanBP3 Are Both Required for Efficient Crm1-mediated Export
To better understand the mechanism by which RanBP3 activates Crm1-dependent export, we generated various RanBP3 mutants ( A). The N domain (residues 1–182) contains an NLS; the F domain (residues 183–293) comprises the central region containing two FxFG motifs; the R domain (residues 294–499) comprises the COOH-terminal low-affinity Ran:GTP-binding domain (Radu et al. 1995
; Mueller et al. 1998
; Welch et al. 1999
). Interestingly, we found that RanBP3 possesses two distinct direct binding sites for Crm1 ( B). The major binding site is located in the F domain. A weaker binding site resides in the N domain. No binding was detected using the isolated R domain.
Figure 5 The FxFG-containing domain and RBD of RanBP3 are required for full export activity. (A) Diagram of deletion mutants, showing domain structure of RanBP3. Mutants are illustrated without the 27-kD amino terminal GST tag. Locations of the NLS, two FxFG repeats, (more ...)
To determine which domains of the RanBP3 protein are responsible for export activity, the export assay was repeated using the recombinant F, R, FR, and NF fragments. Only FR, comprising both the F and R domain, restored full export activity ( C). Remarkably, the F fragment alone had an intermediate activity. However, this activity was suppressed by the presence of the NH2
-terminal region, as the NF fragment had no detectable activity. It has been shown that RanBP1 can also stimulate Crm1-mediated export, by dissociating Crm1 from nuclear pores (Kehlenbach et al. 1999
). However, it is unlikely that RanBP3 operates via this mechanism, for two reasons. First, the RanBP1 effect on nuclear export is apparent only after preincubation of the cells with RanQ69L (Kehlenbach et al. 1999
); second, the R fragment, which contains the RanBP1-like domain, has no activity in this assay ( C).
To determine whether the export activity correlated with the ability of RanBP3 to promote Ran:GTP binding to Crm1, we repeated the RanGAP protection assay with the deletion mutants. As shown in D, the ability of RanBP3 to promote Ran:GTP binding to Crm1 is dependent on both the F and R domains of RanBP3, matching the requirement for full export activity. Note that the RBD, R, is unable in isolation to promote Ran binding to Crm1, probably because it has a very low intrinsic affinity for Ran:GTP (Mueller et al. 1998
Together, these data demonstrate that the stimulation of export by RanBP3 requires its major site for Crm1 binding, the F domain, with maximal activity requiring both the F and R domains.
RanBP3 Exports from the Nucleus in a Crm1-dependent Manner
What is the fate of the Crm1–RanBP3-NES-Ran:GTP complex once it has assembled within the nucleus? We considered two alternate hypotheses. First, the quarternary complex could exit the nucleus as a unit. Second, RanBP3 could act as an assembly catalyst and be removed from the complex before export. The first model predicts that RanBP3 cycles in and out of the nucleus, whereas the second model predicts that it is constitutively nuclear. To distinguish between these models, heterokaryon fusion assays were performed, to test the ability of RanBP3 to shuttle.
For this assay, we used a triple hemagglutinin–tagged RanBP3 (HA3
-RanBP3) (Welch et al. 1999
). After transfection of BHK21 cells, they were trypsinized and replated with an equal number of GSN2 cells, which express a GFP–streptavidin–NLS fusion protein as a non-shuttling nuclear marker (Black et al. 1999
). The two cell types were fused, fixed, and stained. HA3
-RanBP3 shuttled from BHK21 nuclei into GSN2 nuclei, as evidenced by the costaining of the GSN2 nuclei ( A). The GFP–streptavidin–NLS fusion protein remained within the GSN2 acceptor nucleus. We conclude that RanBP3 is a shuttling protein and, in principle, could be exported as a component of a Crm1–RanBP3–NES–Ran:GTP complex.
Figure 6 RanBP3 exports from the nucleus in a Crm1-dependent manner. (A) Triple HA-tagged RanBP3 (HA3-RanBP3) was transiently expressed in BHK21 cells, which were trypsinized and replated with an equal number of GSN2 cells and then fused by addition of polyethyleneglycol. (more ...)
If this hypothesis were correct, RanBP3 export should be Crm1 dependent. To test this prediction, we used the Crm1-specific inhibitor, LMB. Heterokaryon fusion assays were performed with HA3
-RanBP3 and, as a control, with GFP–GFP–RanBP1 (GGRanBP1). RanBP1 is a shuttling protein that contains a LMB-sensitive NES (Richards et al. 1996
; Plafker and Macara 2000a
). The additional size imparted by the two GFPs prevents passive diffusion of the protein through the nuclear pores. Therefore, upon the addition of LMB, this construct accumulates irreversibly in the nucleus (Plafker and Macara 2000a
HA3-RanBP3 and GGRanBP1 were cotransfected into BHK21 cells that were plated at high density. Adjacent cells were fused, and their cytoplasms were allowed to mix. As expected, GGRanBP1 was cytoplasmic, whereas HA3-RanBP3 was nuclear ( B). When LMB was added after fusion, GGRanBP1 translocated into all of the nuclei of the multikaryon cell. However, when LMB was added before fusion, GGRanBP1 is present only in the donor nuclei, demonstrating that LMB completely prevents the export of a Crm1-dependent cargo. Under these conditions, RanBP3 is unable to shuttle ( B), indicating that export of RanBP3 is LMB sensitive. These data demonstrate that export of RanBP3 is dependent on formation of productive Crm1 export complexes.
The Crm1–RanBP3–NES–Ran:GTP Complex Interacts with NPCs
Since RanBP3 export was Crm1 dependent, we hypothesized that the Crm1–RanBP3–NES–Ran:GTP complex could interact with nucleoporins. To test this hypothesis, we first asked if RanBP3 could compete for Crm1 binding to nucleoporins. Cells were permeabilized with digitonin, washed to remove soluble components, and then solubilized with 1% NP-40 (Kehlenbach et al. 1999
). The soluble extract, enriched in nucleoporins, was incubated with Crm1 and/or RanBP3, RanQ69L, and NES peptide. Crm1 was precipitated with anti-Crm1 antibodies, and associated proteins were detected by immunoblotting. When supplemented with RanQ69L or RanQ69L and NES peptide, Crm1 bound both Nup214 and p62 ( A), as reported previously (Kehlenbach et al. 1999
). When RanBP3 was added in large excess, it did not reduce nucleoporin binding, suggesting that RanBP3 does not compete with Nup214 or p62 for Crm1 in the presence of Ran:GTP. This result was of interest because the F domain contains FxFG motifs that are both representative of nucleoporins and have been shown to directly bind to transport receptors (Radu et al. 1995
; Bayliss et al. 2000
). Moreover, the F domain is a principal Crm1 binding site on RanBP3.
Figure 7 The Crm1–RanBP3–NES–Ran:GTP complex interacts with the NPC. (A) Crm1 immunoprecipitations were performed in the presence or absence of excess RanBP3. 4 × 108 digitonin-permeabilized HeLa cells were solubilized, after which (more ...)
To determine whether nucleoporins can bind to a Crm1–RanBP3 complex, we used GST-RanBP3 on glutathione–Sepharose beads. In the absence of Crm1, very little Nup214 associated with the beads, suggesting that RanBP3 alone does not bind with high affinity to this nucleoporin. Similarly, when Crm1 was added, it bound RanBP3 without increasing Nup214 binding. However, addition of Crm1 and RanQ69L significantly increased Nup214 binding to the beads. Binding did not occur to beads coupled to GST alone (not shown). These results suggest that RanBP3 and Nup214 can bind simultaneously to Crm1, and this interaction is stabilized by Ran:GTP.
Detergent solubilization destroys the integrity of the NPC, and may obscure low affinity interactions with nucleoporins. We therefore tested the ability of Crm1 to bind intact NPCs in the presence of various transport factors ( and ) (Fornerod et al. 1997b
; Kehlenbach et al. 1999
). In the absence of RanBP3, binding of Crm1 to NPCs was readily detectable under all conditions (Kehlenbach et al. 1999
) ( C, top). Unexpectedly, RanBP3 inhibited the association of Crm1 with NPCs, and this inhibition was relieved by addition of RanQ69L with or without NES peptide. Conversely, RanBP3 was found to associate with NPCs only in the presence of RanQ69L or RanQ69L and NES peptide ( C, bottom). These data suggest that Ran:GTP stimulates binding of the Crm1–RanBP3 complex to NPCs.
To test this hypothesis, we studied the binding of LMB-treated Crm1 to NPCs. In addition to preventing Crm1 from binding to NES-containing proteins, it also prevents high-affinity binding of Crm1 to Ran:GTP (Fornerod et al. 1997a
). Since RanBP3 can bind LMB-treated Crm1 ( A), RanBP3 should inhibit binding of LMB-treated Crm1 to NPCs even in the presence of RanQ69L or RanQ69L and NES peptide. This effect was observed ( C, bottom, and D), demonstrating that high-affinity Ran binding to Crm1 stimulates the Crm1–RanBP3 complex to bind NPCs.
How might Ran:GTP modulate the binding of the Crm1–RanBP3 complex to the NPC? One interesting possibility is that Ran might alter the conformation of Crm1 such that it interacts with RanBP3 in a different manner, exposing a surface on Crm1 involved in NPC binding. To study the Crm1–RanBP3 interaction further, we first determined its affinity by ELISA and quantitative immunoblotting. Each of these methods produced nearly identical values (data not shown). By ELISA, the apparent dissociation constant of the RanBP3-Crm1 complex was 64 ± 14 nM; this affinity was reduced only slightly by Ran:GTP and NES (106 ± 23 nM) (data not shown). A control peptide that does not bind Crm1 ( A) does not mediate this reduction. The cause of this change in affinity became evident when we tested the effect of Ran:GTP on binding of Crm1 to isolated domains of RanBP3. Ran:GTP plus NES substantially reduced binding of the F domain of RanBP3 to Crm1, although it increased binding of the R domain ( E). Together, these data suggest that, in the absence of Ran:GTP and NES, RanBP3 binds to Crm1 principally through the N and F domains, whereas in the presence of Ran:GTP and NES, the interaction occurs primarily through the N and R domains. Therefore, although the affinity of full length RanBP3 for Crm1 does not change substantially, there is a significant change in the mode of binding. This change in binding mode could explain how Ran alters the ability of the RanBP3-Crm1 complex to interact with NPCs (see Discussion).
The Crm1–RanBP3–NES–Ran:GTP Complex Is Disassembled by RanBP1 and RanGAP
Upon translocation to the cytoplasm, Ran:GTP within the Crm1–RanBP3-NES-Ran:GTP complex must be removed to release NES cargo. What factors mediate this event? It is clear that RanGAP alone is insufficient to mediate disassembly of either the Crm1–Ran:GTP-NES complex (Askjaer et al. 1999
) or the Crm1–RanBP3–NES–Ran:GTP complex (). Since RanBP1 has been shown to stimulate export by removing Ran from Crm1–Ran:GTP–NES complexes and subsequent presentation to RanGAP (Askjaer et al. 1999
; Kehlenbach et al. 1999
), we asked whether it might have a similar role for the Crm1–RanBP3–NES–Ran:GTP complex.
To test this hypothesis, RanGAP was added to preassembled export complexes with or without RanBP1. RanBP1 and RanGAP, but not RanGAP alone, were able to hydrolyze Ran:GTP bound within both the Crm1–Ran:GTP–NES complex as well as the Crm1–RanBP3–NES–Ran:GTP quaternary complex ( A).
Figure 8 The Crm1–RanBP3–NES–Ran:GTP complex is dissembled by RanBP1 and RanGAP. (A) GST-RanGAP (30 nM) or GST-RanGAP plus RanBP1 (1.0 μM) were added to complexes of Ranγ[32P]GTP (40 nM), GST-RanBP3 (1.0 μM), Crm1-H (more ...)
What is the mechanism by which RanBP1 disassembles the quaternary complex? The RBDs of RanBP1 and RanBP3 are related structurally, and it is likely that RanBP1 would compete with the RBD of RanBP3 within the complex to gain access to Ran:GTP. To test this idea, glutathione–Sepharose beads coated with GST–RanBP3 were incubated with excess Crm1, RanQ69L, and NES peptide ( B). Under these conditions, no detectable RanQ69L is able to bind RanBP3 in the absence of Crm1 (data not shown). After complex assembly, the beads were washed then resuspended in either buffer alone or buffer containing RanBP1 (1 μM), and the bound and unbound fractions were analyzed at 1 and 10 min. At 1 min, a slightly greater amount of Crm1 was released from the beads in the presence of RanBP1 (ratio bound/total = 0.59 vs. 0.52). At 10 min the difference was greater, with the ratio decreasing from 0.52 to 0.39. The effect of RanBP1 on the release of Ran from this complex was more pronounced. At 10 min, ~90% of the RanQ69L was released with RanBP1, whereas in contrast only ~50% was released without RanBP1. These results indicate that RanBP1 can remove Ran:GTP from the Crm1–RanBP3-NES-Ran:GTP quaternary complex.