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INTRODUCTION |
It is well known that the nucleocytoplasmic transport of
macromolecules occurs through
NPCs1 and that the selective
nuclear import and export of proteins occur via a signal- and
temperature-dependent pathway (1, 2). Conventional nuclear
localization signal (NLS)-containing proteins target the nuclear pores
via the formation of a stable complex, termed the nuclear
pore-targeting complex (PTAC), in the cytoplasm (3-5). This complex is
composed of a karyophilic protein and two cytosolic components. One of
these (PTAC58/importin-
/karyopherin-
/human SRP1/Srp1/Kap60p)
functions as an NLS receptor (2). The other component
(PTAC97/importin-
/karyopherin-
/p97/Kap95p) mediates the targeting
of the NLS substrate bound to the NLS receptor to the nuclear pores by
binding both the NLS receptor and components of the NPC (2). The
NPC-binding domain of importin-
is able to migrate from the
cytoplasm to the nucleus through the NPC, suggesting that importin-
is translocated through the NPC by binding directly to nucleoporins (6,
7). After targeting of the trimeric complex to the NPC, the
translocation step through the NPC occurs in an
energy-dependent manner and requires additional soluble
factors, namely a small GTPase Ran and its interacting protein,
p10/NTF2 (2). The direct binding of RanGTP to importin-
causes the
dissociation of the importin-
/
heterodimer (6, 8, 9). This event
is generally thought to occur on the nucleoplasmic side of the NPC,
resulting in the release of NLS substrate into the nucleus (6).
After the translocation of the NLS substrate into the nucleus, its
carrier molecules, importin-
and -
must return to the cytoplasm
in order to transport the next NLS substrate into the nucleus. CAS has
recently been identified as a nuclear export mediator of importin-
(10). CAS is also related to importin-
and binds simultaneously to
both importin-
and RanGTP. In addition, it has been reported that
Kap95p, which is an Saccharomyces cerevisiae homologue of
importin-
, requires a leucine-rich NES in the molecule for its
nuclear export in mammalian and yeast cells (11). Recently, an export
receptor (CRM1/exportin 1/XPO1) for a leucine-rich NES has been
identified (12-15). CRM1 is related to importin-
and binds directly
to the leucine-rich NES and RanGTP, and this trimeric complex is then
translocated to the cytoplasm through the NPC (12). However, it is not
yet known whether or not the nuclear export of mammalian importin-
requires a leucine-rich NES in mammalian cells.
Ran is a key component in the nucleocytoplasmic transport of proteins
(2, 16). Ran is an abundant, small GTPase of the Ras superfamily, which
is predominantly localized in the nucleus (17, 18). Like other GTPases,
Ran is thought to function as a molecular switch by cycling between a
GDP- and a GTP-bound state. This cycle is catalyzed mainly by two
molecules, RCC1 and RanGAP1 (19). RCC1 was first identified
as a causative gene for the temperature sensitivity of tsBN2 cells
derived from hamster BHK21 cells (20). RCC1 is located on the chromatin
(21) and functions as a guanine nucleotide exchanging factor of Ran,
which enhances the rate of guanine nucleotide exchange on Ran by about
100,000-fold (17, 22). In addition, RanGAP1 is located in the cytoplasm and on the cytoplasmic fibers extending from the NPC (23, 24) and
functions as a GTPase-activating protein for Ran, which enhances the
rate of GTP hydrolysis on Ran by about 100,000-fold (22, 25). The
asymmetric distribution of these two factors across the nuclear
envelope suggests that nuclear Ran may be predominantly the GTP-bound
form and that cytoplasmic Ran is the GDP-bound form. Furthermore, it
has been proposed that the asymmetric distribution of RanGTP across the
nuclear envelope assures the directional movement of proteins between
the cytoplasm and the nucleus (6, 26).
In this study, we show that mouse importin-
is exported from the
nucleus to the cytoplasm through the NPC in living mammalian cells and
that this nuclear export of importin-
is
temperature-dependent. Moreover, we demonstrate that the
nuclear export of mammalian importin-
depends on the NPC-binding
domain of this molecule and is not mediated by CRM1. Experiments using
a GTPase-deficient Ran mutant show that the nuclear export of
importin-
does not require Ran-dependent GTP hydrolysis.
Further, microinjection experiments using tsBN2 cells cultured at the
nonpermissive temperature show that importin-
can be transported
from the nucleus in a nuclear RanGTP-independent manner. The
requirement of Ran on nucleocytoplasmic shuttling of importin-
is discussed.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
MDBK, BHK21, and tsBN2 cells were incubated in
Dulbecco's modified Eagle's minimum essential medium supplemented
with 5% fetal bovine serum at 37 °C (MDBK, BHK21) or 33.5 °C
(tsBN2). Cultured cells were grown on coverslips for 48 h at
37 °C (BHK21) or 39.5 °C (tsBN2) prior to use in microinjection experiments.
Cell Fusion by the Hemagglutinating Virus of Japan (HVJ, Sendai
Virus)--
BHK21 or tsBN2 cells were fused by HVJ as described
previously (27). Homokaryons were incubated for 3 h at 37 °C
(BHK21) or 39.5 °C (tsBN2) prior to microinjection. In experiments
using leptomycin B, BHK21 cells were fused by HVJ after preincubation with leptomycin B (2 ng/ml in Dulbecco's modified Eagle's minimum essential medium supplemented with 5% fetal bovine serum) for 3 h
at 37 °C.
Purification of Recombinant Proteins--
Expression and
purification of recombinant mouse importin-
, the amino acid (aa)
448-876 mutant, and importin-
were performed as described
previously (7, 28, 29). To construct the expression vector of the aa
145-449 mutant, the region of aa 145-449 of mouse importin-
was amplified by PCR using the synthetic oligonucleotides (5'-TTGGGGAGCTCGAGCATATGAAAGAGTCCACATTGG-3' and
5'-GAAAAGGTACCAGGTAGACATCGTTGATGGCGGCTTC-3'), and this PCR
product was inserted into BamHI and KpnI sites of the pRSETA vector (Invitrogen Corp.). The green fluorescent protein (GFP)-fragment amplified by PCR was then inserted into a
BamHI site. Recombinant GFP-(145-449) protein was expressed
by 0.5 mM isopropyl-1-thio-
-D-galactopyranoside for 18 h at
20 °C in Escherichia coli strain BL21(DE3) and purified
with Ni-nitrilotriacetic acid resin (Qiagen Inc.) and a MonoQ column
(Amersham Pharmacia Biotech). The recombinant proteins were dialyzed
against 20 mM Hepes (pH 7.3), 110 mM potassium
acetate, 2 mM dithiothreitol (DTT), and 1 µg/ml each of
aprotinin, leupeptin, and pepstatin.
The human RanGAP1 gene (30) was amplified from a HeLa cell cDNA
library by PCR using the synthetic oligonucleotide primers 5'-GTTCCGGGATCCATGGCCTCGGAAGACATTGCCAAG-3' and
5'-GTTGCAGAATTCCTAGACCTTGTACAGCGTCTGCAG-3'. The PCR product was
inserted into the BamHI and EcoRI sites of pGEX-2T vector (Amersham Pharmacia Biotech). Recombinant glutathione S-transferase (GST)-RanGAP1 protein was expressed by 1 mM isopropyl-1-thio-
-D-galactopyranoside for
12 h at 20 °C in E. coli strain BL21(DE3). E. coli cells were harvested by centrifugation and resuspended in 25 ml of TE buffer (50 mM Tris-HCl, pH 8.3, 1 mM
EDTA, 2 mM DTT) containing 500 mM NaCl and 1 mM phenylmethylsulfonyl fluoride. Cells were disrupted by
means of a French press. The extract was clarified by centrifugation (30 min at 15,000 × g) and incubated with 1.5 ml of
glutathione-Sepharose. The recombinant protein, which was trapped in
glutathione-Sepharose, was eluted with TE buffer containing 100 mM NaCl and 10 mM glutathione. The GST portion
of the GST-RanGAP1 fusion protein was cleaved off by a 2-h incubation
at room temperature with 1 National Institutes of Health unit of
thrombin/100 µg of protein. GST and thrombin were separated from the
recombinant protein on a MonoQ column at a flow rate of 0.5 ml/min with
a linear gradient from 0.05-1.0 M NaCl in 20 mM Hepes (pH 7.3) and 2 mM DTT. RanGAP1 eluted
with 600 mM NaCl. Peak fractions containing RanGAP1 were
pooled and dialyzed against 20 mM Hepes (pH 7.3), 110 mM potassium acetate and 2 mM DTT.
The mouse RanBP1 gene was amplified by PCR using the synthetic
oligonucleotide primers 5'-CCTACGGATCCATGGCGGCCGCCAAGGACA-3' and
5'-CCACTGAATTCTCATTGTTTCTCCTCAGACTTCTC-3'. The PCR product was inserted
into the BamHI and EcoRI sites of pGEX-2T vector. Expression in Escherichia coli and purification of the
recombinant fusion protein was performed in the same manner as for
RanGAP1. GST and thrombin were separated from the recombinant protein
on a MonoQ column at a flow rate of 0.5 ml/min with a linear gradient from 0 to 1.0 M NaCl in 20 mM Hepes (pH 7.3)
and 2 mM DTT.
An expression vector of GST-GFP (containing the S65A/Y145F mutation)
fusion protein, pGEX-6P-2-hGFP, was kindly provided by Dr. S. Kuroda
(Institute of Scientific and Industrial Research, Osaka University,
Japan). Expression in E. coli and purification of the
recombinant fusion protein was performed in the same manner as for
RanGAP1. GST-GFP binding to glutathione-Sepharose was cleaved by
PreScission Protease (Amersham Pharmacia Biotech). Free GFP protein was
dialyzed against 20 mM Hepes (pH 7.3), 110 mM
potassium acetate, and 2 mM DTT.
These resultant plasmids were sequenced to confirm the fidelity of the
region amplified by PCR and in frame ligation of the fused region.
Aliquots of each recombinant protein were frozen in liquid nitrogen and
stored at
80 °C.
Ran(G19V) (31) and GST-NES-GFP proteins (32) were prepared as described previously.
Conjugation of Texas Red with Bovine Serum Albumin
(TR-BSA)--
Bovine serum albumin (BSA) was dissolved at 5 mg/ml in
0.1 M NaHCO3, 0.5 mg of Texas Red (TR) was
added per 5 mg of BSA, and the solution was incubated for 2 h at
room temperature. Free fluorophore was removed by gel filtration on a
PD10 (Amersham Pharmacia Biotech) equilibrated with 10 mM
Hepes (pH 7.3), 110 mM potassium acetate. Peak fractions
containing TR-BSA were collected and dialyzed against 10 mM
Hepes (pH 7.3), 110 mM potassium acetate.
Microinjection--
Microinjection experiments were performed
essentially as described previously (33). After microinjection and
incubation, cells were fixed with 3.7% formaldehyde in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM
Na2HPO4, 1 mM
KH2PO4, pH 7.2) for 30 min at room temperature.
The injected fluorescence-labeled proteins were detected by Axiophot 2 microscopy (Carl Zeiss, Inc.).
Ran Overlay Assay--
RanGTP overlay assay was performed as
described previously (7). Recombinant importin-
, its aa 145-449
mutant, and RanBP1 were separated by 12.5% SDS-polyacylamide gel
electrophoresis and transferred to nitrocellulose and then incubated,
first in buffer containing 20 mM Hepes (pH 7.3), 100 mM sodium acetate, 5 mM magnesium acetate,
0.25% Tween 20, 0.5% BSA, and 5 mM DTT and then
preincubated for 30 min at room temperature in binding buffer (20 mM Hepes (pH 7.3), 100 mM potassium acetate, 5 mM magnesium acetate, 0.05% Tween 20, 0.5% BSA, and 5 mM DTT) in the presence of 100 µM GTP. Blots
were rinsed with binding buffer and then overlaid with 0.126 pmol/µl
[
-32P]GTP-Ran (specific activity 15400 cpm/pmol) in
binding buffer for 30 min at room temperature.
Preparation of an NLS-containing Transport Substrate
(T-APC)--
Allophycocyanin (Calbiochem) was chemically conjugated to
synthetic peptide containing the amino acid sequence of SV40 large T-antigen NLS (CYGGPKKKRKVEDP), as described previously (28).
Cell-free Import Assay--
Digitonin-permeabilized MDBK cells
were prepared as described previously (28, 34). Digitonin-permeabilized
cells were incubated with transport buffer (20 mM Hepes (pH
7.3), 110 mM potassium acetate, 2 mM magnesium
acetate, 5 mM sodium acetate, 1 mM
glycoletherdiaminetetraacetic acid, 2 mM DTT, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin) containing test proteins, in 10 µl, under the conditions indicated in the figure legends. After
incubation, cells were fixed with 3.7% formaldehyde in transport buffer.
Antibodies--
Rabbit anti-mouse importin-
and anti-mouse
importin-
antibodies were prepared as described previously (28,
29).
Indirect Immunofluorescence--
Cells were washed twice in PBS
and fixed with 3.7% formaldehyde in PBS for 30 min at room
temperature. After permeabilization with 0.5% Triton X-100 in PBS for
5 min at room temperature, cells were incubated with 10 µg/ml of
affinity-purified antibodies for 1.5 h at room temperature. Rabbit
antibodies were detected with fluorescein isothiocyanate-conjugated
goat antibodies to rabbit IgG (TAGO). The samples were examined using
an Axiophot 2 microscope (Carl Zeiss Inc.).
 |
RESULTS |
Nuclear Export of Importin-
in Homokaryons of Mammalian
Cells--
It is proposed that importin-
is recycled back to the
cytoplasm after termination of translocation of NLS protein-carrier complexes via the binding of RanGTP to importin-
at the
nucleoplasmic side of the NPC. However, the precise mechanism involved
in the recycling of importin-
remains obscure. Therefore, we
attempted to better understand the mechanism of how importin-
is
exported from the nucleus to the cytoplasm after dissociating from the cargo in the nucleus. To accomplish this, we constructed homokaryons by
using the HVJ, injected recombinant GFP-fused mouse importin-
proteins into a nucleus of multinucleated cells, and then examined the
intracellular localization of GFP-importin-
. As shown in Fig.
1C, GFP-importin-
was
exported from the nucleus to the cytoplasm and then reimported into all
of the nuclei in the homokaryons within 30 min at 37 °C.

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Fig. 1.
Nuclear export of mouse
importin- in mammalian culture cells.
A, constructions of deletion mutants of importin- . The
binding activity of wild type and each mutant form of importin- to
importin- and GTPase Ran and the nuclear migration activity are
summarized on the right. B, the aa 145-449
mutant of importin- contains NPC-binding domain, but not Ran-binding
domain. Left, RanGTP-binding activity of aa 145-449 mutant
determined by overlay assay. 100 pmol of WT importin- , its aa
145-449 mutant, and RanBP1 were subjected to SDS-polyacylamide gel
electrophoresis, transferred to a nitrocellulose sheet, and incubated
with [ -32P]GTP-Ran. [ -32P]GTP-Ran
bound to the recombinant proteins was detected by autoradiography.
Right, digitonin-permeabilized cells were incubated with 10 µl of testing solution containing 6 pmol of importin- , 6 pmol of
importin- , and T-APC (50 µg/ml) in the presence or absence of 150 pmol of aa 145-449 mutant for 20 min on ice. After incubation, the
cells were fixed with 3.7% formaldehyde in transport buffer, and T-APC
was detected by Axiophot microscopy (Carl Zeiss Inc.). C,
recombinant GFP-WT importin- (30 µM), GFP-(145-449)
(25 µM) or GFP-(448-876) (10 µM) was
injected with TR-BSA (10 µM) into a nucleus in
homokaryons formed from BHK21 cells by HVJ. After incubation for 30 min
at 37 °C, the cells were fixed with 3.7% formaldehyde in PBS, and
the localization of GFP fusion proteins was examined by Axiophot
microscopy (Carl Zeiss Inc.). TR-BSA shows an injection site.
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We next examined whether the export of importin-
is
temperature-dependent. After the BHK21 cells were
fused by HVJ, the homokaryons were preincubated for 10 min on ice and
the recombinant GFP-importin-
proteins were then injected into a
nucleus. After incubation for 30 min on ice, the injected
GFP-importin-
proteins were largely detected only in the injected
nucleus (Fig. 2A). This
finding indicates that importin-
is exported from the nucleus in a
temperature-dependent manner. We further examined whether
wheat germ agglutinin (WGA) inhibits the nuclear export of
importin-
. As shown in Fig. 2C, the nuclear export of
importin-
was inhibited by the co-injection of WGA (2 mg/ml in a
needle), whereas the passive diffusion of GFP (about 27 kDa) from the
nucleus was not (Fig. 2B). These results indicate that the
nuclear export of importin-
observed herein does not result from the
passive diffusion of the degradation products of GFP-importin-
fusion proteins.

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Fig. 2.
Temperature-dependent and
WGA-sensitive export of importin- from the
nucleus. A, nuclear export of importin- does not
occur in chilled cells. BHK21 cells fused by HVJ were preincubated for
10 min on ice, after which GFP-WT importin- (30 µM) or
GFP-(145-449) (25 µM) was injected into a nucleus in the
homokaryons. Injected cells were incubated for 30 min on ice.
B, WGA does not inhibit the passive diffusion of small
molecules. Recombinant GFP proteins (27 kDa) (35 µM)
alone or with WGA (2 mg/ml) were injected into nuclei in BHK21 cells.
Injected cells were incubated for 30 min at 37 °C. C,
nuclear export of importin- is inhibited by WGA. GFP-WT importin-
(25 µM) or GFP-(145-449) (20 µM) was
injected into a nucleus in homokaryons of BHK21 cells in the presence
of WGA (2 mg/ml). Injected cells were incubated for 30 min at
37 °C.
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Nuclear Export of Importin-
Is Not Inhibited by Leptomycin
B--
Recently, an export receptor (CRM1) for leucine-rich NES was
identified (12-15). Meanwhile, it was reported that Kap95p, a yeast
homologue of importin-
, contains a region (amino acid residues 55-65) that is similar to the leucine-rich NES motifs and that this
region of Kap95p is sufficient to mediate the active nuclear export of
Kap95p (11). More recently, an antibiotic, leptomycin B was
demonstrated to specifically inhibit the leucine-rich NES-mediated nuclear export by binding directly to CRM1 (35-37). Therefore, we
investigated whether nuclear export of mouse importin-
is inhibited
by leptomycin B. BHK21 cells were preincubated with leptomycin B at 2 ng/ml in a culture medium for 3 h at 37 °C and then fused by
HVJ. As reported previously, nuclear export of GST-NES-GFP was
inhibited in these homokaryons, which had been preincubated with
leptomycin B (Fig. 3A).
However, the nuclear export of the importin-
was completely
unaffected by leptomycin B. These results strongly suggest that mouse
importin-
is not carried by CRM1 and does not possess a leucine-rich
NES such as those in Rev and PKI (protein kinase A inhibitor)
(discussed below).

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Fig. 3.
Nuclear export of
importin- is not mediated by CRM1.
A, leucine-rich NES-mediated nuclear export is inhibited by
leptomycin B. GST-NES-GFP (45 µM) was injected into a
nucleus in BHK21 homokaryons treated or not treated with leptomycin B
(2 ng/ml). Injected cells were incubated for 30 min at 37 °C.
B, nuclear export of importin- is not inhibited by
leptomycin B. GFP-WT-importin- (30 µM) or
GFP-(145-449) (25 µM) was injected into a nucleus in
BHK21 homokaryons treated with leptomycin B (2 ng/ml). Injected cells
were incubated for 30 min at 37 °C.
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We next attempted to determine the region required for the nuclear
export of importin-
. Since it was previously found that importin-
contains the NPC-binding domain (amino-terminal region) as well as the
importin-
binding domain (carboxyl-terminal region) (7), we
constructed two deletion mutants containing the NPC-binding region (aa
145-449) and the importin-
binding region (aa 448-876). As
expected, the aa 145-449 mutant failed to bind the importin-
(data
not shown) and Ran (Fig. 1B, left), but migrated
into the nucleus in vivo and in vitro (summarized
in Fig. 1A), consistent with our previous results (7).
Further, in the digitonin-permeabilized cells, NPC-targeting of
NLS-containing proteins was competitively inhibited by the addition of
the aa 145-449 mutant, indicating that the aa 145-449 mutant contains
a domain that interacts with the NPC (Fig. 1B,
right).
It was found that, similar to the case of wild type (WT) importin-
,
the aa 145-449 mutant was exported from the nucleus and reimported
into the nuclei (Fig. 1C). Moreover, it was also shown that
the nuclear export of the aa 145-449 mutant is
temperature-dependent, WGA-sensitive, and unaffected by
leptomycin B treatment, similar to WT importin-
(Figs.
1C, 2A, 2C, and 3B). In
contrast, the NPC binding-deficient mutant, aa 448-876, was not
exported from the nucleus at all (Fig. 1C). These results
indicate that the NPC-binding domain of importin-
is involved in not
only nuclear import but also the nuclear export of importin-
.
Importin-
Can Be Exported in the Absence of RanGTP as Well as
Its GTP Hydrolysis--
Richards et al. (38) showed that a
leucine-rich NES-mediated nuclear export requires RanGTP but not the
GTP hydrolysis of Ran. In order to determine if the hydrolysis of
RanGTP is required for the nuclear export of importin-
, we
investigated the effect of a Ran mutant, Ran(G19V), which is known to
be a GTPase-deficient mutant (39). Consistent with the previous results
on the leucine-rich NES-mediated nuclear export (38), the nuclear
export of WT importin-
was not inhibited by co-injection with
Ran(G19V)GTP (Fig. 4), indicating that
the GTP hydrolysis of Ran is not required for the export of
importin-
. Moreover, the nuclear export of the aa 145-449 mutant,
which does not bind Ran, was not inhibited by co-injection with
Ran(G19V)GTP (Fig. 4).

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Fig. 4.
Nuclear export of
importin- is not inhibited by
Ran(G19V)GTP. GFP-WT-importin- (25 µM) or
GFP-(145-449) (20 µM) was injected with Ran(G19V)GTP
(200 µM) into a nucleus of BHK21 homokaryons. Injected
cells were incubated for 30 min at 37 °C.
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In order to determine whether the nuclear export of importin-
actually requires the GTP-bound form of Ran, we used tsBN2 cells (40,
41). When tsBN2 cells were incubated for 3 h at the nonpermissive
temperature (39.5 °C), RCC1 could not be detected immunocytochemically, and the degree of cytoplasmic staining of Ran
substantially increased (data not shown), which is consistent with
previous reports (18, 27, 42). We first examined the localization of
endogenous importin-
in tsBN2 cells cultured for 24 h at
39.5 °C. As shown in Fig. 5, staining
of importin-
in tsBN2 cells cultured for 24 h at 39.5 °C was
not substantially different from that in tsBN2 cells cultured at the
permissive temperature (33.5 °C), and significant accumulation of
importin-
in the nuclei of tsBN2 cells cultured at 39.5 °C was
not observed. However, importin-
, which is known to be exported by
CAS in a nuclear RanGTP-dependent manner, accumulated at
considerably higher levels in the nuclei of the tsBN2 cells cultured
for 24 h at 39.5 °C compared with those cultured at 33.5 °C.
These results suggest that importin-
, but not importin-
, can be
exported in the absence of nuclear RanGTP.

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Fig. 5.
Subcellular localization of
importin- and importin-
in tsBN2 cells cultured at permissive temperature (33.5 °C) or
nonpermissive temperature (39.5 °C). After incubation for
24 h at 33.5 or at 39.5 °C, tsBN2 cells were stained with
affinity-purified anti-mouse importin- or anti-mouse importin-
antibodies.
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To support this hypothesis, we investigated the effect of co-injection
of RanGAP1 with importin-
in the nuclei of tsBN2 cells cultured at
the nonpermissive temperature. As shown in Fig.
6, the nuclear export of importin-
was
not inhibited by co-injection of RanGAP1, while the export of the
leucine-rich NES-containing substrates was strongly inhibited. In
addition, the nuclear export of importin-
occurred in tsBN2 cells
incubated for 24 h at 39.5 °C followed by treatment of 35.5 µM cycloheximide for 3 h at 39.5 °C to prevent
the resynthesis of RCC1 (data not shown). Moreover, it was also found
that the nuclear export of the Ran binding-deficient mutant, aa
145-449, was not inhibited under the same assay conditions (Fig. 6).

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Fig. 6.
Importin- can be
exported in the absence of RanGTP. After incubation for 24 h
at 39.5 °C, tsBN2 cells were fused by HVJ. GFP-WT-importin- (25 µM), GFP-(145-449) (20 µM), or GST-NES-GFP
(35 µM) was injected with RanGAP1 (10 µM)
into a nucleus of the homokaryons. Injected cells were incubated for 30 min at 39.5 °C.
|
|
These results indicate that mouse importin-
can be exported from the
nucleus through the NPC without the support of RanGTP as well as its
GTP hydrolysis in living mammalian cells, while the possibility that
importin-
bound to RanGTP is also exported without its GTP
hydrolysis cannot be excluded. The actual role of Ran in the export of
importin-
is discussed below.
 |
DISCUSSION |
Nuclear Export of Importin-
Is Temperature-dependent
and WGA-sensitive--
The NLS substrate targets to the NPC through
nuclear pore-targeting complex formation with the importin-
/
heterodimer and is then translocated through the NPC into the nucleus
in an energy-dependent manner. After translocation of the
NLS substrate-carrier complex into the nucleus, carrier molecules are
required to return to the cytoplasm.
As expected, microinjection experiments showed that importin-
was
exported from the nucleus to the cytoplasm in living mammalian cells
(Fig. 1C). Nuclear export of importin-
did not occur in chilled cells (Fig. 2A), which is consistent with a previous
report (11). The nuclear export of importin-
was inhibited by WGA (Fig. 2C), while the passive diffusion of small molecules
was not affected (Fig. 2B). These results exclude the
possibility that the degradation products of GFP fusion proteins
passively diffuse out of the injected nucleus. Moreover, although the
precise mechanism of the inhibitory effects of WGA remains unclear, it is generally assumed that some glycoproteins of the NPC can function, not only in selective nuclear import but also in the nuclear export of
importin-
.
Nuclear Export of Importin-
Is Dependent on the NPC-binding
Domain--
Importin-
mediates the targeting of the NLS substrate
bound to importin-
by directly binding to both importin-
and
components of the NPC. Previously, we showed that importin-
alone is
translocated through nuclear pores from the cytoplasm into the nucleus,
when it does not carry the NLS substrate-importin-
complex (7). This
nuclear migration of importin-
depends on the domain of importin-
for binding to components of the NPC and does not require the binding
domains for importin-
and GTPase Ran.
In this study, it was found that the aa 145-449 mutant containing the
region for NPC binding involved in the nuclear import of importin-
also possesses nuclear export activity (Fig. 1C). In
addition, numerous other studies have shown that several
importin-
-related proteins are likely to associate with components
of the NPC, suggesting that the binding of importin-
family members
to components of the NPC is critical in order for the member to
function as a nuclear transport factor. These data strongly suggest
that the nuclear export of importin-
is promoted by direct
association with components of the NPC.
Recently, it was reported that the nuclear export of proteins
containing a leucine-rich NES is mediated by CRM1 (12-15). Leptomycin B is a specific inhibitor of CRM1 (35-37). However, the nuclear export
of importin-
was insensitive to leptomycin B treatment (Fig.
3B), suggesting that importin-
is not carried back to the cytoplasm by CRM1. Iovine and Wente (11) previously showed that Kap95p,
which is a S. cerevisiae homologue of importin-
, contains a region similar to a leucine-rich NES in the amino terminus (amino acid residues 55LEGRILAALTL65) and that the
NES-like motif-mutated Kap95p abolished the binding activity to
glycine-leucine-phenylalanine-glycine. (GLFG) repeat regions of
nucleoporins Nup116p and Nup100p. Moreover, they found that the
NES-like motif-containing substrate moved to the cytoplasm in a
temperature-dependent manner after injection into the
nuclei of cultured mammalian cells. From these findings, they proposed a model in which the recycling of Kap95p is mediated by the interaction of an NES-like motif with GLFG repeat regions. In contrast, in this
study, we found that the aa 145-449 mutant of mouse importin-
, which lacks the region (52VARVAAGLQI61)
corresponding to the NES-like motif of yeast Kap95p, is fully capable
of migrating from the nucleus to the cytoplasm through the NPC (Fig.
1C). Although we cannot clearly explain this contradiction, the differences may result from the source of importin-
family molecules used in these experiments.
A putative vertebrate homologue of yeast Nup116p is
human/rat/Xenopus Nup98, which localizes at the
nucleoplasmic side of the NPC (43, 44). At present, Nup98 is the only
identified vertebrate GLFG repeat-containing nucleoporin. It has been
reported that importin-
is able to bind to Nup98 in overlay assays
(45). Powers et al. showed that nuclear injection of
anti-Xenopus Nup98 antibodies did not inhibit nuclear
protein import but did inhibit the nuclear export of RNAs, including
small nuclear RNAs, 5 S RNA, large ribosomal RNAs, and mRNA (46),
although it has not been determined whether the nuclear export of
importin-
is inhibited by the nuclear injection of
anti-Xenopus Nup98 antibodies. Further studies are required
to elucidate the function of GLFG repeat-containing nucleoporins in the
nuclear export of importin-
.
The Roles of Ran in Nuclear Export of Importin-
--
In this
study, we found that the nuclear export of importin-
is not
inhibited by nuclear injection of Ran(G19V)GTP (Fig. 4), which is
consistent with a previous report concerning NES-mediated nuclear
export (38). Moreover, it appeared that the recycling of importin-
,
but not importin-
, occurred in tsBN2 cells cultured at the
nonpermissive temperature (Fig. 5), in which endogenous mutated RCC1 is
inactive and the level of nuclear RanGTP would be expected to be quite
low (18). The export of importin-
was not inhibited by nuclear
co-injection of RanGAP1, which lowers the level of free RanGTP and, in
turn, elevates that of RanGDP, into the tsBN2 cells at nonpermissive
temperature (Fig. 6). In addition, we showed that the aa 145-449
mutant, which lacks the Ran-binding domain, is capable of migrating
from nucleus to cytoplasm through the NPC (Figs. 1C,
3B, and 6). These results suggest that nuclear RanGTP is not
essential for the nuclear export of importin-
. In contrast,
Izaurralde et al. (26) demonstrated that the nuclear export
of human importin-
was significantly, but not completely, inhibited
by the injection of Rna1p, a Saccharomyces pombe homologue of RanGAP1, into Xenopus oocyte nuclei. However, their data
showed that a considerable amount of importin-
was exported into the cytoplasm, even when Rna1p was injected into Xenopus oocyte
nuclei at higher concentration than that which nearly completely
inhibited the nuclear export of importin-
. These findings suggest
that at least part of importin-
can be exported via a pathway that is unaffected by Rna1p. Alternatively, since we did not examine the
export kinetics of importin-
, we cannot exclude the possibility that
the decrease of nuclear RanGTP may affect export efficiency and that
the export rate of importin-
may be decreased by nuclear injection
of RanGAP1 in mammalian cells as in Xenopus oocytes.
It should be noted that the nuclear reimport of WT importin-
was
considerably inhibited when Ran(G19V)GTP was co-injected in a nucleus
of multinucleated cells, whereas the aa 145-449 mutant lacking the
Ran-binding domain was not inhibited (Fig. 4). From these findings, we
speculate that the WT importin-
-Ran(G19V)GTP complex formed in the
nucleus is exported to the cytoplasm, and the disassembly of the
importin-
-RanGTP complex through GTP hydrolysis of Ran is involved
in the reimport of WT importin-
. Moreover, these results suggest
that WT importin-
, which is complexed with RanGTP in the nucleus,
can be exported from the nucleus without GTP hydrolysis of Ran.
From these and other findings, we propose that importin-
is recycled
from the nucleus in two distinct ways: 1) in the form of a complex with
RanGTP and 2) alone in a Ran-independent manner. In either case, the
export does not require the GTP hydrolysis of Ran. Since importin-
binds RanGTP with high affinity and nuclear Ran would be expected to be
the predominant GTP-bound form, it is plausible that nuclear RanGTP
binds importin-
to dissociate the nuclear pore-targeting complex
after its translocation through the NPC and that the
importin-
-RanGTP complex recycles back to the cytoplasm without
hydrolysis of its GTP. However, in our previous study, it was
demonstrated that importin-
is translocated alone into the nucleus
through the NPC in a Ran-independent manner when it does not carry the
importin-
-NLS substrate complex (7). Furthermore, we found that the
NPC-binding domain of importin-
, which had been shown to be involved
in nuclear import of importin-
, was also required for the nuclear
export. Therefore, we speculate that importin-
traverses the NPC
into and out of the nucleus in two ways;
"Ran-dependent" and "Ran-independent." As a result, we assume that importin-
, which is imported independently from the
NLS substrates, may be exported without the binding of RanGTP, although
the biological significance for this type of shuttling of importin-
is not known at present. Further studies of this Ran-dependent and Ran-independent nucleocytoplasmic
shuttling of importin-
will provide new insights in our
understanding of the directionality for movement and the requirement of
Ran and energy in nucleocytoplasmic protein transport.