An Assay for an Additional Nuclear Export Factor(s)
We previously found that the NES receptor CRM1 and the small GTPase Ran reconstitute efficient nuclear export of GFP-NFAT in digitonin-permeabilized HeLa cells that have been preincubated with ATP at 30°C in order to deplete shuttling export factors (Kehlenbach et al., 1998
). To identify additional cytosolic export factors besides CRM1 and Ran, we had to find conditions to make them rate limiting in our assay. We therefore compared the ability of CRM1 and cytosol to promote NFAT export in HeLa cells after various preincubation steps that might more completely deplete export factors from the permeabilized cells.
After our standard preincubation with ATP alone, we found that either CRM1 alone or cytosol alone stimulated nuclear export of GFP-NFAT to equivalent levels in the presence of an excess of wild-type Ran (Fig. a), consistent with our previous work (Kehlenbach et al., 1998
). We then examined the effects of preincubation with RanQ69L, a mutant form of Ran that is insensitive to RanGAP and therefore is predominantly in the GTP-bound form (Klebe et al., 1995
). RanQ69L has been shown to promote nuclear export of substrates carrying a leucine-rich NES (Richards et al., 1997
; Kehlenbach et al., 1998
) and to strongly inhibit NLS-mediated nuclear protein import (Dickmanns et al., 1996
; Palacios et al., 1996
). After preincubation of permeabilized cells with ATP and RanQ69L, the ability of CRM1 plus wild-type Ran to promote nuclear export was clearly reduced (Fig. b). In contrast, cytosol still retained its strong stimulatory effect on nuclear export. We next examined the effect of including an excess of NES substrate in the preincubation, which would be expected to more fully mobilize the endogenous export factors. For the NES substrate, we coupled the Rev NES to cytochrome c, a 12-kD protein that is expected to enter the nucleus by passive diffusion. Consistent with this possibility, the Rev-NES conjugate (cc-NES), but not a conjugate containing an export-defective NES, was able to compete for nuclear export of GFP-NFAT in vitro (data not shown). After preincubation of permeabilized cells with ATP, RanQ69L, and cc-NES, CRM1 and Ran were no longer sufficient to support any nuclear export of GFP-NFAT (Fig. c). Under these conditions, the GFP-NFAT remained in the nucleoplasm (see Fig. b). Again, the addition of cytosol after this preincubation resulted in a strong stimulation of nuclear export. However, preincubation of cells with ATP and cc-NES alone did not affect the ability of CRM1 and Ran to support nuclear export (data not shown).
Figure 1 Preincubation of cells with RanQ69L and cc-NES reveals a new cytosolic export factor. Permeabilized cells were preincubated with ATP (a), ATP and RanQ69L (b), or ATP, RanQ69L, and cc-NES (c). Subsequent export reactions were performed with increasing (more ...)
Figure 2 RanQ69L targets CRM1 to the cytoplasmic periphery of the NPC. (a) Permeabilized HeLa cells expressing GFP-NFAT were incubated on ice or at 30°C in either the absence or presence of a combination of RanQ69L and cc-NES. The amount of CRM1 associated (more ...)
These results clearly demonstrate that cytosol contains an activity distinct from CRM1 and Ran that is required for nuclear export of GFP-NFAT after preincubation of permeabilized cells with RanQ69L. The export substrate cc-NES potentiates this requirement for an additional factor when added with RanQ69L in the preincubation. The cytosolic activity that stimulates export under these conditions could be a nucleocytoplasmic shuttling factor that is lost from the nuclei during the preincubation. Alternatively, RanQ69L and cc-NES could impose a block on nuclear export that is efficiently relieved by a cytosolic factor. In an attempt to distinguish between these possibilities, we decided to characterize the localization of GFP-NFAT and of CRM1, the NES receptor for NFAT, after incubation of permeabilized cells with RanQ69L.
RanQ69L Targets CRM1 to the Cytoplasmic Periphery of the NPC
An export substrate containing a leucine-rich NES has been shown to bind cooperatively with RanGTP to CRM1 in vitro (Fornerod et al., 1997a
), forming a putative nuclear export complex. Preincubation of permeabilized cells with an excess of RanQ69L and cc-NES substrate should efficiently mobilize CRM1 into export complexes and perhaps trigger the transport of these complexes out of the nucleus. As we observed previously (Kehlenbach et al., 1998
), incubation of permeabilized cells at 30°C with ATP alone (Fig. a, − Q69L/cc-NES) led to a depletion of CRM1 from the cells, as compared with the 0°C control incubation (Fig. a, 0°C). In contrast, incubation with RanQ69L (Fig. a, + Q69L/cc-NES), but not with wild-type Ran (data not shown), largely prevented the loss of CRM1 from the permeabilized cells. The high level of CRM1 retained by preincubation with RanQ69L could result either from inhibition of export of CRM1 out of the nucleus or from facilitated recycling/reimport back into the nucleus. Since incubation of the same number of cells in a 30-fold larger volume at the same concentration of RanQ69L did not change the level of CRM1 compared with the standard RanQ69L incubation as detected by immunoblotting (data not shown), RanQ69L appears to act by inhibiting export of CRM1, rather than by promoting its reimport. This notion is further validated by the data shown below. Thus, although RanQ69L promotes efficient nuclear export of NES-containing substrates in vivo (Richards et al., 1997
), and also a moderate level of nuclear export in vitro (Kehlenbach et al., 1998
), the release of the nuclear export receptor CRM1 from the permeabilized cells is impaired by RanQ69L.
We next used immunocytochemical approaches to examine the localization of CRM1 in the nucleus with and without preincubation of the cells with RanQ69L. In previous work, CRM1 was found to be localized throughout the nucleoplasm as well as at the cytoplasmic and nucleoplasmic sides of the NPC in cultured cells (Fornerod et al., 1997b
). Consistent with this, by indirect immunofluorescence microscopy, we detected CRM1 predominantly in the nucleoplasm of digitonin-permeabilized cells (Fig. b, control). Incubation of cells with RanQ69L, but not with wild-type Ran (data not shown), led to a strong increase in staining at the NE together with a decrease in intranuclear staining (Fig. b, top). The localization of the export substrate GFP-NFAT did not substantially change upon incubation with RanQ69L (Fig. b, middle). Very similar results were obtained when the cells were incubated with a combination of RanQ69L and cc-NES (Fig. b, bottom). When the Triton permeabilization was omitted after the fixation step before antibody labeling, a similar level of CRM1 was detected at the NE, even though the NE remained intact, as demonstrated by the inaccessibility of nuclear lamins to anti-lamin antibodies (data not shown). This suggests that CRM1 resides at the cytoplasmic side of the NPC after the RanQ69L cc-NES incubation.
To further examine the localization of CRM1 under these conditions, we performed immunogold labeling of digitonin-permeabilized cells that had been incubated either at 0°C in buffer, or at 30°C with RanQ69L and cc-NES (Fig. c). The nuclei were permeabilized by freeze-thawing before immunolabeling. Under these conditions, the nucleoplasmic side of the NPC was freely accessible to gold probes, as demonstrated by strong labeling of the nucleoplasmic side of the NPC with antibodies to Tpr (data not shown), which is found on the nuclear side of the NPC (Cordes et al., 1997
). In the control cells, some anti-CRM1 gold labeling was seen throughout the nucleoplasm (not shown) and gold particles were occasionally found at the cytoplasmic side of the NPC (e.g., Fig. c, control, rightmost arrow). Strikingly, however, after cells were incubated with RanQ69L, the cytoplasmic periphery of every NPC was strongly decorated with gold, while essentially no labeling of the nucleoplasmic side of the NPC was evident. Labeling was specific, because it was completely abolished by preincubating the anti-CRM1 antibody with the peptide against which it had been raised. These results indicate that CRM1 crosses the NE and accumulates at the cytoplasmic periphery of the NPC when RanQ69L is present. Hence, RanQ69L appears to inhibit export by inducing the stable trapping of CRM1 at the cytoplasmic periphery of the NPC. Since this accumulation is observed only in the presence of RanQ69L but not wild-type Ran, the release of CRM1 from this binding site is likely to require GTP hydrolysis on Ran and/or a cytosolic release factor that is absent from the permeabilized cells.
Can/Nup214 Is the Major Binding Site for CRM1 at the Cytoplasmic Site of the NPC
We next carried out biochemical analyses to identify the NPC proteins with which CRM1 associates in the presence of RanQ69L. Previous work showed that immunoprecipitation of an epitope-tagged version of the nucleoporin Can/Nup214 from an extract of cultured HeLa cells resulted in the coprecipitation of some CRM1 (Fornerod et al., 1997). Can/Nup214 is one of the FG-repeat–containing nucleoporins that is detected by the monoclonal antibodies RL1 (Snow et al., 1987
) and mAb414 (Davis and Blobel, 1986
). In addition to Can/Nup214, RL1 detects the nucleoporins RanBP2, Nup153, Pom121, Nup98, p62, p58, and p54 (Snow et al., 1987
As shown in Fig. a, on silver-stained gels, we detected two proteins with apparent molecular weights of ~70 and ~220 kD (marked by asterisks) that specifically coprecipitated with CRM1 from a cell lysate after preincubation with RanQ69L at 30°C. These bands were not seen after preincubation at 30°C without RanQ69L (when the level of precipitated CRM1 was reduced due to the loss of CRM1 from the permeabilized cells under this condition; compare with Fig. a). Addition of the peptide that had been used to prepare the anti–CRM1-antibody abolished the precipitation of CRM1 and also led to the loss of the coprecipitated proteins, indicating that the latter are in a complex with CRM1.
Figure 3 RanQ69L promotes binding of CRM1 to Can/ Nup214 and p62. (a) Permeabilized HeLa cells were incubated at 30°C in the absence (−) or presence (+) of RanQ69L. Proteins were immunoprecipitated from an NP40 lysate (containing 300 (more ...)
In a similar experiment we used immunoblotting with the RL1 monoclonal antibody to analyze the NPC proteins that coprecipitated with CRM1 (Fig. b). As a control, we examined the proteins that coprecipitated with the import receptor importin β. No RL1-reactive proteins were detectable in the anti–CRM1 immunoprecipitate from cells that had been incubated in buffer at 0°C. In contrast, after incubation with ATP, RanQ69L, and cc-NES at 30°C, two RL1-positive proteins with the same apparent molecular weights as the coprecipitated proteins detected on silver-stained gels (~70 and ~220 kD; compare with Fig. a) were coprecipitated with CRM1. The latter was identified as Can/Nup214, using a specific antibody against this protein (data not shown). The 70-kD protein is p62, as judged by its mobility on SDS-PAGE and by the specificity of RL1. The monoclonal antibody mAb414 detected the same two proteins as RL1 in these immunoprecipitates (data not shown). The p58, p54, and p45 subunits of the p62 complex were not detected in these immunoprecipitation experiments by either RL1 or mAb414 with our immunoblotting conditions, probably because larger amounts of these proteins are required for detection compared with p62 (see Davis and Blobel, 1986
; Snow et al., 1987
). However, since these proteins remain tightly associated with p62 under similar extraction conditions (Guan et al., 1995
), they most likely are present in the immunoprecipitates with p62. The level of immunoprecipitated CRM1 itself in this experiment was the same with or without the RanQ69L preincubation (Fig. b, bottom left; note the difference from Fig. a, where both, the + and − RanQ69L samples were incubated at 30°C), consistent with the finding that CRM1 is retained in the permeabilized cells during the RanQ69L treatment.
As a control, we examined whether RL1 antigens coimmunoprecipitated with the import receptor importin β under these conditions. In contrast to the results obtained with CRM1, a distinct set of nucleoporins coprecipitated with importin β in this experiment. RanBP2 and Nup153 were the only detectable nucleoporins that appeared in anti–importin β immunoprecipitates from extracts of cells that had been incubated at 0°C (Fig. b). When cells were preincubated with ATP, RanQ69L, and cc-NES at 30°C, Nup153 no longer coprecipitated with importin β, although the amount of coimmunoprecipitated RanBP2 did not change significantly. The amount of importin β in the immunoprecipitate (Fig. b, bottom right) was clearly reduced after the RanQ69L incubation, indicating that importin β, unlike CRM1, is depleted from the nucleus under these conditions. Thus, the export receptor CRM1 and the import receptor importin β behave differently during preincubation with RanQ69L: RanQ69L promotes the association of CRM1 with a subset of cytoplasmic nucleoporins, but does not promote its release from nuclei, whereas RanQ69L induces the dissociation of importin β from a nucleoplasmic nucleoporin (Nup153) and stimulates release of importin β from the permeabilized cells.
To investigate the binding of CRM1 to proteins of the NPC under defined in vitro conditions, we immobilized CRM1 on anti–CRM1 antibodies coupled to Protein A beads. The beads were then incubated with an NP40 lysate derived from purified rat liver NEs in the presence of RanGDP or RanGMP-PNP, a nonhydrolyzable GTP analogue. In some reactions, we added cc-NES, which enhances the binding of RanGMP-PNP to CRM1 (data not shown). Proteins bound to the CRM1 beads were detected on immunoblots using the RL1 antibody. In the absence of added cc-NES, the nucleoporins p62 and Nup153 and a faint band migrating below Nup153 bound to the beads in the presence of RanGDP (Fig. c). Very similar levels of these proteins bound when no exogenous Ran was added (data not shown). When the incubation contained Ran that had been loaded with GMP-PNP, Can/Nup214 and an increased level of p62 bound to the beads. Neither the level of Nup153 nor of the minor band below Nup153 changed upon addition of RanGMP-PNP. Although addition of cc-NES to the incubations had no effect when the samples contained RanGDP, cc-NES increased the amount of Can/Nup214 and p62 bound to the beads in the presence of RanGMP-PNP. As a control for the specificity of binding, we used Protein A beads that had been coupled to rabbit IgG instead of anti–CRM1 antibody, but were otherwise treated identically. Only a very faint band corresponding to Nup153 could be detected (data not shown), indicating that the binding of nucleoporins to the beads was specific for CRM1. The differential binding of Nup153 to CRM1 in Fig. , b and c, probably results from the different experimental approaches: in the immunoprecipitation experiment (Fig. b), we examined CRM1 that is associated with proteins of intact NPCs at the end point of a transport reaction in a permeabilized cell assay. This reaction leads to accumulation of CRM1 on the cytoplasmic side of the NPC, whereas Nup153 is localized to the nucleoplasmic side. In contrast, in the in vitro binding experiment (Fig. c), solubilized proteins of the NPC are allowed to bind to immobilized CRM1.
We next investigated the binding of partially purified CRM1 to recombinant proteins of the p62 complex (p54, p58, and p62) and to a COOH-terminal fragment of Can/ Nup214 that has previously been shown to bind to CRM1 in vivo (Fornerod et al., 1996
). In this experiment, the nucleoporins (instead of CRM1) were immobilized on affinity matrices for the binding reactions. As shown in Fig. d, RanGMP-PNP, but not RanGDP or GMP-PNP alone, strongly promoted binding of CRM1 to all three proteins of the p62 complex. The binding was further increased if an NES peptide was included in the reaction. We detected substantial binding of CRM1 to the COOH-terminal fragment of Can/Nup214 in the presence of RanGDP or free GMP-PNP (i.e., in the absence of Ran), and only a modest increase in binding when RanGMP-PNP was added. No binding was detected when we used Ni beads that had not been coupled with the Can/Nup214 fragment (data not shown).
In the two different binding reconstitution experiments (Fig. , c and d), RanGMP-PNP stimulated the nucleoporin/CRM1 interactions to different levels, depending on the source of nucleoporins and the nature of the affinity matrix. It strongly enhanced the interaction between Can/Nup214 and CRM1 when solubilized NEs were used as a source of nucleoporins (Fig. c), but only modestly enhanced the interaction when a recombinant fragment of Can/Nup214 was used (Fig. d). Conversely, RanGMP-PNP only modestly enhanced the association of p62 with CRM1 when p62 came from solubilized NEs, but strongly enhanced the association when recombinant p62, p58, and p54 were used (Fig. d). Aside from the different nature of the affinity matrices in the two cases (see Fig. , legend), these differences may be due to the presence of components in the solubilized NEs that differentially affect the binding of Can/Nup214 versus p62 to CRM1. In addition, the short recombinant fragment from the COOH terminus of Can/Nup214 might be folded in a way that results in a different binding activity of the fragment compared with the full-length protein from NEs.
Despite these quantitative differences, the results of these in vitro binding reconstitution experiments are in agreement with the results of the immunoprecipitation experiments involving extracts of permeabilized cells (Fig. , a and b), which showed that RanQ69L induces the stable binding of CRM1 to Can/Nup214 and p62 in permeabilized cells. The stimulation of binding of CRM1 to these nucleoporins by RanQ69L/RanGMP-PNP suggests that these interactions reflect the association of an export complex separately with Can/Nup214 and p62. It remains possible that a ternary complex of CRM1, p62, and Can/ Nup214 can be formed. However, in light of our EM data, we think it unlikely that the end point of the transport reaction in the presence of RanQ69L, which is analyzed in the immunoprecipitation experiments (Fig. , a and b), involves such a complex. This is because much of the CRM1 was found at the periphery of the NPC where Can/Nup214 is localized (Panté et al., 1994
), which is distinct from the central location where p62 is found (Guan et al., 1995
Significantly, the above results imply that RanGTP not only promotes the formation of an export complex, but also targets it to specific sites in the NPC during transport. Since Can/Nup214 is located in a more peripheral cytoplasmic location in the NPC than the p62 complex, it is likely that the association of the export complex with Can/ Nup214 represents a late step in nuclear export. Release from this site is mediated by a cytosolic factor, at least in cells that have been preincubated with RanQ69L, thereby allowing efficient nuclear export in a subsequent reaction (see Fig. ).
RanBP1 Promotes Nuclear Export of GFP-NFAT
To identify the cytosolic factor that restores export of GFP-NFAT after preincubation of permeabilized cells with RanQ69L and cc-NES, we fractionated cytosol by column chromatography and tested individual fractions for their ability to promote export in the presence of exogenous Ran and partially purified CRM1. With analysis by ion exchange chromatography using a Mono Q column, we obtained a major peak of export activity in fraction 20, which eluted from the column at ~280 mM NaCl (Fig. a). This peak fraction was further analyzed by gel filtration on an S200 column. A small peak of export activity was observed in fraction 19, at the position of a globular protein of ~30 kD (Fig. b). A much larger peak eluting at the same position was obtained when total cytosol instead of a Mono Q fraction was used for gel filtration (data not shown). The apparent size of the active protein eluting from the gel filtration column prompted us to probe fractions of both purification steps for the presence of RanBP1, a previously characterized cytosolic RanGTP-binding protein of 28 kD (Coutavas et al., 1993
; Bischoff et al., 1995b
). As shown in Fig. , a and b (insets), the peak of RanBP1 detectable by immunoblotting coincides with the peak of export activity on both the Mono Q and the gel filtration columns. When the S200 samples were analyzed by silver staining, a protein with the same mobility as RanBP1 was the only species that peaked in fraction 19 (data not shown). This protein accounted for ~10% of the total protein in that fraction.
Figure 4 RanBP1 and RanBP1-related domains promote nuclear export in vitro. (a) Activity profile of Mono Q fractions. HeLa cytosol was chromatographed on a Mono Q ion exchange column. 20 μl of individual fractions was dialyzed against transport buffer (more ...)
We next tested the ability of recombinant RanBP1 to stimulate nuclear export of GFP-NFAT after the RanQ69L cc-NES preincubation. We found that a combination of wild-type Ran and RanBP1 strongly stimulated nuclear export (Fig. c). Neither Ran nor RanBP1 alone had a significant effect. No further stimulation of export was obtained by adding purified CRM1 to Ran and RanBP1 in the reconstitution (data not shown). In a titration experiment (data not shown), we found that 50% of the maximal stimulation was obtained with ~2 μg/ml RanBP1. RanBP1 was maximally active at a concentration of ~15 μg/ml, whereas a higher concentration resulted in inhibition of nuclear export. These results demonstrate that RanBP1 stimulates nuclear export in cells that have been preincubated with RanQ69L and cc-NES, indicating that it relieves the block imposed by these reagents. This raises the possibility that RanBP1 or related proteins have a role in nuclear export under normal conditions.
As discussed above (see Fig. ), Ran and CRM1 support efficient nuclear export in permeabilized cells that have been preincubated with ATP alone. Nevertheless, RanBP1 does have a modest stimulatory effect on nuclear export in nonpreincubated cells, as demonstrated by the time course experiment shown in Fig. d (note that RanQ69L-preincubated cells were used in all other panels of Fig. ). Here, incubation of the permeabilized cells for 30 min reduced the nuclear fluorescence from 100 to 37 U in the presence of RanBP1, but only to 51 U in its absence. This RanBP1-mediated stimulation of nuclear export by ~30% was obtained in four independent experiments.
The observation that RanBP1 is not absolutely required for nuclear export under these conditions raised the possibility that another factor might provide RanBP1-like functions when the cells have not been preincubated with RanQ69L. RanBP2 is a giant nucleoporin that has four RBDs very similar to that of RanBP1 (Yokoyama et al., 1995
). RanBP2 is localized in close proximity to Can/ Nup214 on the cytoplasmic side of the NPC (Wilken et al., 1995
; Wu et al., 1995
; Yokoyama et al., 1995
) and is well situated to interact with an export complex bound to Can/ Nup214. In RanQ69L preincubated cells, the RBDs of RanBP2 are likely to be stably occupied by RanQ69L, which cannot be readily released because the RanQ69L mutant is insensitive to RanGAP. This situation would necessitate the addition of exogenous RanBP1 to promote export, even though the RBDs of RanBP2 would be available under normal conditions.
To test whether the RBDs of RanBP2 can function like RanBP1 in our assay, we expressed them as 6× His-tagged fusion proteins and tested their ability to stimulate nuclear export of GFP-NFAT after preincubation of permeabilized cells with RanQ69L and cc-NES. As shown in Fig. e, the combination of RBD1 of RanBP2 and Ran stimulates nuclear export to a similar extent as RanBP1 and Ran (compare Fig. c). RBDs 2–4 of RanBP2 were equally effective (data not shown). These results show that either RanBP1 or the RBDs of RanBP2 can release the block of nuclear export imposed by preincubating cells with RanQ69L and cc-NES.
RanBP1 and RBDs of RanBP2 Release CRM1 from Can/Nup214 at the Cytoplasmic Periphery of the NPC
RanBP1 has been shown to promote the dissociation of RanGTP from the importin β-related proteins transportin and CAS, as well as from importin β itself (in the presence of importin α; Bischoff and Görlich, 1997
). To test whether RanBP1 has a similar effect on the RanGTP-CRM1 complex, we bound purified CRM1 to GST-RanGMP-PNP and examined whether RanBP1 was able to release the CRM1 in a subsequent incubation. As shown in Fig. a, RanBP1 released the majority of CRM1 into the supernatant (compare the levels of released and bound CRM1), whereas very little CRM1 was released in the absence of RanBP1.
Figure 5 RanBP1 and RBDs of RBP2 release CRM1 from the NE. (a) Release of CRM1 from GST-Ran by RanBP1. Purified CRM1 was bound to GST-RanGMP-PNP. Beads were either kept on ice (0°C) or incubated at 30°C in the absence (−) or presence (more ...)
We next tested whether RanBP1 might have a similar effect on CRM1 that is trapped in a complex with Can/ Nup214 at the cytoplasmic side of the NPC as a consequence of incubation of cells with RanQ69L and cc-NES. Since the binding of CRM1 to Can/Nup214 is strongly promoted by RanGTP, the dissociation of RanGTP from CRM1 would be expected to promote the release of the latter from the NPC. Indeed, after a first incubation of permeabilized cells with RanQ69L, a second incubation with RanBP1 strongly reduced the amount of CRM1 at the NE as seen by immunofluorescence microscopy (Fig. b). Without RanBP1, cells exhibited a strong nuclear rim staining for CRM1, very similar to the one obtained without a second incubation (compare with Fig. b). The release of CRM1 from NE of RanQ69L-preincubated cells was analyzed also by immunoblotting (Fig. c). Whereas a major proportion of CRM1 remained associated with the cells (pellet, P) in the absence of RanBP1, the addition of RanBP1 resulted in the recovery of most CRM1 in the released fraction (supernatant, S). A similar effect was obtained with RBD1 of RanBP2 (Fig. c).
Taken together, these results suggest that RanBP1, and probably the RBDs of RanBP2, are involved in the disassembly of the export complex at a terminal nucleoporin site (Can/Nup214). RanBP1 and RanBP2 apparently act by releasing RanGTP from CRM1, thereby promoting release of the export complex from the NPC and dissociation of the cargo-receptor complex. If the RBDs of RanBP2 are occupied by RanQ69L, exogenously added RanBP1 (or cytosolic RanBP1 in intact cells) or soluble RBDs of RanBP2 are required to fulfill this function.