Department of Anatomy and Cell Biology, Osaka University Medical School, Suita, Osaka 565-0871, Japan
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Abstract |
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A small GTPase Ran is a key regulator for
active nuclear transport. In immunoblotting analysis, a
monoclonal antibody against recombinant human Ran,
designated ARAN1, was found to recognize an epitope
in the COOH-terminal domain of Ran. In a solution
binding assay, ARAN1 recognized Ran when complexed with importin , transportin, and CAS, but not
the Ran-GTP or the Ran-GDP alone, indicating that
the COOH-terminal domain of Ran is exposed via its
interaction with importin
-related proteins. In addition, ARAN1 suppressed the binding of RanBP1 to the
Ran-importin
complex. When injected into the nucleus of BHK cells, ARAN1 was rapidly exported
to the cytoplasm, indicating that the Ran-importin
-related protein complex is exported as a complex
from the nucleus to the cytoplasm in living cells. Moreover, ARAN1, when injected into the cultured cells induces the accumulation of endogenous Ran in the
cytoplasm and prevents the nuclear import of SV-40
T-antigen nuclear localization signal substrates. From
these findings, we propose that the binding of RanBP1
to the Ran-importin
complex is required for the dissociation of the complex in the cytoplasm and that the
released Ran is recycled to the nucleus, which is essential for the nuclear protein transport.
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Introduction |
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THE intracellular transport of macromolecules between the nucleus and the cytoplasm takes place
through the nuclear pore complexes (NPCs)1 which
permit the passive diffusion of molecules smaller than 20-
40 kD and also support an active and receptor-mediated
transport. The active protein import pathway which has
been studied most intensively is mediated by a basic-type
nuclear localization signal (NLS) which contains a short
amino acid stretch rich in basic amino acids. The basic-type NLS of the import substrate binds to the heterodimeric importin /
to form a nuclear pore-targeting complex. Importin
provides the NLS-binding site, whereas
importin
accounts for the targeting of the complex to the
NPC (for reviews see Görlich and Mattaj, 1996
; Koepp
and Silver, 1996
; Görlich, 1997
; Nigg, 1997
; Yoneda, 1997
).
In addition to the basic-type NLS mediated import pathway, it is known that the import of heterogeneous nuclear
RNA-binding protein A1 (hnRNP A1) is mediated by a
signal comprised of 38 amino acids, which is referred to as
M9, and which is rich in glycine and aromatic residues. M9
directly binds to an importin
-related transport factor,
transportin (Nakielny et al., 1996
; Pollard et al., 1996
). It has
also been demonstrated that the nuclear export of some
proteins depend on nuclear export signal (NES), which is
rich in hydrophobic residues (for review see Nakielny and
Dreyfuss, 1997
). Nuclear export of proteins containing NES
is mediated by an importin
-related export receptor, CRM1 (Fornerod et al., 1997b
; Fukuda et al., 1997
; Ossareh et al., 1997
; Stade et al., 1997
). Although the export signal of importin
has not been identified, its export is also mediated
by another importin
-related export receptor, cellular apoptosis susceptibility gene (CAS) (Kutay et al., 1997
). These
transport factors, importin
, transportin, CAS, and CRM1
constitute a protein superfamily which has a Ran-GTP-
binding motif (Fornerod et al., 1997a
; Görlich et al., 1997
).
A small GTPase Ran is known to be essential for the active transport pathways including the import of the basic-type NLS and M9 containing proteins or snRNPs, or the
export of importin , NES-containing proteins, tRNA,
UsnRNA, and several mRNAs (Melchior et al., 1993a
;
Moore and Blobel, 1993
; Koepp et al., 1996
; Nakielny et
al., 1996
; Izaurralde et al., 1997
; Kutay et al., 1997
; Richards et al., 1997
; for reviews see Avis and Clarke, 1996
;
Koepp and Silver, 1996
; Görlich, 1997
; Goldfarb, 1997
).
Ran is predominantly, but not exclusively, a nuclear protein (Bischoff and Ponstingl, 1991a
). The Ran GTPase cycle is regulated by the GDP-GTP exchange factor, RCC1,
which charges Ran with GTP (Bischoff and Ponstingl, 1991b
), the GTPase-activating protein, RanGAP1, which
converts Ran-GTP into Ran-GDP (Bischoff et al., 1994
;
Becker et al., 1995
; Corbett et al., 1995
), and RanBP1 (or
RanBP2) whose binding to Ran-GTP further stimulates
the GTPase-activating activity of RanGAP1 (Coutavas et
al., 1993
; Bischoff et al., 1995
; Richards et al., 1995
). RCC1
is a chromatin-associated nuclear protein (Ohtsubo et al.,
1989
), whereas RanGAP1 and its coactivator, RanBP1, are exclusively cytoplasmic, and RanBP2 is located on the cytoplasmic fibrils of the NPC (Hopper et al., 1990
; Melchior
et al., 1993b
; Bischoff et al., 1994
, 1995
; Wu et al., 1995
;
Yokoyama et al., 1995
; Matunis et al., 1996
; Richards et al.,
1996
; Mahajan et al., 1997
). Because of this asymmetric distribution of RCC1, RanGAP1, and RanBP1/RanBP2, it
has been predicted that a steep Ran-GTP gradient across
the nuclear envelope exists (very low levels of cytoplasmic
Ran-GTP and high levels inside the nucleus).
In the case of nuclear import of proteins which contain
the basic-type classical NLS, it has been proposed that
Ran is involved in several different steps in the process. At
least a portion of the energy required for translocation
through the NPC is supplied via GTP hydrolysis by Ran
(Melchior et al., 1993a), although other GTPases may also
be involved (Sweet and Gerace, 1996
). In addition, nuclear
Ran-GTP functions to terminate the import reaction. After the translocation of the trimeric complex, which is
comprised of importin
/
and a karyophile with the classical NLS, through the nuclear envelope, the direct binding of
nuclear Ran-GTP to importin
causes the dissociation of
the trimeric complex thus releasing the cargo (Rexach and
Blobel, 1995
; Görlich et al., 1996; Moroianu et al., 1996
). In
the same manner, transportin binds to its cargo only in the
absence of Ran-GTP, i.e., in the cytoplasm and releases it
in the nucleus. For the next rounds of nuclear import, importin
and transportin are required to return to the cytoplasm and dissociate from Ran-GTP. In contrast, the binding
of the export receptors such as CRM1 and CAS to their cargos is enhanced by the simultaneous binding of Ran-GTP,
which occurs in the nucleus. The trimeric cargo-export receptor-Ran-GTP complex is then transferred to the cytoplasm. Thus, the binding of Ran-GTP to importin
-related
transport factors regulates their interaction with cargos or
adapter molecules. Therefore, it has been suggested that this
Ran-GTP concentration gradient is a critical determinant in
the directionality of nucleocytoplasmic transport, ensuring
that the transport factors carry their cargoes unidirectionally (Görlich et al., 1996, 1997; Izaurralde et al., 1997
).
It is also noteworthy that the Ran-GTP-importin complex is very stable and resistant to the Ran GTPase activating activity of RanGAP1 (Floer and Blobel, 1996
).
Biochemical analyses have suggested that RanBP1 directly promotes the dissociation of Ran-GTP from importin
, CAS, and transportin (Bischoff and Görlich, 1997
;
Lounsbury et al., 1997). Nuclear injection of RanGAP1
significantly inhibited the export of injected importin
and
, transportin, and NES-containing proteins in Xenopus laevis oocyte (Izaurralde et al., 1997
). As a result, it
has been proposed that the importin
-related transport
factors are probably exported to the cytoplasm as complexes with Ran-GTP, and that these complexes dissociate and Ran is then converted to the GDP-bound form by
RanGAP1 in conjunction with RanBP1 in the cytoplasm
(Bischoff and Görlich, 1997
; Izaurralde et al., 1997
). However, direct in vivo evidence for this proposal is currently lacking.
Unlike other small GTPases, Ran has negatively charged
amino acid residues at the COOH terminus instead of consensus sequences for lipid modification. To better understand the biological significance of the COOH-terminal domain, the COOH terminus-deleted mutant Ran, referred to
as DE-Ran, has been used (Lounsbury et al., 1994
, 1996a
,b;
Ren et al., 1995
; Richards et al., 1995
; Carey et al., 1996
; Chi
et al., 1997
). This mutant is capable of supporting the nuclear import of basic-type NLS-containing substrates in digitonin-permeabilized semi-intact cells, whereas the
DE-Ran, which is expressed in cultured cells has a dominant-negative phenotype for both nuclear protein import and
RNA export (Ren et al., 1995
; Richards et al., 1995
; Carey et
al., 1996
). This mutant protein has a lower affinity for
RanBP1 than the wild-type (wt) Ran and binds to importin
with higher affinity than wild-type Ran in an overlay assay
(Richards et al., 1995
; Lounsbury et al., 1996b
). These results suggest that the deletion of the COOH-terminal portion of Ran may be the cause of the defect in the Ran GTPase cycle. However, the issue of how the COOH-terminal
domain is involved in the nucleocytoplasmic transport or the
Ran GTPase cycle is not known with certainty, since the deletion in COOH-terminal domain may cause drastic conformational changes in Ran.
In this study, in order to identify functional domains of
Ran including the COOH-terminal domain, we produced
anti-Ran monoclonal antibodies (mAbs). By using one of
the anti-Ran mAbs, we provide evidence that the COOH-terminal domain of Ran is not exposed to the surface of
the molecule until Ran interacts with importin or importin
-related transport factors, CAS and transportin. This
observation suggests that the exposed COOH-terminal acidic sequence of Ran may be essential for the binding of RanBP1
to the Ran-GTP complexed with importin
-related transport factors. Furthermore, we show in vivo evidence that
Ran/importin
can be exported in the form of a complex
from the nucleus to the cytoplasm. Our results indicate
that the binding of RanBP1 to the Ran/importin
complex in the cytoplasm, which appears to be blocked by injected mAb, is essential for the recycling of Ran and nuclear protein import.
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Materials and Methods |
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Production of mAbs
mAbs were obtained essentially according to the procedure of Köhler and
Milstein (1975). 50 µg of denatured recombinant human Ran was initially
intraperitoneally administered with Freund's complete adjuvant to a
16-wk-old BDF1 mouse (Japan SLC), followed by three subsequent injections at 3-wk intervals with the same dose in Freund's incomplete adjuvant. 1 mo after the fourth injection, the mouse was given a booster injection of the same dose. 4 d later, spleen cells isolated from the mouse were
fused with the mouse myeloma cell line P3U1 using standard methods.
Screening was performed by ELISA and immunoblotting using the recombinant human Ran. Ran-specific mAbs were typed by using mouse
monoclonal antibody isotyping kit (Amersham). Ascites fluid was produced from a BDF1 × BALB/3T3 mouse (Japan SLC) which had been implanted with the hybridoma. The IgG fraction was obtained by precipitation with 50% saturated ammonium sulfate followed by chromatography on a protein A affinity column.
Antibodies
Rabbit anti-Ran polyclonal antibodies were produced as described previously (Sekimoto et al., 1996). Chromatographically purified normal mouse
IgG was purchased from Zymed. Rabbit anti-importin
polyclonal antibodies were prepared as described previously (Kose et al., 1997
). Mouse
anti-human CAS monoclonal antibody and mouse anti-human transportin monoclonal antibody were purchased from Transduction Laboratories.
Expression and Purification of Recombinant Proteins
Expression and purification of recombinant importin were performed
as described previously (Kose et al., 1997
). The human CAS gene was amplified from a HeLa cDNA library via the polymerase chain reaction
(PCR) using the synthetic oligonucleotide primers, 5'-TTTTTTGGATCCATGGAACTCAGCGATGCAAATCTGCAA-3' and 5'-TTTTTTCTCGAGTTAAAGCAGTGTCACACTGGCTGCCTG-3'. The PCR
product was inserted into BamHI and XhoI sites of pGEX-6P-2/hGFP vector, which was an expression vector of glutathione-S-transferase (GST)-
fused green fluorescent protein (containing the S65A/Y145F mutation)
(hGFP). This construct was transformed to express the recombinant
GST-hGFP-CAS fusion protein in Escherichia coli strain BL21. The expressed GST-hGFP-CAS fusion protein was purified on glutathione-
Sepharose (Pharmacia Biotech). After the digestion of GST portion with
PreScission Protease (Pharmacia Biotech), hGFP-CAS protein was purified by ion-exchange chromatography on Hi-TrapQ (Pharmacia Biotech,
Inc.), and then was dialyzed against 20 mM Hepes, pH 7.3, 110 mM potassium acetate, 2 mM DTT, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin.
Human transportin/karyopherin 2 gene (Pollard et al., 1996
; Bonifaci
et al., 1997
) was amplified from HeLa cell cDNA library by the PCR using
the synthetic oligonucleotide primers (5'-CTCAGCGGATCCATGGAGTATGAGTGGAAACCTGAC-3' and 5'-CTCAGCGGTACCTTAAACACCATAAAAAGCTGCAAG-3'). The PCR product was inserted into
the BamHI and KpnI sites of a modified pGEX-6P-3 (Pharmacia) and verified by DNA sequencing. Recombinant GST-transportin was expressed
and purified as described for hGFP-CAS.
E. coli strains expressing wild-type Ran were prepared as described previously (Sekimoto et al., 1996). Mutation (Q69L) within Ran was created
using QuikChange site-directed mutagenesis kit (Strategene). RanQ69L
fragment was inserted into NcoI and BamHI sites of pET3d. Recombinant
RanQ69L protein was expressed in E. coli and purified in the same manner as wild-type Ran. Recombinant wild-type and Q69L Ran were expressed, purified, and then charged with GTP or GDP according to the
method of Bischoff and Ponstingl (1995)
and Melchior et al. (1995)
with
slight modifications. In brief, expression was induced by addition of 1 mM
isopropyl-
D-thiogalactopyranoside (IPTG) and incubation for 14 h at 20°C. The E. coli cells were lysed in buffer C (50 mM Tris-HCl, pH 8.0, 75 mM
NaCl, 1 mM MgCl2, 0.1 mM PMSF, 1 mM DTT, 1 mg/ml each of aprotinin,
leupeptin, and pepstatin) by freeze-thaw. The clarified lysates were applied to DEAE-Sepharose FF column (Pharmacia) and flow through fractions were collected. After 60% saturate ammonium sulfate precipitation,
2 mM GTP or GDP was added and incubated for at least 1 h in buffer D
(50 mM phosphate buffer, pH 7.0, 1 mM 2-mercaptoethanol, 10% glycerol,
2 mM GTP or GDP) on ice. The samples were applied to HiPrep Sephacryl
S-200 HR FPLC column (Pharmacia) equilibrated with buffer D, and
peak fractions containing Ran proteins were pooled. GTP- and GDP-form
of Ran were further separated on Fractogel EMDSO3
650 (s) column
(Merck) with linear gradient of buffer D containing 50 mM phosphate to
500 mM phosphate. GDP-bound form of Ran was eluted between 200 and
250 mM phosphate. GTP-bound form was eluted between 350 and 400 mM phosphate. The purified GTP- or GDP-form of Ran was then desalted with PD10 column (Pharmacia) equilibrated with transport buffer
(see below), concentrated with centricon 30 (Amicon), and then frozen in
small aliquots.
The guanine nucleotides bound to purified Ran were determined by FPLC (Pharmacia) after denaturation. The Ran preparations were heated at 95°C for 2 min. After filtration, the solutions were applied to a monoQ HR5/5 (Pharmacia) column and then the guanine nucleotides were eluted with a linear gradient from 10 mM potassium phosphate buffer, pH 8.0, to 50 mM potassium phosphate buffer, pH 8.0, containing 250 mM NaCl. Nucleotides were detected at 254 nm and quantified against a standard GDP/ GTP solution. The bound nucleotides were found to be virtually 100% GDP for the GDP-bound preparations and 95% GTP for the GTP-bound preparations.
All GST-fused deletion mutants of human Ran were prepared using
PCR with appropriate oligonucleotides. The PCR product was inserted
into the BamHI/EcoRI site of pGEX-2T vector. For the production of the
GST-fused COOH-terminal domain of Ran, the synthesized oligonucleotide, corresponding to the COOH-terminal region of human Ran,
TALPDEDDDL, was inserted into the BamHI/EcoRI site of pGEX-2T
vector (Pharmacia). These resultant plasmids were sequenced to confirm
the fidelity of the region, which had been amplified by PCR and in-frame
ligation of the fused region. Recombinant GST-fused proteins were expressed by incubation with 1 mM IPTG for 6 h at 37°C in E. coli strain
BL21(DE3). The purification of the GST-fused COOH-terminal domain of
Ran with glutathione-Sepharose was performed as described for importin .
The protein was then dialyzed against 20 mM Hepes, pH 7.3, 110 mM potassium acetate, and 2 mM DTT.
Mouse RanBP1 was expressed as GST fusion protein. A fragment
which is encoding the entire amino acid sequence of this protein was amplified from mouse Ehrlich ascites tumor cell cDNA by PCR using synthetic oligonucleotides (5'-CCTACGGATCCATGGCGGCCGCCAAGGACA-3' and 5'-CCACTGAATTCTCATTGTTTCTCCTCAGAC-3') and
cloned into the BamH1-EcoRI sites of pGEX-2T, to produce an in-frame
fusion with GST. Expression in BL21(DE3), lysis of bacteria, and purification of the fusion protein with glutathione-Sepharose were performed as
described for importin . The GST portion of chimeras was cleaved off by
a 2-h incubation at room temperature with 1 NIH U of thrombin (Sigma)
per 100 µg of chimeras. GST and thrombin were separated from recombinant protein on a MonoQ column (Pharmacia Biotech) at flow rate of 0.5 ml/min with a linear gradient from 0.025 to 1.0 M NaCl in 20 mM Hepes,
pH 7.3, 2 mM DTT, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin. Free RanBP1 protein was dialyzed against 20 mM Hepes, pH 7.3, 110 mM potassium acetate, 2 mM DTT, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin.
Preparation of Ehrlich Ascites Tumor Cells Cytosolic Extract
Cytosolic extract was prepared from Ehrlich ascites tumor cells as described previously (Imamoto et al., 1995).
Gel Electrophoresis and Immunoblotting
One-dimensional SDS-PAGE was based on the method of Laemmli
(1970) and the gels were stained with Coomassie brilliant blue. For immunoblotting, after electrophoresis, proteins were electrophoretically transferred from gels to nitrocellulose sheets. After blocking with 3% skim
milk in PBS, the sheets were then incubated with the first antibody followed by alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG
(Bio-Rad).
Immunofluorescence
BHK cells, plated on coverslips, were washed twice in PBS and fixed with 3.7% formaldehyde in PBS (at room temperature for 20 min) followed by permeabilization with 0.5% Triton X-100 in PBS (at room temperature for 5 min). After blocking with 3% skim milk in PBS, the cells were incubated with ARAN1 (3 µg/ml) or affinity-purified rabbit anti-Ran polyclonal antibodies (3 µg/ml) overnight at 4°C and detected with Cy3-conjugated goat anti-mouse IgG (Amersham) or FITC-conjugated goat anti-rabbit IgG (Tago). The samples were examined using a Zeiss Axiophot fluorescent microscope (Carl Zeiss).
Immunoprecipitation Using Recombinant Proteins
50 pmol of Ran (GTP-bound or GDP-bound) and/or 50 pmol of importin
were incubated with 50 pmol of ARAN1 in total 300 µl of transport
buffer (TB: 20 mM Hepes, pH 7.3, 100 mM potassium acetate, 2 mM magnesium acetate, 5 mM sodium acetate, 1 mM glycoletherdiaminetetraacetic acid [EGTA], 2 mM DTT, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin) for 1 h at 4°C. Protein A-bound agarose beads (Calbiochem-Novabiochem) were then added and the mixture was incubated for
1 h at 4°C. The beads were collected by centrifugation and washed five
times with TB. The bound fraction and unbound fraction were analyzed by SDS-PAGE and immunoblotting.
150 pmol of transportin or hGFP-CAS and 150 pmol of Ran-GTP were
incubated with 150 pmol of ARAN1 in the presence or absence of 150 pmol of importin in TB for 1 h at 4°C. The proteins which bound to
ARAN1 were then analyzed using the same procedure as above.
Immunoprecipitation Using Cytosolic Extract
To preclear the extract, anti-mouse IgG-conjugated agarose beads (American Qualex) were added to the Ehrlich ascetics tumor cell cytosolic extract and rotated for 1 h at 4°C. The beads were then removed by centrifugation. 300 pmol of ARAN1 and 300 pmol of Q69L Ran-GTP was added to 200 µl of the precleared cytosolic extract followed by 1 h of incubation at 4°C. 15 µl of protein A-agarose beads were then added to the mixture, which was then incubated for 1 h. After washing five times with TB, the proteins bound to the protein A-conjugated agarose beads were resuspended in SDS-PAGE sample buffer containing DTT and analyzed by immunoblotting using antibodies indicated in the figure legend.
Solution Binding Assay
Solution binding assays were performed in TB. 50 pmol of GST-importin
and 50 pmol of Ran-GTP was incubated with 10 µl of a packed volume
of glutathione-Sepharose for 1 h at 4°C. After washing the beads twice
with TB, 250 pmol or 2,500 pmol of ARAN1 was added to the mixture followed by a 30-min incubation, 50 pmol of RanBP1 was then added and the
mixture incubated for an additional 30 min at 4°C. The beads were washed
with TB and proteins bound to the beads were resuspended in SDS-PAGE sample buffer containing DTT and analyzed by SDS-PAGE.
50 pmol of GST-importin , 50 pmol of Ran-GDP, and 250 pmol of
ARAN1 was incubated with 10 µl of a packed volume of glutathione- Sepharose for 1 h at 4°C. After washing the beads twice with TB, 50 pmol
of RanBP1 was added to the beads and the mixture incubated for 1 h. For
a control, 50 pmol of GST-importin
, Ran-GDP, and RanBP1 were incubated with glutathione-Sepharose for 1 h. The beads were then washed
with TB and proteins bound to the beads were resuspended in SDS-PAGE sample buffer containing DTT, and then analyzed by SDS-PAGE.
Preparation of Fluorescein-labeled NLS-containing Transport Substrates
BSA was fluorescein-labeled and then chemically conjugated to the synthetic peptides which contained the amino acid sequence of SV-40 large
T-antigen NLS (CYGGPKKKRKVEDP), for the transport substrates
termed FITC-T-BSA, as described previously (Tachibana et al., 1994).
Microinjection
BHK cells were grown on coverslips. Microinjection of proteins was performed as described previously (Yoneda et al., 1987). After incubation for
the indicated time in each figure at 37°C, the cells were fixed with 3.7%
formaldehyde in PBS. The injected fluorescently labeled proteins were
detected by fluorescent microscopy (model Axiophot 2; Carl Zeiss).
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Results |
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A Monoclonal Antibody Specific for Ran
To study the functional domain(s) of Ran, monoclonal antibodies were produced against recombinant human Ran. The screening of the monoclonal antibodies was based on two criteria: (a) that they react with only Ran protein in immunoblotting; and (b) that they show the same staining pattern with polyclonal anti-Ran antibodies in immunofluorescence microscopy. Through this screening procedure, we isolated only one clone, designated ARAN1, for further characterization. ARAN1 was typed using a mouse monoclonal antibody isotyping kit as an immunoglobulin kappa G2b. On an immunoblot, ARAN1 detected recombinant human Ran and specifically reacted with a single 25-kD band in cell extracts from mouse Ehrlich ascites tumor cells, BHK21 cells, human embryonic lung (HEL) cells (Fig. 1 A), Madin-Darby bovine kidney-derived epithelial cells (MDBK), and Xenopus laevis oocytes (data not shown), which was also detected by affinity-purified rabbit anti-Ran polyclonal antibodies. Furthermore, ARAN1 recognized a single spot by immunoblotting analysis of HeLa total cell extracts after two-dimensional electrophoresis, which was also detected with the anti-Ran polyclonal antibodies (data not shown). Immunofluorescent image with ARAN1 in BHK cells showed the same typical staining pattern as that with the anti-Ran polyclonal antibodies (Fig. 1 B). From these findings, it was concluded that the monoclonal antibody ARAN1 specifically binds to Ran.
|
ARAN1 Recognizes an Epitope at the COOH-terminal Domain of Ran
To determine the specific epitope recognized by ARAN1,
we prepared a series of truncated forms of recombinant
Ran fused with GST (Fig. 2 A) and analyzed the interaction of these with ARAN1 by immunoblotting. Although
anti-GST polyclonal antibodies showed that all of the Ran
mutants were appropriately expressed and transferred to
nitrocellulose (Fig. 2 B), ARAN1 recognized only the full-length of GST-Ran. Removal of the COOH-terminal seven
amino acids (210-216) of Ran completely abolished the reactivity with ARAN1 in immunoblotting, suggesting that
the COOH-terminal domain, which consists of seven amino
acid residues in Ran, represents the epitope for ARAN1. To confirm this, we prepared the 10-mer peptide of the
COOH-terminal domain, 207-216, of Ran fused to GST
(Fig. 3 A). As shown in Fig. 3, B and C, ARAN1 clearly
reacted with the residues between 207 and 216 of human
Ran not only in immunoblotting but also in a solution binding assay. From these findings, it was concluded that the
epitope of ARAN1 is located in the COOH-terminal portion of Ran, which is highly negatively charged (DEDDDL)
and conserved among species (Fig. 3 D).
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|
ARAN1 Binds to the Ran-Importin Complex But Not
the Ran Molecule Alone in Solution Binding Assay
It is known that the stable Ran-GTP-importin complex
causes the inhibition of GTP hydrolysis on Ran stimulated
by RanGAP1. In contrast, Ran-GDP shows a much lower
affinity to importin
than Ran-GTP. To determine which
form of Ran is recognized by ARAN1 in solution, immunoprecipitation analysis of ARAN1 using recombinant
proteins was performed. Purified recombinant human Ran-GTP or Ran-GDP was incubated with ARAN1 in the
presence or absence of importin
followed by the precipitation with protein A-conjugated agarose beads. The results showed that, whereas ARAN1 precipitated neither
Ran-GTP nor Ran-GDP alone, it reacted with both Ran-GTP and Ran-GDP only in the presence of importin
and precipitated both Ran and importin
. To clearly confirm
that ARAN1 can't recognize Ran molecule alone, precipitated proteins were analyzed by immunoblotting with anti-Ran polyclonal antibodies (Fig. 4 A). The molar ratio of
the Ran and importin
precipitated with ARAN1 was estimated to be ~1:1 (data not shown). Since ARAN1 did
not bind to importin
directly in this assay, these results indicate that ARAN1 reacts only with Ran when bound to
importin
, and suggest that Ran changes conformation
when bound to importin
which, in turn, exposes its
COOH-terminal portion.
|
ARAN1 Also Binds to the Ran-Transportin Complex
and the Ran-CAS-Importin Complex
It is well known that Ran interacts with some import and
export receptors, e.g., transportin, CAS, and CRM1, which
are members of the superfamily of Ran-GTP-binding protein/importin (Kutay et al., 1997
; Görlich et al., 1997
;
Fornerod et al., 1997a
). Tranportin forms a complex with
Ran-GTP and CAS simultaneously binds to both Ran-GTP and importin
(Kutay et al., 1997
). Judging from the
conservation of the Ran-binding domain in this family, it is
likely that the COOH-terminal tail of Ran may also be exposed as the result of the interaction with these molecules.
To confirm this, we performed the solution binding assay
with different importin
-related transport factors, transportin, and CAS. The results clearly showed that ARAN1
binds to both Ran-GTP/transportin complex and Ran-GTP-CAS-importin
complex (Fig. 4 B), suggesting that importin
-related transport factors might induce a general conformational change in Ran through their interaction. In the case of CAS, it should be noted that a slight
recognition of a complex between CAS and Ran-GTP by
ARAN1 can also be observed (Fig. 4 B). This suggests that
CAS directly binds to Ran-GTP in the absence of importin
, although the affinity is very low.
ARAN1 Suppresses the Ternary Complex Formation of
Ran, Importin , and RanBP1 in a Solution
Binding Assay
Both Ran-GTP and Ran-GDP form a ternary complex
with RanBP1 and importin in a solution binding assay.
Although it has been assumed that RanBP1 plays a role in
the dissociation of the Ran-GTP-importin
complex, the
biological significance of the ternary complex has not yet
been established. The RanBP1-Ran-GDP-importin
complex has been proposed to play a role in nuclear protein import as a ternary complex (Chi et al., 1997
), although it has
not yet demonstrated how the complex functions in the nuclear protein import. We therefore examined the effects of
ARAN1 on the interaction of RanBP1 with both Ran-GTP-
importin
and Ran-GDP-importin
complexes in the solution binding assay. In the absence of ARAN1, GST-tagged
importin
efficiently precipitated both Ran-GTP and Ran-GDP together with RanBP1 in a molar ratio of 1:1 (Fig. 5, A
and B, lane 1). In contrast, in the presence of ARAN1, the
amount of RanBP1 precipitated with GST-tagged importin
apparently decreased, although the Ran was efficiently
precipitated. These results indicate that RanBP1 is not able
to interact with the Ran-GTP-importin
and Ran-GDP-
importin
complexes, when bound to ARAN1. That is, the
binding of ARAN1 to the Ran-importin
complex suppresses the ternary complex formation of both RanBP1-
Ran-GTP-importin
and RanBP1-Ran-GDP-importin
in solution.
|
Ran-Importin -related Transport Factor Complex Is
Exported from the Nucleus to the Cytoplasm
As described above, it has been proposed that the Ran-GTP-
importin complex and Ran-GTP-importin
-related transport factor (such as transportin and CAS) complex are
formed in the nucleus and exported to the cytoplasm. To
determine whether these complexes are actually formed in
the nucleus and migrate to the cytoplasm as a single entity
in living cells, ARAN1, which binds to only the Ran complexed with importin
and importin
-related transport factors in solution, was injected into the cytoplasm or nucleus of cultured BHK cells. It was found that nuclear-injected
ARAN1 was efficiently exported to the cytoplasm within
30 min of incubation (Fig. 6 A), whereas control mouse
IgG was retained in the injected nucleus (data not shown).
Since due to its size (Mr = 160,000), mouse IgG cannot
passively traverse the nuclear envelope (Wen et al., 1995
),
these results indicate that ARAN1 migrates to the cytoplasm in a piggyback fashion. These findings suggest that it
is most likely that ARAN1 binds to the Ran-GTP complexed with importin
-related transport factors in the nucleus, in which Ran would exist as the GTP-bound form,
and is transported to the cytoplasm as a complex with them.
That is, these results suggest that the Ran-GTP-importin
-related transport factor complex moves to the cytoplasm as a single entity without dissociation. On the other
hand, cytoplasmically injected ARAN1 remained in the cytoplasm even after 3 h of incubation (data not shown).
|
To determine whether ARAN1 actually recognizes the
Ran-GTP-importin -related transport factor complex in
a crude cell extract, we performed immunoprecipitation
analysis with mouse Ehrlich ascites tumor cell cytosolic extract in the presence of Q69L Ran-GTP, which should stabilize the complexes of importin
-related transport factors and Ran-GTP in the crude lysate. As shown in Fig. 6
B, importin
, CAS, and transportin were coprecipitated
with the Ran from the cell lysate by ARAN1. This result
supports the view that nuclear-injected ARAN1 binds to
Ran-GTP-importin
-related factors and is exported from
the nucleus to the cytoplasm in vivo.
ARAN1 Affects the Distribution of Endogenous Ran
Ran is localized predominantly in the nucleus. If Ran continuously shuttles between two compartments, the nucleus
and cytoplasm, and the binding of RanBP1 to the Ran-GTP-importin complex in the cytoplasm is required for
the recycling of Ran to the nucleus, ARAN1 would be expected to affect the distribution of Ran. To examine this
hypothesis, ARAN1 was introduced into the cell and the distribution of endogenous Ran was observed. As shown in Fig. 6, 30 min after injection into either the nucleus or the cytoplasm, injected ARAN1 localized in the cytoplasm. 30 min
after injection of ARAN1 into either the nucleus or the cytoplasm, the subcellular localization of endogenous Ran became apparently dominant in the cytoplasm of the ARAN1-injected cells, whereas control mouse IgG had no effect on
this distribution (Fig. 7 A for cytoplasmic injection and data
not shown for nuclear injection). The relocalization of Ran
was caused irrespective of the injection sites of ARAN1.
|
ARAN1 Inhibits the Nuclear Import of Proteins Containing the Classical NLS
Lastly, we examined the effect of ARAN1 on the nuclear
protein import in living cells. For this, ARAN1 or control
mouse IgG was injected into nucleus or cytoplasm before
the cytoplasmic injection of FITC-labeled BSA conjugated
with the NLS peptide of SV-40 T antigen (FITC-T-BSA).
In both cases, in which ARAN1 was injected into the cytoplasm or the nucleus, the nuclear import of FITC-T-BSA,
which was injected afterwards was clearly inhibited (Fig. 7
B for cytoplasmic injection and data not shown for nuclear injection). In both cases, the injected ARAN1 was confirmed to be localized in the cytoplasm as expected from
the results of Fig. 6 (data not shown). In contrast, the simultaneous cytoplasmic injection of ARAN1 with FITC-
T-BSA failed to inhibit the nuclear import of the FITC-
T-BSA (data not shown). These results suggest that the
injected ARAN1 interrupts the Ran cycle in the cytoplasm probably by preventing RanBP1 from interacting with the
Ran-importin complex, which causes an abnormal distribution of Ran and further the suppression of nuclear
protein import.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The COOH-terminal Acidic Domain of Ran Is Exposed
to the Molecular Surface as the Result of Its Interaction
with Importin or Importin
Families
In this study we obtained a monoclonal anti-Ran antibody,
ARAN1, which recognizes mono-specific Ran as confirmed
by immunoblotting of total cell extract and immunofluorescent analysis of cultured cells. An epitope of ARAN1 was
found to lie in the acidic COOH-terminal domain of Ran. A
solution binding assay revealed that ARAN1 interacts only
with Ran, when it is complexed with importin , transportin, or CAS-importin
but not Ran alone. These results indicate that the COOH-terminal domain of Ran is exposed
to the molecular surface probably as the result of a conformational change only when importin
-related transport
factors bind to Ran.
Although ARAN1 recognizes only Ran complexed with
importin or its related transport factors in solution, it
shows the same immunofluorescence pattern as a polyclonal anti-Ran antibodies (Fig. 1). Since it was previously
reported that importin
is located not only in the cytoplasm but also within the nucleus (Kose et al., 1997
), it is
likely that the apparent nucleoplasmic staining by ARAN1
means the complex formation of Ran with importin
and
importin
-related transport factors in the nucleus. Alternatively, this can be explained by a similar degree of Ran
denaturation during cell fixation as in immunoblotting.
It was found that ARAN1 binds to Ran-GDP-importin
complex as well as Ran-GTP-importin
complex,
whereas it is known that importin
binds to Ran-GDP with
much lower affinity than to Ran-GTP. Chi et al. (1996)
demonstrated that the addition of RanBP1 with Ran-GDP
increased the affinity of Ran-GDP for importin
. By analogy, it is speculated that ARAN1 may stabilize the interaction of Ran-GDP with importin
like RanBP1. In both
cases, whereas ARAN 1 cannot reach the COOH-terminal
domain of Ran-GTP or Ran-GDP alone, the interaction of
Ran with importin
appears to induce a conformational change of Ran so that ARAN1 is able to recognize the
epitope domain. Richards et al. (1995)
demonstrated that
a polyclonal antibody against a peptide corresponding to
residues 196-207 of Ran, which is adjacent to the acidic
211DEDDDL-COOH216 domain, preferentially recognizes
the GTP-bound form of Ran. They therefore proposed that the COOH-terminal domain is structurally flexible and
undergoes a nucleotide-dependent conformational change,
which is not inconsistent with the results herein.
Due to the characteristic amino acid sequences and conformational flexibility, it is likely that the COOH-terminal
domain has some functions. Richards et al. (1995) also
proposed from their biochemical analysis using
DE-Ran
that the DEDDDL sequence stabilizes the GDP form of
Ran, possibly by folding into the guanine nucleotide binding
pocket and mimicking the negative charge on the
-phosphate of GTP. In addition, it was suggested that the COOH
terminus of Ran is implicated in the regulation of interaction between Ran and RanBP1 (Richards et al., 1995
).
Consistent with their suggestion, we showed that the binding of ARAN1 to the COOH-terminal tail suppresses the
interaction of RanBP1 with the Ran-importin
complex.
Although
DE-Ran is able to bind to RanBP1, its affinity to RanBP1 is lower than wild-type Ran (Richards et al.,
1995
). Therefore, we speculate that exposure of the COOH-terminal tail may increase the accessibility of RanBP1 not
only to the Ran-importin
complex but also to the Ran-
importin
-related transport factors.
A similar sequence in importin (335DENDDDW342)
with the COOH-terminal domain of Ran is immediately
adjacent to sequences required for binding Ran-GDP/
RanBP1, but not Ran-GTP (Chi et al., 1997
). They proposed that this sequence in importin
substitutes for the
COOH-terminal Ran sequence in the nucleotide binding
pocket of Ran when GDP is bound and that this rearrangement would expose the COOH terminus of Ran for
binding to RanBP1, stabilizing the trimeric complex. Their
proposal is consistent with our result that the COOH-terminal tail of Ran-GDP was also exposed by the binding of
importin
.
The Export of Ran-Importin -related Transport
Factor Complexes from the Nucleus
Although it has been proposed that the Ran-importin
-related transport factor complexes form in the nucleus,
are exported to the cytoplasm, and then dissociate in order
to recycle (Bischoff and Görlich, 1997
; Izaurralde et al.,
1997
), this proposal has not been experimentally verified
in vivo. Recently, Izaurralde et al. (1997)
showed that the
depletion of nuclear Ran-GTP inhibits the export of importin
and
, transportin and NES-containing proteins
in Xenopus laevis oocyte, suggesting that nuclear Ran-GTP is required for the export of importin
and
, transportin, and NES-containing proteins from the nucleus.
However, their results do not necessarily mean that the
Ran-importin
-related transport factor complexes are
translocated from the nucleus to the cytoplasm as a complex. Thus, in regard to the dynamic behavior of Ran
through the nuclear envelope, little concrete experimental
data exist. In this study, we found that a monoclonal anti-Ran antibody ARAN1 failed to inhibit the export of Ran
from the nucleus and inhibited reimport into the nucleus,
and that nuclearly injected ARAN1 was efficiently exported from the nucleus. Our results indicate that the
Ran-importin
-related transport factor complexes efficiently migrate from the nucleus to the cytoplasm as a complex in living cells, even when complexed with ARAN1. Although it can not be excluded that the nuclear export of
ARAN1 injected into the nucleus could be due to complex
formation with Ran-GDP-importin
-related factors rather
than Ran-GTP-importin
-related factors, the current model
of a steep Ran-GTP gradient across the nuclear membrane (see introduction) and the immunoprecipitation experiments with ARAN1 in the presence of Q69L Ran-GTP
(Fig. 6 B) support that ARAN1 is exported from the nucleus as a complex with Ran-GTP and importin
-related factors.
The Role of RanBP1 in the Recycling of Ran
After the translocation of the Ran-importin complex
into the cytoplasm, it is predicted that the complex is disassembled for the next round of nuclear import and the recycling of Ran. The Ran-GTP-importin
complex is kinetically very stable (the dissociation constant is ~1 nM, when
nucleotide exchange and GTP hydrolysis are blocked)
(Görlich et al., 1996; Izaurralde et al., 1997
). Injected
ARAN1 induced the relocalization of endogenous Ran in
living cells. That is, endogenous Ran was found predominantly in the cytoplasm within 30 min after injection (Fig.
7). Since the injected ARAN1 was also localized in the cytoplasm, irrespective of the injection site (Fig. 6), it is concluded that ARAN1 prevented the event(s) from occurring in the cytoplasm but not in the nucleus. Our in vitro
biochemical experiments (Figs. 4 and 5) strongly suggest
that the injected ARAN1 efficiently interacts with the Ran-importin
complex in the nucleus or the cytoplasm
and prevents the binding of RanBP1 to the Ran-importin
complex in the cytoplasm. Furthermore, it was found
that the COOH-terminal tail of Ran is also exposed via interaction with other importin
family molecules. Therefore, we conceive that Ran probably remains in the cytoplasm complexed with importin
or its related transport
factors due to the inhibition by ARAN1 on the binding of
RanBP1 to Ran-importin
or its related transport factor
complexes in the cytoplasm, although we did not examine
in this study that ARAN1 actually inhibits the binding of
RanBP1 to the complex of Ran with importin
-related
transport factors other than importin
. At least part of
our conclusion is consistent with the recent biochemical
analysis by Bischoff and Görlich (1997)
. They showed that
the importin
and RanBP1 cooperatively relieved the
GAP resistance of the Ran-GTP-importin
complex, suggesting that RanBP1 is critical for the release of Ran-GTP
from importin
. Moreover, in our study, it was found that
ARAN1 competes much better for RanBP1 complexed to
Ran-GDP-importin
than to Ran-GTP-importin
, which
means that RanBP1 has much higher affinity for Ran-GTP-
importin
complex than for Ran-GDP-importin
complex. This is consistent with the idea that RanBP1 efficiently
binds to Ran-GTP-importin
complex in the cytoplasm
to promote the dissociation of the complex.
RanBP2 contains four Ran-binding domains which are
functionally equivalent to RanBP1 (Lounsbury et al.,
1994; Yokoyama et al., 1995
). It is located at the cytoplasmic filaments of the NPC (Wu et al., 1995
; Yokoyama et al.,
1995
) and a fraction of RanGAP which is modified by the
addition of a small ubiquitin-like peptide binds to RanBP2
(Matunis et al., 1996
; Mahajan et al., 1997
; Saitoh et al.,
1997
). Therefore, RanBP2 may play the same role as
RanBP1 in the recycling of Ran, although further experiments will be required to better understand the role(s) of
RanBP2 on the nucleocytoplasmic transport of macromolecules as well as to understand the effect of ARAN1 on RanBP2.
Nuclear Protein Import and Recycling of Ran and
Importin
This study showed that ARAN1, when injected into cultured cells inhibits nuclear protein import mediated by basic-type classical NLS irrespective of the injection site, i.e.,
nucleoplasm or cytoplasm. It has been recently reported
that RanBP1 is required for nuclear protein import and
RNA export in vivo (Schlenstedt et al., 1995; Richards et al.,
1996
) and stimulates the protein import in a permeabilized
cell-free assay (Chi et al., 1996
). Furthermore, it has been
shown that the COOH terminus deleted Ran (
DE-Ran) represents the dominant-negative effect on the nuclear
protein import. In this study, we found that ARAN1 inhibits the interaction of RanBP1 (and probably RanBP2)
with the Ran-importin
complex. There are two possible
explanations for the inhibitory effect of ARAN1 on the
nuclear import of the classical NLS substrates. One possibility is that ARAN1 may inhibit the interaction of RanBP1 to
Ran-GTP-importin
complex, which leads to the suppression of the disassembly of the complex. Alternatively,
consistent with the suggestion of Chi et al. (1996)
that
RanBP1 promotes nuclear protein import by stabilizing the
interaction of Ran-GDP with importin
, ARAN1 may
block the function of RanBP1 on the interaction of Ran-GDP with importin
by binding to the Ran-GDP-importin
complex stably and, as a result, affect nuclear protein import.
Thus, it seems most likely that the inhibition of the function of RanBP1 (and probably RanBP2) blocks the disassembly of Ran-importin complex in the cytoplasm,
which causes the depletion of nuclear Ran, an inhibition in
the recruitment of importin
, and further, the decline of
the recycling of importin
/
to the cytoplasm by the suppression of the dissociation of the nuclear pore-targeting complex (importin
/
bound to a karyophile) in the nucleus following the depletion of nuclear Ran. It appears
that such an imbalance in the import factors totally inhibits the nuclear protein import. Further studies will be necessary in order to understand whether ARAN1 also affects import and export pathways other than the classical NLS import pathway.
![]() |
Footnotes |
---|
Address correspondence to Y. Yoneda, Department of Anatomy and Cell Biology, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: (81) 6-6879-3210. Fax: (81) 6-6879-3219. E-mail: yyoneda{at}anat3.med.osaka-u.ac.jp
Received for publication 30 July 1998 and in revised form 29 December 1998.
M. Hieda and T. Tachibana contributed equally to this work.
T. Tachibana's present address is Department of Neurochemistry and
Neuropharmacology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
We thank S. Kuroda (Institute of Scientific and Industrial Research, Osaka University, Japan) for the gift of pGEX-6P-2-hGFP vector.
This work was supported by a Grant-in-Aid for Scientific Research on Priority Area (07282103), a Grant-in-Aid for Scientific Research (B) (08458229), and a Grant-in-Aid for COE Research (07CE2006) from the Japanese Ministry of Education, Sciences, Sports and Culture, the Nissan Science Foundation, and the Naito Foundation. M. Hieda, T. Tachibana, F. Yokoya, and S. Kose are Research Fellows of the Japanese Society for the Promotion of Science.
![]() |
Abbreviations used in this paper |
---|
BSA, bovine serum albumin; CAS, cellular apoptosis susceptibility gene; GFP, green fluorescent protein; GST, glutathione-S-transferase; NES, nuclear export signal; NLS, nuclear localization signal; NPC, nuclear pore complex; RanBP, Ran-binding protein; TB, transport buffer.
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