Stimulation of Nuclear Export and Inhibition of Nuclear Import by a Ran Mutant Deficient in Binding to Ran-binding Protein 1*

Ralph H. KehlenbachDagger §, Ralf Assheuer, Angelika KehlenbachDagger , Jörg Becker, and Larry GeraceDagger

From the Dagger  Departments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 and the  Max-Planck-Institut für Molekulare Physiologie, 11 Otto-Hahn-Strasse, Dortmund 44227, Germany

Received for publication, December 11, 2000, and in revised form, January 16, 2001




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Receptor-mediated nucleocytoplasmic transport is dependent on the GTPase Ran and Ran-binding protein 1 (RanBP1). The acidic C terminus of Ran is required for high affinity interaction between Ran and RanBP1. We found that a novel Ran mutant with four of its five acidic C-terminal amino acids modified to alanine (RanC4A) has an ~20-fold reduced affinity for RanBP1. We investigated the effects of RanC4A on nuclear import and export in permeabilized HeLa cells. Although RanC4A promotes accumulation of the nuclear export receptor CRM1 at the cytoplasmic nucleoporin Nup214, it strongly stimulates nuclear export of GFP-NFAT. Since RanC4A exhibits an elevated affinity for CRM1 and other nuclear transport receptors, this suggests that formation of the export complex containing CRM1, Ran-GTP, and substrate is a rate-limiting step in export, not release from Nup214. Conversely, importin alpha /beta -dependent nuclear import of bovine serum albumin, coupled to a classical nuclear localization sequence is strongly inhibited by RanC4A. Inhibition can be reversed by additional importin alpha , which promotes the formation of an importin alpha /beta complex. These results provide physiological evidence that release of Ran-GTP from importin beta  by RanBP1 and importin alpha  is critical for the recycling of importin beta  to a transport-competent state.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molecular transport between the cytoplasm and the nucleus occurs through nuclear pore complexes (NPCs),1 large supramolecular structures that span the nuclear envelope (for review, see Refs. 1 and 2). Small molecules such as ions and metabolites cross the NPC by passive diffusion. In contrast, most macromolecules are transported through the NPC by signal- and energy-dependent processes. Much of the signal-dependent nucleocytoplasmic transport is mediated by nucleocytoplasmic shuttling receptor proteins of the importin beta /karyopherin beta  family. These receptors are thought to transfer their cargoes between the nucleus and the cytoplasm by sequentially interacting with a series of NPC proteins (nucleoporins; for review, see Refs. 3 and 4).

Transport mediated by importin/karyopherin beta -type transport receptors is dependent on the small GTPase Ran, which shuttles between the cytoplasm and the nucleus (3). Due to the segregation of the RanGEF in the nucleus and the RanGAP in the cytoplasm, the GTP-bound form of Ran is thought to be concentrated in the nucleus and the GDP-bound form in the cytoplasm. Ran-GTP, which binds to all importin beta -type import receptors, promotes the association of cargoes with export receptors and the dissociation of cargoes from import receptors in vitro and thus appears to regulate receptor loading and unloading in the nucleus (see below).

The best characterized nuclear import pathway involves cargo proteins carrying a basic amino acid-rich nuclear localization sequence (NLS). A trimeric import complex is formed by the binding of the cargo to the adapter protein importin alpha , which interacts with the import receptor importin beta  via its importin beta -binding (IBB) domain (3). After the import complex is translocated to the nucleoplasmic side of the NPC, Ran-GTP is thought to dissociate importin alpha  from importin beta  and importin beta  from nucleoporins. The resulting Ran-GTP-importin beta  complex is exported back to the cytoplasm, whereas importin alpha  is recycled to the cytoplasm by the export receptor CAS (5).

In the most extensively studied nuclear protein export pathway, Ran-GTP and a cargo protein containing a leucine-rich nuclear export sequence (NES) bind cooperatively to the export receptor CRM1 (6, 7). After translocation of this trimeric export complex to the cytoplasmic side of the NPC, RanBP1 and the related Ran-GTP binding domains of the nucleoporin Nup358/RanBP2 are thought to disassemble the export complex (8, 9) as well as release it from a terminal cytoplasmic nucleoporin in the pathway, Nup214/CAN (10).

RanBP1 is a cytosolic protein that further enhances the rate of GTP hydrolysis on free Ran-GTP that is stimulated by the GTPase-activating protein RanGAP (11, 12). When Ran-GTP is complexed with import or export receptors, such as occurs when import receptors are recycled or export complexes are translocated to the cytoplasm, the Ran-GTP is GAP-insensitive. In this case, RanBP1 strongly promotes GTPase stimulation by RanGAP because it releases Ran-GTP from the receptors (8, 9). Thus, RanBP1 may play a crucial role in the termination of nuclear export reactions in the cytoplasm by promoting the dissociation of Ran from the receptors and, as a consequence, the hydrolysis of Ran-GTP.

An acidic stretch at the C terminus of Ran is important for its interaction with RanBP1, since a mutant in which the last six amino acids of Ran are removed (RanDelta DEDDDL; RanDelta C) does not interact with Ran-GTP and RanBP1 in the yeast two-hybrid system (13) and on blot overlays (13, 14). Moreover, RanDelta C has a greatly reduced affinity for RanBP1 in solution (15, 16). Whereas the acidic tail of Ran strongly promotes the interaction with RanBP1, it is inhibitory for binding to importin beta  (14, 16), perhaps due to competition with an acidic loop in importin beta  for binding to a basic patch on Ran (17).

We have characterized a novel Ran mutant, RanC4A, which has four of its five C-terminal acidic residues exchanged with alanine (18), to examine the role of Ran-RanBP1 interactions in nuclear import and export in vitro. As RanDelta C, RanC4A has a reduced affinity for RanBP1 and an increased affinity for importin beta  and also for CRM1. Using well established in vitro nuclear transport systems, we show that RanC4A strongly stimulates CRM1-mediated nuclear export of GFP-NFAT, whereas it inhibits importin alpha -dependent nuclear import by importin beta . Our findings shed light on rate-limiting steps of CRM1-mediated export and provide physiological evidence for a role of RanBP1 in the recycling of import receptors.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- HeLa cells were grown on either plastic dishes or coverslips in Dulbecco's modified Eagle's medium or in suspension in Joklik's modified S-MEM. Both media contained 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. All tissue culture reagents were from Life Technologies, Inc.

Recombinant Transport Factors-- The expression plasmid (pET3d (19)) for C-terminally mutated RanC4A (DEDDDL mutated to AAADAL, amino acids 211-216) used for the experiments in Table I and Figs. 1-3 was constructed by polymerase chain reaction using appropriate oligonucleotides containing NcoI/BamHI sites for cloning. Wild-type and mutant Ran were purified as described (20). Purified Ran was >95% pure, as judged by gel electrophoresis. Its quality was assessed by determining the concentration of GDP bound to Ran using reverse-phase HPLC analysis on a C-18 HPLC column (21).

The wild-type Ran and RanQ69L used for all experiments except Table I were purified as described (22), with minor modifications. RanC4A used for the experiments in Figs. 4 and 5 was expressed in Escherichia coli BL21 (DE3) as a GST fusion protein. After purification using glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech), the GST tag was cleaved with biotin-coupled thrombin (Novagen), as described by the manufacturer, followed by the removal of thrombin with streptavidin-agarose (Sigma) and of cleaved GST with glutathione-Sepharose 4B beads. RanDelta C was expressed as a GST fusion protein and thrombin-cleaved as described above. Proteins were dialyzed against transport buffer (20 mM Hepes, pH 7.3, 110 mM KOAc, 2 mM Mg(OAc)2, 1 mM EGTA). No functional difference was evident between RanC4A obtained by cleavage from a GST fusion protein as compared with expression as an unfused protein.

Importins alpha  and beta  (23), CRM1 (24), and mouse RanGAP (25) were prepared as described and dialyzed against transport buffer containing 2 mM dithiothreitol and 1 µg/ml each of leupeptin, pepstatin, and aprotinin. RanBP1 (kindly provided by Dr. Iris Schmitt) was expressed in E. coli from pET11d. After precipitation of proteins from a bacterial lysate with ammonium sulfate (50-75% cut), RanBP1 was further purified by gel filtration and ion exchange chromatography using a MonoQ column and dialyzed against transport buffer. GST-RanBP1 (15), Rna1p (26), and RCC1 (RanGEF; Ref. 27) were purified as described. All proteins were frozen in liquid nitrogen and stored at -80 °C.

Guanine Nucleotide Exchange-- To exchange the Ran-bound GDP with GTP or the fluorescently labeled mant-GDP (28, 29), Ran (3-5 mg) was incubated for 3 h at room temperature in the presence of a 50-200-fold molar excess of GTP or mant-GDP in 50 mM Tris, pH 7.4, 20 mM EDTA, 5 mM MgCl2, 5 mM dithioerythritol. The nucleotide exchange of Ran-bound GDP to Gpp(NH)p or mant-Gpp(NH)p was performed in the presence of alkaline phosphatase (5 units/mg Ran) and a 5-fold molar excess of Gpp(NH)p or mant-Gpp(NH)p in 50 mM Tris, pH 7.4, 200 mM (NH4)2S04, 100 nM ZnCI2, 5 mM dithioerythritol, for 3 h at room temperature. Unbound nucleotide was removed by gel filtration over two PD10 columns (Amersham Pharmacia Biotech) in 50 mM Tris, pH 7.4, 5 mM MgCl2, 5 mM dithioerythritol or in 20 mM K2HPO4/KH2PO4, pH 7.4, 5 mM MgCl2, 5 mM dithioerythritol. The concentration of the nucleotide-loaded Ran was measured by HPLC (21).

Intrinsic GTPase Activity-- The intrinsic GTPase activity was measured by reverse-phase HPLC analysis (21). 400 µM Ran-GTP was incubated at 37 °C in 50 mM Tris, 5 mM MgCl2, 5 mM dithioerythritol, pH 7.4, at 37 °C. Aliquots were removed at regular intervals and applied to HPLC analysis to measure the protein-bound GTP and GDP concentration. The HPLC data were expressed as ln((GTP + GDP)/GTP) and plotted as a function of time. The GTP hydrolysis rate, kcat (min-1), was calculated by linear regression of the data with Kaleidagraph 3.0.5 (Synergy Software).

Interaction of Ran and Ran Mutants with Regulatory Proteins-- The RanGAP-stimulated GTP hydrolysis of Ran analyzed in Table I was measured by radioactive filter assays as described previously (30) using different concentrations of the RanGAP of Schizosaccharomyces pombe, Rnalp.

The GST-RanBP1-Ran interaction was analyzed by determining the dissociation constant Kd = koff/kon using fluorescence spectroscopy (15). The association kinetics were measured under pseudo-first-order conditions with 200 nM Ran-mant-Gpp(NH)p and increasing concentrations of GST-RanBP1 (1, 2, 3, 4, 5, and 6 µM). The signal of the Ran-bound fluorescence-labeled nonhydrolyzable GTP analog mant-Gpp(NH)p (excitation, 350 nm; emission, 440 nm) was followed in a SX16MV stopped flow system (Applied Photophysics). The kinetic constant kon of the Ran-GST-RanBP1 association was calculated with Kaleidagraph 3.0.5 (Synergy Software) as described previously (15). For the measurement of the dissociation kinetics (koff), a 1:1 complex between 200 nM Ran-mant-Gpp(NH)p and GST-RanBP1 was generated in 20 mM K2HPO4/KH2PO4, pH 7.4, 5 mM MgCl2, 5 mM dithioerythritol, in a titration experiment at 25 °C. The titration was followed in a SPEX Fluoromax fluorescence spectrometer (excitation, 350 nm; emission, 440 nm; Ref. 15). After formation of the GST-RanBP1-Ran complex, it was dissociated by adding a 50-fold molar excess of unlabeled RanGpp(NH)p. The data of the dissociation reaction were fitted as first-order exponentials with Kaleidagraph 3.0.5 to calculate the kinetic constant koff. The two kinetic constants were used to calculate the equilibrium constant Kd = koff/kon, which characterizes the affinity between Ran and RanBP1.

For the analysis of the Ran-RCC1 interaction, different Ran-GDP concentrations (0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 7, and 10 mM) were incubated in the presence of 5 nM RCC1 and 200 µM mant-GDP at 25 °C in 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 5 mM dithioerythritol. The reaction rates of the RCC1-catalyzed GDP dissociation on Ran were measured as a decrease in fluorescence (excitation, 295 nm; emission, 335 nm) due to energy transfer, using a PerkinElmer Life Sciences LS SOB fluorescence spectrophotometer (31). The kinetic constants of the Ran-RCC1 interaction (kcat, Km) were calculated using Kaleidagraph 3.0.5 (Synergy Software) as described previously (31).

To analyze the RanGAP activity for all experiments other than in Table I, Ran was loaded with [gamma -32P]GTP as described (10). RanGAP assays (except for Table I; see above) were performed essentially as described (32), using 30-60 nM total Ran, 4 nM RanGAP, 400 µM GTP, and, in some reactions, a 20 µM concentration of the NES peptide from the minute virus of mouse NS2 protein (CVDEMTKKFGTLTIHDTEK (32, 33)) or a 20 µM concentration of the NLS peptide from the SV40 large T antigen (CGGGPKKKRKVED). After 10 min at 20 °C, reactions were stopped by adding 1 ml of stop buffer (7% charcoal, 10% ethanol, 0.1 M HCl, 10 mM NaH2PO4). After centrifugation, the released [32P]phosphate in 700 µl of the supernatant was measured by scintillation counting. Background values of samples with radioactive Ran added immediately to the stop buffer were subtracted, and GTP hydrolysis was expressed as the percentage of the maximal value of recovered radioactivity, as obtained in the absence of inhibiting transport receptors.

Nuclear Transport Assays-- For nuclear import assays on adherent cells, HeLa cells were grown on coverslips and permeabilized with 30 µg/ml digitonin (Calbiochem) in transport buffer containing 2 mM dithiothreitol and 1 µg/ml each of leupeptin, pepstatin, and aprotinin. The permeabilized cells were incubated in nuclear import reactions for 30 min at 30 °C as described (34), except the import substrate was a fusion protein between the amino terminus of human SRPalpha and beta -galactosidase (IBB-beta Gal; 112 µg/ml; modified from Ref. 35; kindly provided by Dr. S. Lyman). Cells were fixed with 3.7% formaldehyde in phosphate-buffered saline, permeabilized with 0.2% Triton X-100 in phosphate-buffered saline, and then analyzed by indirect immunofluorescence microscopy using a monoclonal antibody against beta -galactosidase (Promega) to detect the IBB-beta Gal. Nuclear import was visualized by confocal microscopy using a Bio-Rad MRC1024 confocal unit attached to a Zeiss Axiovert S100TV microscope. All digital data sets were processed identically using Adobe Photoshop (Adobe). When suspension HeLa cells were used for import assays, the reactions were performed as described in Ref. 36, containing 2.5 mg/ml cytosol, unless otherwise indicated. The nuclear import substrate for suspension assays was Cy5-BSA-NLS, which was prepared as described (37). Flow cytometry was performed using a dual laser cytometer (FACSCaliber, Becton Dickinson). Reactions were standardized by assigning a fluorescent value of 100 arbitrary units to a reaction resulting in optimal import. The nuclear export assay using GFP-NFAT-transfected HeLa cells was carried out as described (37).

Antibodies, Immunoprecipitations, and Western Blotting-- The anti-CRM1 peptide antibody was prepared and affinity-purified as described (37). The monoclonal antibody RL2 is described elsewhere (38). The monoclonal anti-importin beta -antibody (ascites fluid; Affinity BioReagents, Inc.) used for immunoprecipitations was diluted 1:1 with 5 mg/ml BSA in transport buffer and stored at -20 °C. Monoclonal anti-Ran antibody was from Transduction Laboratories. The polyclonal anti-importin beta  and anti-importin alpha  antibodies were raised in rabbits against recombinant proteins and used after affinity purification.

For immunoblotting, proteins were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose using standard methods. Blots were blocked with 5% milk powder in TBST (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05% Tween 20) overnight. Horseradish peroxidase-coupled goat-anti-mouse or donkey-anti-rabbit IgG (Pierce; 1:10,000 in 10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05% Tween 20) was used as a secondary antibody. The enhanced chemiluminescence system (Pierce) was used for visualization of proteins.

For coimmunoprecipitation of nucleoporins, 3 × 106 digitonin-permeabilized cells were solubilized for 20 min on ice in 1 ml of Nonidet P-40 buffer (1% Nonidet P-40, 50 mM Tris-HCl, pH 8, 300 mM NaCl, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl2, 60 mM beta -glycerophosphate, 100 µM KF, 100 µM NaVO4, 2 mM dithiothreitol, 1 µg/ml each of leupeptin, pepstatin, aprotinin). After centrifugation at 14,000 × g for 20 min at 4 °C, the lysate was precleared with protein G-agarose beads (Life Technologies) for 30 min, and anti-CRM1 antibody (10 µg/ml) was added to the supernatant. Samples were incubated at 20 °C for 1.5 h, and immunoreactive proteins were collected with protein G-agarose. The beads were washed four times with Nonidet P-40 buffer, and precipitated proteins were analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting. For coimmunoprecipitation of importin beta , 2 × 106 permeabilized cells were incubated with cytosol, import ligand, and an ATP-regenerating system (i.e. under the conditions used in nuclear import reactions). After centrifugation (1,000 × g for 1 min and 14,000 × g for 5 min), the pelleted nuclei were discarded, and the supernatant was precleared with protein G-agarose beads for 30 min. Anti-importin alpha  antibody was added to 10 µg/ml. Samples were then incubated for 1.5 h at 20 °C, and immunoreactive proteins were collected with protein G-agarose. The beads were washed four times with transport buffer, and precipitated proteins were analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Biochemical Characterization of RanC4A-- The acidic C terminus of Ran promotes the interaction between Ran and RanBP1 (15, 16, 39), whereas it appears to impede the interaction between Ran and importin beta  (16, 40). To investigate the importance of this Ran region for nuclear import and export, we have characterized a novel Ran mutant, RanC4A, which has its C-terminal sequence DEDDDL (amino acids 211-216) mutated to AAADAL. We expected that this mutant, where the acidic C terminus is largely neutralized, would be structurally more similar to wild-type Ran than the previously described C-terminal deletion mutant RanDelta C, where different effects on nuclear import have been described in different assay systems (13, 41). Since RanC4A exhibited a much stronger effect on nuclear export as compared with RanDelta C (see below), we have focused our functional analysis on RanC4A, although RanDelta C was also analyzed in most experiments to attempt to resolve the discrepancies previously reported with functional assays (13, 41).

The biochemical characteristics of RanC4A, as compared with wild-type Ran (RanWT) and the previously analyzed RanQ69L mutant (the standard for our export inhibition studies; see below), are summarized in Table I (see also Ref. 18). As described before, RanQ69L had a strongly reduced intrinsic GTPase activity and was largely insensitive to RanGAP (31). RanC4A had an about 3-fold increased GTPase activity, when stimulated by RanGAP (0.261 min-1 as compared with 0.074 min-1 for RanWT). The kcat/Km of RanC4A for the exchange factor RanGEF (RCC1), on the other hand, was decreased 3-fold as compared with RanWT. A more dramatic change was observed in the affinity of RanC4A for RanBP1. As a result of an increased off-rate, the apparent affinity of RanC4A is ~20-fold lower, as compared with wild-type Ran (74.4 versus 3.7 nM). Thus, the mutation in RanC4A, which neutralizes the acidic C terminus, most strongly affects the binding of the protein to RanBP1. Very likely, RanC4A also has a strongly diminished affinity for the Ran-binding domains of Nup358/RanBP2. Our findings are consistent with the strong decrease in affinity of the C-terminal deletion mutant RanDelta C for RanBP1 that was reported previously (15, 16, 39).


                              
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Table I
Biochemical characterization of RanC4A as compared to RanWT and RanQ69L

CRM1 Accumulates at Nup214 in the Presence of RanC4A-- We used the RanC4A mutant to test the model that RanBP1 promotes the release of the export complex containing CRM1, Ran, and substrate from Nup214 at the cytoplasmic side of the NPC (10). This model was based on the finding that CRM1 accumulates at Nup214 in permeabilized cells preincubated with the RanQ69L mutant, reflecting an apparent transport arrest at a terminal nucleoporin binding site. Since CRM1 can be released from the NPC in these cells with exogenous RanBP1, the export inhibition is presumed to be due to RanQ69L-mediated occlusion of the Ran-binding domains of Nup358/RanBP2 that normally mediate the release step.

Permeabilized cells were incubated with RanWT, RanQ69L, or RanC4A and subsequently analyzed for CRM1 and Ran distribution by indirect immunofluorescence microscopy. As shown in Fig. 1A, after incubation of cells with RanWT, CRM1 was largely depleted from the nuclei. In contrast, CRM1 accumulated at the nuclear envelope in the presence of RanQ69L, as described before (10). Incubation of cells with RanC4A also leads to the accumulation of CRM1 at the nuclear envelope, although to a lesser extent than with RanQ69L. Also, more CRM1 remained in the nucleoplasm in the presence of RanC4A, as compared with RanWT or RanQ69L. RanWT was largely lost from nuclei after the reaction (and after washing the cells), whereas RanQ69L became concentrated at the nuclear envelope, as described before (42). RanC4A, on the other hand, was detectable at a high level throughout the nucleus after the reaction. This retention of RanC4A in the nucleus probably resulted from an increased affinity for intranuclear importin beta -related transport receptors (see below).



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Fig. 1.   CRM1 accumulates at Nup214 in the presence of RanC4A. A, indirect immunofluorescence detecting CRM1 (left panel) or Ran (right panel) after incubation of permeabilized cells in the presence of 40 µg/ml RanWT (WT), RanQ69L (Q69L), or RanC4A (C4A). B, coimmunoprecipitation of solubilized nuclear envelope proteins using the anti-CRM1 antibody after incubation of permeabilized cells in the presence of 20 µg/ml RanWT (WT), RanQ69L (Q69L), or RanC4A (C4A). Immunoprecipitated Nup214 (top) or CRM1 (bottom) were detected by Western blotting using the RL2 antibody or the anti-CRM1 antibody, respectively.

To identify the nucleoporins associated with CRM1 in cells treated with the Ran mutants and RanWT, we solubilized cells and carried out coimmunoprecipitation analysis with anti-CRM1 antibodies. As shown previously (10), Nup214 coprecipitated with CRM1 after incubation of permeabilized cells with RanQ69L, but not after incubation with RanWT (Fig. 1B, top). Incubation with RanC4A also resulted in coprecipitation of CRM1 with Nup214, although to a somewhat lesser extent than with RanQ69L. Immunoprecipitation of CRM1 was very similar under the different conditions (Fig. 1B, bottom). No other nucleoporins were detected in immunoprecipitates with CRM1 after pretreatment of cells with RanC4A, when samples were analyzed by Western blotting with the RL2 monoclonal antibody that reacts with a group of FG repeat nucleoporins (data not shown). Incubation of permeabilized cells with the C-terminal deletion mutant RanDelta C also resulted in increased coprecipitation of Nup214 (data not shown).

Thus, these data indicate that CRM1 accumulates at Nup214 in the presence of RanC4A, which is strongly deficient in its interaction with RanBP1 and (by extension) the Ran-binding domains of Nup358/RanBP2. These findings provide independent support for our previous conclusion that the interaction of RanBP1 and/or the Ran-binding domains of Nup358/RanBP2 with the export complex is required for efficient release of CRM1 from Nup214 in a terminal step of export (10).

Stimulation of Nuclear Export by RanC4A-- We next tested the effect of RanC4A and RanDelta C on in vitro nuclear export mediated by CRM1. Surprisingly, RanC4A did not inhibit nuclear export of GFP-NFAT when transport was assayed in the presence of cytosol, which contains RanWT (data not shown). This is in contrast to RanQ69L, which strongly inhibited nuclear export under similar conditions (37). Furthermore, RanC4A strongly stimulated nuclear export in permeabilized cells when the analysis was done without added cytosol and under conditions where Ran is the only rate-limiting factor (Ref. 37; Fig. 2A). Stimulation of nuclear export by RanC4A was even more efficient than by RanWT, since high levels of export were obtained with much lower levels of RanC4A as compared with RanWT (Fig. 2A). The C-terminal deletion mutant RanDelta C also promoted nuclear export to some extent. RanDelta C, however, did not yield the degree of stimulation of RanWT or RanC4A, even at saturating concentrations (Fig. 2A). Release of GFP-NFAT from the nucleus in the presence of RanC4A still followed a physiological pathway, since export could be inhibited by wheat germ agglutinin (43), which occludes the NPC; by leptomycin B (44), which inhibits cargo binding to CRM1; and by a short peptide corresponding to the nuclear export sequence of the minute virus of mice (32), which competes for binding to CRM1 (data not shown). The stimulation of nuclear export obtained with RanC4A suggests that the release of the nuclear export complex from the cytoplasmic side of the NPC, which appears to be impaired with RanC4A, is not a rate-limiting step in export in our assay (see "Discussion").



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Fig. 2.   Stimulation of nuclear export of GFP-NFAT. A, nuclear export reactions were performed in the absence of cytosol with increasing concentrations of RanWT (open squares), Ran C4A (closed circles), or RanDelta C (open triangles). Under the conditions of this experiment, the CRM1 in the permeabilized cell is sufficient to support efficient export, and Ran is the only rate-limiting factor. Note that the highest concentration of RanC4A in this experiment was 40 µg/ml. The corresponding data point for RanWT overlaps with the one for RanC4A. B, GAP assays were performed in the presence of RanWT (open squares) or RanC4A (closed circles), both loaded with [gamma -32P]GTP, 20 µM NS2 peptide, and increasing concentrations of His-tagged CRM1.

It has been observed previously by blot overlay analysis that RanDelta C binds more strongly to importin beta  (and potentially to other importin beta  family members) than does RanWT (16, 40). We therefore tested whether the greater stimulation of nuclear export by RanC4A, as compared with RanWT, resulted from an increased affinity of the Ran mutant for CRM1, which could enhance the formation of a nuclear export complex. Since the binding of nuclear transport receptors to Ran-GTP inhibits GTP hydrolysis stimulated by RanGAP, RanGAP resistance can be used to determine the apparent affinities of receptors for Ran-GTP (8). In the case of CRM1, a trimeric GAP-resistant complex is formed by export substrate, Ran-GTP and CRM1 (32). We used a peptide comprising the strong nuclear export sequence of the NS2 protein of minute virus of mice (32) to generate a CRM1 export complex. As shown in Fig. 2B, half-maximal inhibition of GTP hydrolysis could be obtained at much lower concentrations of CRM1 in reactions containing RanC4A, as compared with reactions containing RanWT (~2 nM versus 40 nM). The apparent affinity of CRM1 for RanWT that we measured is consistent with the one obtained previously under similar conditions (32). Almost no inhibition of GTP hydrolysis was observed when the NES peptide was omitted in the reaction (data not shown). In contrast to CRM1, we did not detect a significant difference in the affinity of RanWT and RanC4A for the export receptor CAS (data not shown).

These data indicate that RanC4A can increase the rate of formation of a CRM1 export complex, as compared with RanWT. The ability of RanC4A to stimulate nuclear export at lower concentrations than RanWT suggests that the initial formation of the export complex containing export substrate, CRM1, and Ran-GTP is the rate-limiting step in export. Although the release of the export complex from the cytoplasmic side of the NPC is impaired with RanC4A, this step is not rate-limiting in the export cycle in our conditions. Apparently, the 20-fold reduced affinity of RanC4A for RanBP1 (or the Ran-binding domains of Nup358/RanBP2) still suffices for release of the export complex from the cytoplasmic side of the NPC.

Inhibition of Nuclear Import by RanC4A-- RanBP1 has been proposed to mediate the release of Ran-GTP from the import receptor importin beta  and from CAS, the export receptor for importin alpha , to promote the recycling of nuclear import receptors (8, 9). Consistent with the prediction that RanBP1 has an important role in the nuclear import cycle, expression of a C-terminal deletion mutant of Ran has been found to inhibit nuclear import in vivo (41). By contrast, the same mutant was found to stimulate nuclear import similar to RanWT under certain in vitro conditions (13). To address this discrepancy, we tested the effects of RanC4A and RanDelta C on nuclear import in permeabilized cells. In the presence of cytosol, import of BSA-NLS was strongly inhibited by RanC4A (Fig. 3A), but not by RanWT (data not shown). The C-terminal deletion mutant RanDelta C also strongly inhibited nuclear import in the presence of cytosol (Fig. 3B).



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Fig. 3.   Inhibition of nuclear import by RanC4A and RanDelta C. Nuclear import reactions were performed in the presence of cytosol with increasing concentrations of RanC4A (A) or with or without 16 µg/ml RanDelta C (B). C, GAP assays were performed with increasing concentrations of S-His-tagged importin beta  and RanWT (open squares) or RanC4A (closed circles), both loaded with [gamma -32P]GTP.

The 20-fold reduced affinity of RanC4A for RanBP1 suggests that nuclear import in the presence of RanC4A might be restored to normal levels by the addition of high levels of exogenous RanBP1. Although we found that RanBP1 did not stimulate nuclear import when added together with RanC4A, a meaningful conclusion could not be drawn from this experiment, since RanBP1 itself inhibits nuclear import under similar conditions (data not shown).

To further investigate the basis for inhibition of nuclear import by RanC4A, we used RanGAP assays (as in Fig. 2B) to determine the apparent affinity of RanWT and RanC4A for importin beta . As shown in Fig. 3C, importin beta  has a higher apparent affinity for RanC4A than for RanWT, although the difference is not as pronounced as seen with CRM1. Half-maximal inhibition of GTP hydrolysis was obtained with ~0.4 nM importin beta  and RanC4A, as compared with ~2 nM importin beta  and RanWT. Our results with RanC4A indicate that the C terminus of Ran negatively affects the interaction of the protein with importin beta , confirming previous findings with RanDelta C (16, 40).

A priori, the increased affinity of RanC4A for importin beta  is unlikely to inhibit the nuclear import phase of the import cycle. Consistent with this premise, we found by coprecipitation experiments that the putative Ran-GTP-mediated release of importin beta  from nucleoporins including Nup153 (45, 46) does not appear to be inhibited by RanC4A (data not shown). However, the increased affinity of these proteins may impair efficient recycling of import receptors and their release from Ran-GTP, thereby indirectly inhibiting nuclear import. Impaired receptor recycling might be manifest by an increase in the intranuclear level of the receptors. To examine whether transport receptors accumulate in the nucleus in the presence of RanC4A, cells were incubated in the presence of cytosol with RanWT or RanC4A and subsequently fractionated into a nuclear and a cytosolic pool. RanC4A became somewhat concentrated in the nuclear fraction as compared with RanWT, consistent with the immunofluorescence data in Fig. 1A. However, importin alpha , importin beta , and CAS were largely or exclusively recovered in the cytosolic fraction, irrespective of the type of Ran used during the incubation (data not shown). Hence, RanC4A does not lead to nuclear accumulation (or as a consequence, cytosolic depletion) of either one of those transport factors.

RanC4A also could inhibit the recycling of import receptors by inhibiting the release of Ran-GTP from the Ran-GTP-importin beta  complex that is exported from the nucleus, which apparently is needed to form an import-competent importin alpha /beta complex. In vitro studies have suggested that the release of Ran-GTP from importin beta  is mediated by RanBP1 and importin alpha  (8, 9). To investigate whether RanC4A caused any changes in the cytosolic levels of the importin alpha /beta complex generated by importin beta  recycling, we incubated permeabilized cells with RanWT or RanC4A in the presence of cytosol, sedimented the nuclei by centrifugation, and immunoprecipitated proteins from the supernatant (i.e. the cytosolic fraction) using an antibody against importin alpha . As shown in Fig. 4A, RanC4A strongly decreased the level of coprecipitated importin beta , as compared with an incubation with RanWT, whereas levels of precipitated importin alpha  were similar in both conditions. Therefore, it appears that RanC4A impairs the release of Ran-GTP from cytosolic importin beta , thereby causing a decrease in the amount of the transport-competent importin alpha /beta complex in the transport assays.



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Fig. 4.   A, coimmunoprecipitation of importin beta  with importin alpha . Permeabilized cells were incubated with cytosol, import ligand (BSA-NLS), and 20 µg/ml RanWT (WT) or RanC4A (C4A). Cells were pelleted, and proteins from the supernatant were precipitated using the anti-importin alpha  antibody. Immunoprecipitated importin alpha  and beta  were detected by Western blotting. The band below importin alpha  is unspecific. B, release of inhibition of GTP hydrolysis by importin alpha . GAP assays were performed in the presence of 30 nM importin beta , 500 nM RanBP1, 20 µM NLS peptide, and increasing concentrations of importin alpha  with RanWT (open squares) or RanC4A (closed circles), loaded with [gamma -32P]GTP. If the NLS peptide was omitted, ~4-fold higher levels of importin alpha  were required for maximal stimulation of GTP hydrolysis on RanWT (data not shown).

We next analyzed the ability of importin alpha  to release the inhibition of GTP hydrolysis imposed by importin beta  on either RanWT or RanC4A in vitro. After formation of a complex between importin beta  and RanWT or RanC4A, GAP assays were performed with increasing concentrations of importin alpha  together with a high concentration of RanBP1, so that the latter would not be rate-limiting for stimulation of GTP hydrolysis. As shown in Fig. 4B, much higher concentrations of importin alpha  are required to release inhibition of GTP hydrolysis in the RanC4A-importin beta  complex, as compared with the complex containing RanWT. These results suggest that under conditions where nuclear import is inhibited by RanC4A, importin alpha  may be a rate-limiting nuclear import factor; the cytosolic concentration of importin alpha  may be adequate only to generate reduced levels of the importin alpha /beta complex.

Importin alpha  Restores RanC4A-inhibited Nuclear Import-- We directly tested whether generation of an importin alpha /beta complex was limiting for nuclear import in vitro by adding increasing amounts of importin alpha  to reactions in the presence of cytosol. In the absence of RanC4A, increasing concentrations of exogenous importin alpha  inhibited nuclear import of BSA-NLS, probably because free importin alpha  competed with importin alpha -beta complexes for the cargo (Fig. 5A). In the presence of RanC4A, which strongly inhibited import in the absence of added importin alpha , low concentrations of exogenous importin alpha  restored import nearly to the uninhibited level. These results show that although the level of cytosolic importin alpha , as detected by Western blotting, does not change upon incubation of cells with RanC4A (see above), importin alpha  is rate-limiting for nuclear import when RanC4A is present.



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Fig. 5.   Inhibition of nuclear import by RanC4A can be reversed by importin alpha . A, nuclear import reactions were performed without (open triangles, 0 °C; open squares, 30 °C) or with (closed circles, 30 °C) 30 µg/ml RanC4A and increasing concentrations of importin alpha . All reactions contained 2.5 mg/ml cytosol. B, nuclear import reactions were performed using adherent HeLa cells and IBB-beta Gal as import substrate in the presence of cytosol (cyt) or importin beta  (imp beta ), without additional Ran (buffer) or with 40 µg/ml RanWT (WT) or RanC4A (C4A). C, stimulation of GTP hydrolysis on RanWT by IBB-beta Gal or importin alpha . Reactions contained 30 nM importin beta  and 0.5 µM RanBP1.

A number of proteins have been shown to be imported into the nucleus by direct interaction with importin beta  without the participation of importin alpha  (47, 48). We used IBB-beta Gal (35) as an artificial, importin alpha -independent import substrate. As shown in Fig. 5B, nuclear import of IBB-beta Gal in the presence of cytosol was not inhibited by RanC4A. Furthermore, in the presence of importin beta , strong import of IBB-beta Gal could be obtained by adding either RanWT or RanC4A (Fig. 5B).

These results suggested that IBB-beta Gal was able to function like importin alpha  to release Ran-GTP from the Ran-GTP-importin beta  complex and allow importin beta  recycling to an import-competent state. To directly test this, we examined the inhibition of GTP hydrolysis imposed on RanWT by importin beta . As shown in Fig. 5C, in the presence of RanBP1, IBB-beta Gal stimulated GTP-hydrolysis at similar concentrations as importin alpha . Therefore, the importin beta -interacting domain of importin alpha  alone appears to promote GTP-hydrolysis on Ran-importin beta  complexes. Interestingly, IBB-beta Gal was unable to stimulate nuclear import of BSA-NLS in the presence of RanC4A (data not shown), suggesting that the interaction of the IBB domain with importin beta  directly leads to the formation of a competing import complex.

These results clearly demonstrate that importin alpha  becomes rate-limiting when nuclear import is inhibited by RanC4A. Inhibition appears to result from inefficient release of Ran-GTP from importin beta  by importin alpha  (together with RanBP1) and, as a result, a decreased regeneration of import-competent importin alpha /beta complexes. Since the IBB domain of importin alpha  efficiently stimulates GTP-hydrolysis on Ran-GTP-importin beta  complexes, nuclear import of IBB-beta Gal (and probably of other, importin alpha -independent substrates) is not sensitive to inhibition by RanC4A.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have characterized a novel Ran mutant, RanC4A, which has four out of five of the acidic residues at the very C terminus mutated to alanine. The major biochemical phenotype of this mutant is an ~20-fold reduced affinity of the GTP-bound form for RanBP1, as compared with wild-type Ran. This difference is small compared with the 2,000-10,000-fold reduction in affinity of the C-terminal deletion mutant RanDelta C for RanBP1 (15, 16). In the latter case, the reduced affinity results from a 100-fold decrease of the association rate constant kon, combined with a 100-fold increase in the dissociation rate constant koff (15). For RanC4A, the mutation mainly affects koff, suggesting that the initial recognition of RanBP1 still is functional for RanC4A, although it is impaired in the case of RanDelta C. The one acidic residue that remains unchanged in RanC4A (D214) and/or the C-terminal leucine may contribute to the remaining affinity of this mutant for RanBP1. Another prominent biochemical property of RanC4A is its increased affinity for the transport receptors CRM1 and importin beta . For the latter protein, this may be explained by competition between an acidic loop of importin beta  and the acidic tail of Ran for binding to a basic patch in Ran (17). Since CAS did not show differences in its apparent affinity for RanWT or RanC4A, this binding mechanism apparently does not apply to all importin beta -type receptors.

The high affinity of RanC4A for CRM1, importin beta  and possibly other transport receptors probably accounts for the enhanced retention of RanC4A in the nucleus as seen by immunofluorescence and Western blotting. The increased affinity of RanC4A for CRM1 would favor the formation of a nuclear export complex containing CRM1, Ran-GTP, and substrate, explaining the strong stimulation of export of GFP-NFAT by RanC4A, as compared with RanWT. Release of this complex by RanBP1 or the Ran-binding domains of Nup358/RanBP2 from its terminal binding site on the cytoplasmic site of the NPC appears to be impaired, since CRM1 accumulates at Nup214 as seen by coimmunoprecipitation with the anti-CRM1 antibody. Nevertheless, the submicromolar affinity of RanC4A or even the micromolar affinity of RanDelta C for Ran-binding domains may still allow functional interactions because of the high local concentration of the latter at the NPC. As a result, termination of export can occur, even in the presence of RanDelta C. However, it should be noted that RanDelta C, with its drastically reduced affinity for RanBP1 or Ran binding domains of Nup358/RanBP2, is less efficient in promoting export as compared with RanC4A, with its rather modest decrease in affinity. This discrepancy may result from the difference in affinities of the two proteins for RanBP1 or from other, as yet unidentified, properties of RanDelta C. Taken together, our results suggest that the initial formation of the export complex in the nucleus, which is promoted by RanC4A, is the most rate-limiting step in nuclear export.

Preincubation of permeabilized cells with RanQ69L leads to a similar accumulation of CRM1 at Nup214 as RanC4A, as described previously (10), although it occurs for a different reason. In this case, the Ran-binding sites of Nup358/RanBP2 probably are inactivated by the stable binding of RanQ69L, so that they are no longer available for releasing export complexes from the NPC. Efficient export under this condition depends on adding soluble RanBP1 (10). The fact that we obtain accumulation of CRM1 at Nup214 using two Ran mutants, which negatively affect the RanBP1-Ran interaction by two different mechanisms, strongly supports a role for RanBP1-like domains in release of the CRM1 export complex from Nup214.

Nuclear import of BSA-NLS in permeabilized cells supplemented with cytosol is strongly inhibited by RanC4A or RanDelta C, in agreement with the inhibition of import seen in cultured cells transfected with RanDelta C (41). Our results clearly show that the adapter protein importin alpha  becomes rate-limiting under conditions of import inhibition by RanC4A. In the study of Ren et al. (13), where RanDelta C promoted nuclear import as efficiently as RanWT in vitro, transport in permeabilized cells was reconstituted with a high concentration of partially purified importin alpha , which can explain the discrepancy with our study, involving unfractionated cytosol.

Importin alpha  engages in at least two interactions important for nuclear import. First, it binds to proteins carrying a nuclear localization signal. Second, together with RanBP1 it dissociates Ran-GTP from importin beta , thereby releasing the inhibition of GTP hydrolysis on Ran-GTP-importin beta  complexes, after their export from the nucleus. This reaction is favored in the presence of NLS-substrate, as suggested before (49). The first function should not be affected by RanC4A, and we assume that importin alpha -BSA-NLS complexes are readily formed in the absence or presence of RanC4A. These complexes (or importin alpha  alone), however, appear to inefficiently release RanC4A from importin beta , since higher concentrations of importin alpha  are required for the release reaction, even at saturating concentrations of RanBP1. This probably is due to the tight binding between importin beta  and RanC4A, which impedes the dissociation of this complex and subsequent formation of importin alpha /beta complexes. As a result, their level is reduced in the presence of RanC4A, as demonstrated by coimmunoprecipitation.

We showed that the dissociation of Ran-GTP-importin beta  requires only RanBP1 plus the IBB domain of importin alpha , which interacts with importin beta . A substrate like IBB-beta Gal, therefore, is efficiently imported into the nucleus in permeabilized cells supplemented with cytosol in the presence of RanC4A. Since IBB-beta Gal did not stimulate import of BSA-NLS in the presence of RanC4A, we conclude that the release reaction is directly coupled to the formation of import complexes containing IBB-beta Gal rather than BSA-NLS. Our results on nuclear import in vitro provide physiological evidence for a role of RanBP1 and importin alpha  in the recycling of importin beta  after its export from the nucleus, supporting previous biochemical data (8, 9), where it was shown that importin alpha  together with RanBP1 is required for the release of GTPase-inhibition on Ran-GTP-importin beta  complexes.

Two types of importin beta -dependent nuclear import occur in cells. The first requires an adapter protein such as importin alpha , whereas the second does not, since the import substrate binds directly to importin beta . In the context of intact cells, the two types of import cargoes may compete for importin beta . Cargoes like the human immunodeficiency virus Rev protein, which are adaptor-independent (50), could be kinetically favored in import because of their ability to release Ran-GTP from importin beta  in a fashion that is directly linked to the formation of an importin beta -cargo complex that is poised for nuclear import.

In conclusion, RanC4A is the first Ran mutant described to promote nuclear export of proteins carrying a leucine-rich NES and importin beta -dependent/importin alpha -independent nuclear import but to strongly inhibit importin alpha -dependent transport. This mutant should prove useful for future functional studies of nuclear transport.


    ACKNOWLEDGEMENTS

We are grateful to K. Weis and I. Mattaj for gifts of cDNAs and to A. Wittinghofer for continuous support.


    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM41955 (to L. G.) and Deutsche Forschungsgemeinschaft Grant 1432 2-1 (to J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Universität Heidelberg, Abteilung Virologie, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany. Tel.: 49 6221 561325; Fax: 49 6221 565003; E-mail: Ralph.Kehlenbach@med.uni-heidelberg.de.

Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M011087200


    ABBREVIATIONS

The abbreviations used are: NPC, nuclear pore complex; RanBP1 and RanBP2, Ran-binding protein 1 and 2, respectively; RanGAP, Ran GTPase-activating protein; NLS, nuclear localization sequence; IBB domain, importin beta -binding domain; NES, nuclear export sequence; GST, glutathione S-transferase; Gpp(NH)p, guanosine 5'-(beta ,gamma -imido) triphosphate; HPLC, high pressure liquid chromatography; IBB-beta Gal, IBB domain of importin alpha  fused to beta -galactosidase; BSA, bovine serum albumin; RanWT, wild-type Ran; RanGEF, Ran nucleotide exchange factor; mant, N-methylanthranyloyl; NFAT, nuclear factor of activated T cells.


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ABSTRACT
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RESULTS
DISCUSSION
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