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INTRODUCTION |
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
/karyopherin
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
-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
-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
, which interacts with the import receptor
importin
via its importin
-binding (IBB) domain (3). After the
import complex is translocated to the nucleoplasmic side of the NPC,
Ran-GTP is thought to dissociate importin
from importin
and
importin
from nucleoporins. The resulting Ran-GTP-importin
complex is exported back to the cytoplasm, whereas importin
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 (Ran
DEDDDL; Ran
C) does not interact with
Ran-GTP and RanBP1 in the yeast two-hybrid system (13) and on blot
overlays (13, 14). Moreover, Ran
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
(14, 16), perhaps due to competition with an acidic loop
in importin
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 Ran
C, RanC4A has a reduced affinity for
RanBP1 and an increased affinity for importin
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
-dependent
nuclear import by importin
. 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.
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EXPERIMENTAL PROCEDURES |
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. Ran
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
and
(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 [
-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 SRP
and
-galactosidase (IBB-
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
-galactosidase (Promega) to detect the
IBB-
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
-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
and anti-importin
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
-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
, 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
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 |
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
(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
Ran
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 Ran
C (see below),
we have focused our functional analysis on RanC4A, although Ran
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 Ran
C for RanBP1 that was reported previously (15, 16, 39).
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
-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.
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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 Ran
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 Ran
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 Ran
C also promoted nuclear export to some extent. Ran
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 Ran 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
[ -32P]GTP, 20 µM NS2 peptide, and
increasing concentrations of His-tagged CRM1.
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It has been observed previously by blot overlay analysis that Ran
C
binds more strongly to importin
(and potentially to other importin
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
and from CAS, the export receptor for importin
, 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 Ran
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 Ran
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
Ran C. Nuclear import reactions were
performed in the presence of cytosol with increasing concentrations of
RanC4A (A) or with or without 16 µg/ml Ran C
(B). C, GAP assays were performed with increasing
concentrations of S-His-tagged importin and RanWT (open
squares) or RanC4A (closed circles),
both loaded with [ -32P]GTP.
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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
. As shown in
Fig. 3C, importin
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
and RanC4A, as compared with ~2 nM importin
and RanWT. Our results with RanC4A
indicate that the C terminus of Ran negatively affects the interaction
of the protein with importin
, confirming previous findings with
Ran
C (16, 40).
A priori, the increased affinity of RanC4A for importin
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
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
, importin
, 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
complex
that is exported from the nucleus, which apparently is needed to form
an import-competent importin
/
complex. In vitro
studies have suggested that the release of Ran-GTP from importin
is
mediated by RanBP1 and importin
(8, 9). To investigate whether
RanC4A caused any changes in the cytosolic levels of the importin
/
complex generated by importin
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
. As shown in Fig.
4A, RanC4A strongly decreased
the level of coprecipitated importin
, as compared with an
incubation with RanWT, whereas levels of precipitated importin
were
similar in both conditions. Therefore, it appears that RanC4A impairs
the release of Ran-GTP from cytosolic importin
, thereby causing a
decrease in the amount of the transport-competent importin
/
complex in the transport assays.

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Fig. 4.
A, coimmunoprecipitation of importin with importin . 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 antibody.
Immunoprecipitated importin and were detected by Western
blotting. The band below importin is unspecific. B,
release of inhibition of GTP hydrolysis by importin . GAP assays
were performed in the presence of 30 nM importin , 500 nM RanBP1, 20 µM NLS peptide, and increasing
concentrations of importin with RanWT (open
squares) or RanC4A (closed circles),
loaded with [ -32P]GTP. If the NLS peptide was omitted,
~4-fold higher levels of importin were required for maximal
stimulation of GTP hydrolysis on RanWT (data not shown).
|
|
We next analyzed the ability of importin
to release the inhibition
of GTP hydrolysis imposed by importin
on either RanWT or RanC4A
in vitro. After formation of a complex between importin
and RanWT or RanC4A, GAP assays were performed with increasing concentrations of importin
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
are required to release inhibition of
GTP hydrolysis in the RanC4A-importin
complex, as compared with the
complex containing RanWT. These results suggest that under conditions where nuclear import is inhibited by RanC4A, importin
may be a
rate-limiting nuclear import factor; the cytosolic concentration of
importin
may be adequate only to generate reduced levels of the
importin
/
complex.
Importin
Restores RanC4A-inhibited Nuclear Import--
We
directly tested whether generation of an importin
/
complex was
limiting for nuclear import in vitro by adding increasing amounts of importin
to reactions in the presence of cytosol. In the
absence of RanC4A, increasing concentrations of exogenous importin
inhibited nuclear import of BSA-NLS, probably because free importin
competed with importin
-
complexes for the cargo (Fig.
5A). In the presence of
RanC4A, which strongly inhibited import in the absence of added
importin
, low concentrations of exogenous importin
restored
import nearly to the uninhibited level. These results show that
although the level of cytosolic importin
, as detected by Western
blotting, does not change upon incubation of cells with RanC4A (see
above), importin
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 . 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 . All
reactions contained 2.5 mg/ml cytosol. B, nuclear import
reactions were performed using adherent HeLa cells and IBB- Gal as
import substrate in the presence of cytosol (cyt) or
importin (imp ), without additional Ran
(buffer) or with 40 µg/ml RanWT (WT) or RanC4A
(C4A). C, stimulation of GTP hydrolysis on RanWT
by IBB- Gal or importin . Reactions contained 30 nM
importin and 0.5 µM RanBP1.
|
|
A number of proteins have been shown to be imported into the nucleus by
direct interaction with importin
without the participation of
importin
(47, 48). We used IBB-
Gal (35) as an artificial, importin
-independent import substrate. As shown in Fig.
5B, nuclear import of IBB-
Gal in the presence of cytosol
was not inhibited by RanC4A. Furthermore, in the presence of importin
, strong import of IBB-
Gal could be obtained by adding either RanWT or RanC4A (Fig. 5B).
These results suggested that IBB-
Gal was able to function like
importin
to release Ran-GTP from the Ran-GTP-importin
complex
and allow importin
recycling to an import-competent state. To
directly test this, we examined the inhibition of GTP hydrolysis
imposed on RanWT by importin
. As shown in Fig. 5C, in
the presence of RanBP1, IBB-
Gal stimulated GTP-hydrolysis at similar
concentrations as importin
. Therefore, the importin
-interacting
domain of importin
alone appears to promote GTP-hydrolysis on
Ran-importin
complexes. Interestingly, IBB-
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
directly leads to the formation of a competing import complex.
These results clearly demonstrate that importin
becomes
rate-limiting when nuclear import is inhibited by RanC4A. Inhibition appears to result from inefficient release of Ran-GTP from importin
by importin
(together with RanBP1) and, as a result, a decreased regeneration of import-competent importin
/
complexes. Since the
IBB domain of importin
efficiently stimulates GTP-hydrolysis on
Ran-GTP-importin
complexes, nuclear import of IBB-
Gal (and probably of other, importin
-independent substrates) is not
sensitive to inhibition by RanC4A.
 |
DISCUSSION |
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 Ran
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 Ran
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
. For the latter protein, this may be explained by competition
between an acidic loop of importin
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
-type receptors.
The high affinity of RanC4A for CRM1, importin
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 Ran
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 Ran
C. However, it should
be noted that Ran
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 Ran
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 Ran
C, in agreement with
the inhibition of import seen in cultured cells transfected with
Ran
C (41). Our results clearly show that the adapter protein importin
becomes rate-limiting under conditions of import
inhibition by RanC4A. In the study of Ren et al. (13), where
Ran
C promoted nuclear import as efficiently as RanWT in
vitro, transport in permeabilized cells was reconstituted with a
high concentration of partially purified importin
, which can
explain the discrepancy with our study, involving unfractionated cytosol.
Importin
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
, thereby releasing the inhibition of GTP hydrolysis on
Ran-GTP-importin
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
-BSA-NLS complexes are readily
formed in the absence or presence of RanC4A. These complexes (or
importin
alone), however, appear to inefficiently release RanC4A
from importin
, since higher concentrations of importin
are
required for the release reaction, even at saturating concentrations of
RanBP1. This probably is due to the tight binding between importin
and RanC4A, which impedes the dissociation of this complex and
subsequent formation of importin
/
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
requires only
RanBP1 plus the IBB domain of importin
, which interacts with
importin
. A substrate like IBB-
Gal, therefore, is efficiently imported into the nucleus in permeabilized cells supplemented with
cytosol in the presence of RanC4A. Since IBB-
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-
Gal rather than BSA-NLS. Our results on
nuclear import in vitro provide physiological evidence for a
role of RanBP1 and importin
in the recycling of importin
after
its export from the nucleus, supporting previous biochemical data (8,
9), where it was shown that importin
together with RanBP1 is
required for the release of GTPase-inhibition on Ran-GTP-importin
complexes.
Two types of importin
-dependent nuclear import occur in
cells. The first requires an adapter protein such as importin
, whereas the second does not, since the import substrate binds directly
to importin
. In the context of intact cells, the two types of
import cargoes may compete for importin
. 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
in a fashion that is directly linked
to the formation of an importin
-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
-dependent/importin
-independent nuclear import but
to strongly inhibit importin
-dependent transport. This
mutant should prove useful for future functional studies of nuclear transport.