Department of Cell Biology and Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037
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Abstract |
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We have developed a permeabilized cell assay to study the nuclear export of the shuttling transcription factor NFAT, which contains a leucine-rich export signal. The assay uses HeLa cells that are stably transfected with NFAT fused to the green fluorescent protein (GFP). Nuclear export of GFP-NFAT in digitonin-permeabilized cells occurs in a temperature- and ATP-dependent manner and can be quantified by flow cytometry. In vitro NFAT export requires the GTPase Ran, which is released from cells during the digitonin permeabilization. At least one additional rate-limiting export factor is depleted from permeabilized cells by a preincubation at 30°C in the absence of cytosol. This activity can be provided by cytosolic or nucleoplasmic extracts in a subsequent export step. Using this assay, we have purified a second major export activity from cytosol. We found that it corresponds to CRM1, a protein recently reported to be a receptor for certain leucine-rich export sequences. CRM1 appears to be imported into the nucleus by a Ran-dependent mechanism that is distinct from conventional signaling pathways. Considered together, our studies directly demonstrate by fractionation and reconstitution that nuclear export of NFAT is mediated by multiple nucleocytoplasmic shuttling factors, including Ran and CRM1.
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Introduction |
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NUCLEOCYTOPLASMIC trafficking is mediated by nuclear pore complexes (NPCs)1, large supramolecular structures that span the nuclear envelope (for reviews see Doye and Hurt, 1997; Nigg, 1997
). Molecules smaller than ~20-40 kD can passively diffuse
through aqueous channels in the NPC. In contrast, most
macromolecules traverse the NPC by energy-, temperature-, and signal-dependent mechanisms. The best characterized signals for nuclear import (nuclear localization sequences [NLSs]) are short stretches of amino acids enriched in basic residues that occur as either a single or a bipartite motif (for review see Dingwall and Laskey, 1991
).
The M9 domain of the hnRNP A1 protein contains a qualitatively distinct type of NLS, a 38-amino acid stretch that
is enriched in aromatic and glycine residues (Siomi and
Dreyfuss, 1995
).
The nuclear import of proteins containing basic amino
acid-rich (classical) NLSs is beginning to be characterized
in molecular detail (for review see Görlich and Mattaj,
1996; Nigg, 1997
). Studies with a transport assay using digitonin-permeabilized cells (Adam et al., 1990
) have identified four conserved soluble factors directly involved in
the nuclear import of proteins with classic NLSs: (a) importin
/karyopherin
/NLS receptor/Srp1
, (b) importin
/karyopherin
/p97, (c) the small GTPase Ran/TC4, and
(d) NTF2/p10 (for reviews see Görlich and Mattaj, 1996
;
Nigg, 1997
). Nuclear import appears to involve the stepwise movement of a complex containing the transport substrate, importin
, and importin
through the NPC (Nigg,
1997
). Translocation through the NPC also requires RanGTP
hydrolysis (Melchior et al., 1993
; Moore and Blobel, 1993
; Schlenstedt et al., 1995
; Mahajan et al., 1997
) and NTF2
(Moore and Blobel, 1994
; Paschal and Gerace, 1995
), but
the exact functions of these components are not resolved.
Nuclear import mediated by the M9 domain of hnRNP A1
involves a receptor called transportin (Pollard et al., 1996
)
that is distantly related to importin
, and also is suggested
to involve Ran (Bonifaci et al., 1997
; Izaurralde et al., 1997
).
In comparison to nuclear protein import, considerably
less is known about the mechanisms of nuclear export of
proteins and RNA-protein complexes (for review see Izaurralde and Mattaj, 1995; Görlich and Mattaj, 1996
). An
important advance was the characterization of sequences
in nuclear proteins that are sufficient and necessary for nuclear export, called nuclear export sequences (NESs; for review see Gerace, 1995
). NESs consisting of short amino
acid stretches enriched in leucine residues appear to be the
most prevalent (e.g., Fischer et al., 1995
; Wen et al., 1995
;
Richards et al., 1996
), although a distinct type of NES is
present within the M9 domain of hnRNP A1 (Michael et al.,
1995
) and in a sequence of the hnRNP K protein (Michael
et al., 1997
). It was recently reported that certain leucine-rich NESs interact with a member of the importin
superfamily called CRM1 (Fornerod et al., 1997b
; Fukuda et al., 1997
). CRM1 has been implicated in nuclear export from
in vivo studies involving temperature sensitive CRM1 mutants in yeast (Fukuda et al., 1997
; Stade et al., 1997
), and
the overexpression of CRM1 in Xenopus oocytes (Fornerod et al., 1997b
). Other work has suggested that CRM1
targets the exported substrate to proteins of the NPC
(Neville et al., 1997
). Nuclear export of the Rev protein, which also contains a leucine-rich NES, appears to involve
an additional factor, eIF-5A (Ruhl et al., 1993
; Bevec et al.,
1996
). CRM1-independent nuclear export has been described for importin
(Kutay et al., 1997
), which also contains a leucine-rich NES (Boche and Fanning, 1997
).
Genetic studies in yeast have provided evidence that
Ran has a role in RNA export (e.g., Schlenstedt et al.,
1995). Recent work with vertebrate cells has expanded
upon this, showing that the GTP-bound form of Ran is required for the nuclear export of a protein containing a leucine-rich NES (Richards et al., 1997
) and of various RNAs
(Izaurralde et al., 1997
). However, in contrast to nuclear
import, RanGTP hydrolysis does not appear to be necessary for export under these conditions. Although the precise role of RanGTP in nuclear export in these experiments remains to be elaborated, in vitro studies indicate
that RanGTP stimulates the formation of complexes between certain export receptors and their cargos (Fornerod
et al., 1997b
; Kutay et al., 1997
), and suggests a direct role
for Ran in nuclear export.
The availability of a cytosol-dependent in vitro assay for
nuclear export would greatly facilitate the understanding
of nuclear export by allowing the detailed biochemical
characterization of soluble factors involved in this process.
Arts et al. (1997) have described an in vitro assay for export of RNA from synthetic nuclei, but a cytosolic requirement for export in this system is unclear. Yang et al. (1997)
have shown that the glucocorticoid receptor is exported
from nuclei of digitonin-permeabilized cells in an ATP-
and temperature-dependent manner, but the export is apparently independent of exogenous cytosol under their experimental conditions.
Here we report the development and characterization of
a cytosol-dependent assay for the analysis of nuclear protein export in permeabilized cells. The model export substrate in our system is the shuttling transcription factor
NFAT (nuclear factor of activated T cells; Northrop et al.,
1994), which plays a crucial role in the activation of T lymphocytes by stimulating the transcription of cytokine genes
(for review see Crabtree and Clipstone, 1994
). NFAT contains two regulatable NLSs of the basic amino acid type (Beals et al., 1997a
), as well as a regulatable leucine-rich
NES (Klemm et al., 1997
). In resting cells, NFAT resides
in the cytoplasm. Its nuclear import is triggered by elevated intracellular calcium (Flanagan et al., 1991
; Shibasaki et al., 1996
), which induces the activation of the phosphatase calcineurin, leading to the dephosphorylation of
NFAT and subsequent exposure of its NLSs (Beals et al.,
1997a
). Upon return of calcium to resting levels, NFAT
rapidly reappears in the cytosol (Shibasaki et al., 1996
) by
an export process that requires rephosphorylation of
NFAT by glycogen synthase kinase-3 (GSK-3; Beals et al.,
1997b
).
Using our permeabilized cell assay, we have characterized the cytosolic requirements for nuclear export of NFAT. By biochemical fractionation and reconstitution, we found that Ran and CRM1 are the two major cytosolic activities involved in NFAT export under our conditions. Our studies further suggest that the nuclear import of CRM1 is mediated by a Ran-dependent pathway distinct from those involving conventional NLSs, and provide the basis for developing a detailed understanding of the nucleocytoplasmic shuttling of a major nuclear export receptor.
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Materials and Methods |
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Molecular Cloning of the Green Fluorescent Protein-NFAT Transfection Vector pAD-4
In a first step, the green fluorescent protein (GFP) fragment of the eukaryotic expression vector pS65T-C1 (Clontech Laboratories, Inc., Palo Alto, CA) was replaced with the human codon usage-optimized SGFP- TYG (synthetic GFP containing the amino acid change 65SYG to 65TYG). SGFP-TYG was amplified from the vector blue-SGFP-TYG-nosKS (J. Sheen, Harvard Medical School, Boston, MA) by PCR and then primers were designed to introduce a NheI site and a HindIII site. This NheI-HindIII fragment encoding SGFP-TYG was introduced into the eukaryotic expression vector pS65T-C1 (replacing the original GFP sequence in the vector), creating pAD-3. In a second step, the coding sequence of human NFAT was then PCR amplified out of pSH107c (provided by G.R. Crabtree, Howard Hughes Medical Institute, Stanford, CA), inserted into the BamHI and XbaI sites of pAD-3 (resulting in pAD-4), and then verified by sequencing.
Cell Culture and Transfections
HeLa cells were grown either in DME on plastic dishes, or in suspension in Joklik's modified S-MEM (GIBCO BRL, Gaithersburg, MD). In both cases media contained 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Transfections were performed with lipofectamine (GIBCO BRL, Gaithersburg, MD) using 25 µg of linearized plasmid per ~2 × 106 adherent cells. Approximately 50 G418-resistant clones (GIBCO BRL) were pooled and induced with 250 nM of trichostatin A (Wako BioProducts, Richmond, VA) to express GFP-NFAT. Cells expressing high levels were enriched by FACS® (Becton and Dickinson Co., Mountain View, CA). Positive cells were expanded and stored in aliquots in liquid nitrogen.
Preparation of Cytosol and Nuclear Extract
Approximately 4.5 × 109 HeLa cells (8 liters of cells growing in suspension) were collected by centrifugation at 300 g for 10 min, washed twice
with ice-cold PBS, and then once with washing buffer (10 mM Hepes-
KOH, pH 7.3, 110 mM KOAc, 2mM Mg[OAc]2, 2 mM DTT). After resuspension in one volume of lysis buffer (5 mM Hepes-KOH, pH 7.3, 10 mM
KOAc, 2mM Mg[OAc]2, 2 mM DTT, 1 mM PMSF, 1 µg/ml each of leupeptin, pepstatin, and aprotinin) and swelling for 10 min on ice, the cells
were lysed in a stainless steel Dounce homogenizer. After centrifugation
at 1,500 g for 15 min, the supernatant was cleared by centrifugation at
120,000 g for 1 h and then dialyzed overnight against transport buffer (20 mM Hepes-KOH, pH 7.3, 110 mM KOAc, 2 mM Mg[OAc]2, 1mM
EGTA). The resulting cytosol (~10 mg/ml) was frozen in liquid nitrogen
and stored at 80°C.
To prepare a nuclear extract, the pellet of the 1,500-g spin was washed
in 4 ml of lysis buffer and resuspended in 10 ml of 20 mM Hepes-KOH,
pH 7.9, 25% glycerol, 0.5 M NaCl, 1.5 M MgCl2, 0.2 mM EDTA, 1 mM
PMSF, and 1 mM DTT. The nuclei were lysed by 10 strokes in a stainless
steel Dounce homogenizer, left on ice for 15 min, and then centrifuged at
35,000 g for 30 min. The resulting supernatant was dialyzed overnight
against transport buffer and then centrifuged again at 100,000 g for 30 min. The final supernatant (nuclear extract, ~3.5 mg/ml) was frozen in liquid nitrogen and stored at 80°C.
SDS-PAGE and Western Blotting
Proteins were separated by SDS-PAGE and blotted onto nitrocellulose using standard methods. Blots were blocked with 5% milk powder in PBS overnight. NFAT was detected with the mouse monoclonal antibody 7A6 (1:5,000 in 5% milk in PBS) and HRP-coupled goat anti-mouse IgG (Pierce Chemical Co., Rockford, IL; 1:5,000 in 10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05% Tween 20 [Sigma Chemical Co.]). CRM1 was detected with different anti-CRM1 antibodies (see below; 1:5,000-1:3,000 in 5% milk in PBS) and HRP-coupled donkey anti-rabbit IgG (Pierce Chemical Co.; 1:10,000 in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20). The enhanced chemiluminescence system (Pierce Chemical Co.) was used for visualization of proteins.
Antibodies
The mouse monoclonal anti-NFAT antibody (7A6) is described by
Northrop et al. (1994). Anti-importin
antibody was obtained from Affinity BioReagents, Inc. (clone 3E9; Golden, CO). Before use in the export assay, the ascites fluid was diluted 1:4 with transport buffer containing 5 mg/ml BSA and then dialyzed against transport buffer. Two
different polyclonal antibodies against CRM1 were used. The first is described by Fornerod et al. (1997a)
and was provided by G. Grosveld (St.
Jude Children's Research Hospital, Memphis, TN). The second was raised
in rabbits by injecting a peptide corresponding to the COOH terminus of
CRM1 (GIFNPHEIPEEMCD), coupled with glutaraldehyde to keyhole
limpet hemocyanin (Calbiochem-Novabiochem Corp., La Jolla, CA).
Purification of GST-M9
pGEX-M9 provided by G. Dreyfuss (University of Pennsylvania School of
Medicine, Philadelphia, PA) was transformed into DH5 cells. An overnight culture was diluted 1:50 in Luria-Bertani medium, grown to an
OD600 of 0.7, and then induced with 1 mM isopropylthio-
-
-galactoside for 3 h. Cells were harvested by centrifugation at 5,000 g for 10 min,
washed twice in ice-cold PBS, and then resuspended to 1% of the original
volume in PBS with 5 µg/ml each of aprotinin, leupeptin, and pepstatin.
After cell lysis, Triton X-100 was added to a final concentration of 1%.
The lysate was centrifuged at 10,000 g for 10 min and the supernatant was
incubated with glutathione-Sepharose 4B beads (Pharmacia Biotech. Inc.,
Piscataway, NJ) for 1 h at 4°C. The beads were collected by centrifugation
at 800 g for 5 min and then washed in batch three times in PBS and once in
50 mM Tris-HCl, pH 8.8, 150 mM NaCl before being transferred to a column. GST-M9 was eluted with 50 mM Tris base, 150 mM NaCl, and 15 mM glutathione (reduced). Peak fractions were dialyzed against 100 mM
Hepes-KOH, pH 9.6.
Recombinant Import Factors
Wild-type Ran was prepared as described (Melchior et al., 1995).
RanQ69L and RanT24N were obtained from J. Becker (MPI für molekulare Physiologie, Dortmund, Germany) (Klebe et al., 1995
). Importin
and importin
were prepared as in Hu et al. (1996)
. NTF2 was prepared
as in Paschal and Gerace (1995)
.
Preparation of Fluorescent Import Substrates
To prepare Cy5-NLS-BSA, 2.5 mg of BSA in 1 ml 0.1 M Na2CO3 was coupled to Cy5TM (1 vial, Amersham Pharmacia Biotech. Inc., Piscataway, NJ)
for 40 min at room temperature. The conjugate was separated from the
free dye by chromatography on a PD-10 column (Pharmacia Biotech.
Inc.). The NLS peptide derived from the sequence of the SV-40 large T
antigen was reduced as described (Adam et al., 1990) and coupled to Cy5-BSA that had previously been activated with a 20-fold molar excess of
sulfo-SMCC (Pierce Chemical Co.; Adam et al., 1990
) by incubation at
4°C overnight. The Cy5-NLS-BSA conjugate was then separated from
free peptide by chromatography on a PD-10 column equilibrated with
transport buffer. Peak fractions were pooled and then aliquots were frozen in liquid nitrogen.
To prepare Cy5-GST-M9, 5 mg of GST-M9 was coupled with Cy5TM (1 vial, Amersham Pharmacia Biotech. Inc.) for 30 min at room temperature and then separated from free dye by chromatography on a PD-10 column (Pharmacia Biotech. Inc.). Peak fractions of Cy5-GST-M9 were pooled and diluted with transport buffer to a final concentration of 1 mg/ml. Aliquots were frozen in liquid nitrogen.
Nuclear Export Assay
To assay nuclear export of NFAT, its expression was induced by treating
stably transfected HeLa cells growing on tissue culture dishes with 250 nM
of trichostatin A (Wako BioProducts) overnight. To induce nuclear import, ionomycin (Calbiochem-Novabiochem Corp.) and LiOAc were
added to final concentrations of 1 µM and 30 mM, respectively, and then
the cells were incubated for 30 min at 37°C. They were trypsinized in the
continued presence of ionomycin and LiOAc and then washed once with
10% FBS in transport buffer and once with transport buffer containing
80 mM KOAc, 30 mM LiOAc, 2 mM DTT, 1 mM PMSF, and 1 µg/ml each
of leupeptin, pepstatin, and aprotinin. The cells were collected by centrifugation at 300 g for 5 min at 4°C and then resuspended in transport buffer
(with 30 mM LiOAc) at ~107/ml. After permeabilization with 60 µg/ml
digitonin (Calbiochem-Novabiochem Corp.) for 5 min on ice, the cells
were washed and centrifuged as described above and then resuspended to
a concentration of 107/ml in transport buffer containing KOAc and
LiOAc. A preincubation in the presence of an ATP-regenerating system
was performed for 15 min at 0°C or 30°C. Cells were then washed in transport buffer lacking LiOAc but containing 110 mM KOAc and then resuspended at ~3 x 107/ml. The complete transport mixture (40 µl in transport
buffer) contained ~300,000 cells, an ATP-regenerating system (1 mM
ATP, 5 mM creatine phosphate, 20 U/ml creatine phosphokinase), 2.5 mg/
ml BSA, import ligand (Cy5-NLS-BSA at ~2.5 µg/ml or Cy5-GST-M9 at
~25 µg/ml), 1 µM of annealed oligonucleotide (5' AGA GGA AAA TTT
GTT TCA TA and 5' TAT GAA ACA AAT TTT CCT CT, both prepared and HPLC purified by GIBCO BRL), and cytosol or nuclear extract at various concentrations, as indicated. For reactions in the absence
of ATP, the ATP-regenerating system was replaced by 50 U/ml hexokinase (Sigma Chemical Co.) and 12.5 mM glucose. Reactions were incubated at 30°C for 30 min and stopped by the addition of ice-cold transport
buffer. After centrifugation at 300 g for 5 min at 4°C, the cells were resuspended in a small volume of transport buffer. The average nuclear fluorescence emitted from GFP-NFAT, Cy5-NLS-BSA, or Cy5-GST-M9 in
10,000 cells was measured with a FACScan® flow cytometer (Becton and
Dickinson Co.). Reactions were standardized by arbitrarily assigning a
value of 100 to the nuclear fluorescence of GFP-NFAT in a 0°C control
reaction and, unless otherwise indicated, to the maximal nuclear fluorescence of the imported ligands after incubation at 30°C. For export reactions in which permeabilized cells were not preincubated, treatment of
cells was as described above, except that transport buffer always contained 110 mM KOAc and no LiOAc. For analysis of export by SDS-PAGE, nuclei were collected by centrifugation upon completion of the
export reaction, washed once in transport buffer, and then resuspended in
SDS-sample buffer. The supernatant of the first centrifugation was retained and mixed with an equal volume of 2× SDS-sample buffer and nuclei and supernatant fractions were analyzed by SDS-PAGE and immunoblotting. To perform the export assay with adherent cells growing on
coverslips, cells were treated with trichostatin A and ionomycin as above, permeabilized with digitonin, and then subjected to export reactions in
the same manner as previously described for nuclear import reactions
(Adam et al., 1990). Cells were then fixed with 3.7% formaldehyde in PBS
and analyzed by fluorescence microscopy using an Axiophot (Carl Zeiss,
Inc., Thornwood, NY).
Purification of a Major Cytosolic Activity for Nuclear Export
6 ml of cytosol (containing 60 mg of protein) were dialyzed into triethanolamine buffer (20 mM triethanolamine, pH 7.4, 2 mM MgCl2) and saturated ammonium sulfate was added to a final concentration of 900 mM. The precipitate (6% of total protein) was collected by centrifugation (14,000 g for 5 min) and then resuspended in triethanolamine buffer. The remaining ammonium sulfate was removed by chromatography of the dissolved precipitate on a PD-10 column (Pharmacia Biotech. Inc.) equilibrated in triethanolamine buffer. The sample then was loaded on a FPLC Mono Q column (HR 5/5; Pharmacia Biotech. Inc.) and eluted with a 20-ml linear gradient of 100 to 350 ml NaCl in triethanolamine buffer using a flow rate of 0.4 ml/min and collecting 1-ml fractions. Maximal nuclear export activity was typically recovered at ~230 mM NaCl. 900 µl of the peak fraction (as determined by the nuclear export assay and by immunoblotting) were concentrated to 300 µl by vacuum dialysis applied to a gel filtration column (Superdex 200 HR 10/30; Pharmacia Biotech. Inc.) equilibrated in transport buffer. The sample was chromatographed at 0.5 ml/ min, collecting 0.5-ml fractions. The nuclear export activity eluted with a molecular mass of ~120 kD.
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Results |
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We have developed a method for rapid, quantitative analysis of nuclear export of the shuttling transcription factor
NFAT in permeabilized HeLa cells. For this, we prepared
cells that were stably transfected with a cDNA coding for
NFAT fused to the GFP. The expression of GFP-NFAT
was induced to high levels by overnight treatment of the
cells with the histone deacetylase inhibitor trichostatin A
(Fig. 1) or with sodium butyrate (data not shown; Gorman
et al., 1983; Arts et al., 1995
). The actual level of expression of the GFP-NFAT in the induced cell population varied from cell to cell, although typically about two-thirds of
the cells expressed high levels of GFP-NFAT (Fig. 2 a,
ion).
The range of expression levels in the cell population was
most clearly seen in cells analyzed by flow cytometry (see
Fig. 3 b, 0°C).
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The GFP-NFAT in the transfected cells exhibited the
same nuclear transport behavior and responses to inhibitors as originally described for wild-type NFAT in vivo.
The calcium ionophore ionomycin induced rapid nuclear
import of GFP-NFAT (Flanagan et al., 1991; Shibasaki et al.,
1996
), which was predominantly cytoplasmic in untreated
cells (Fig. 2 a, compare
ion with + ion). GFP-NFAT from ionomycin-treated cells had a higher electrophoretic
mobility on an SDS gel than the protein from untreated
cells (Fig. 1, in vivo, compare
ion and + ion lanes), reflecting a partial dephosphorylation of the protein (Ruff
and Leach, 1995
). The ionomycin-induced import of GFP-
NFAT into the nucleus was inhibited by simultaneously adding cyclosporin A, a specific inhibitor of calcineurin
(Flanagan et al., 1991
; Liu et al., 1991
; data not shown).
When the ionomycin-treated cells were washed and incubated at 37°C in the presence of cyclosporin A, GFP-
NFAT was rapidly exported from the nucleus (Shibasaki et al., 1996
; data not shown). Finally, export of GFP-
NFAT was inhibited by the addition of 30 mM of lithium
acetate to the medium, which inhibits its rephosphorylation by inactivating GSK-3 (Klein and Melton, 1996
; data
not shown). Considered together, these in vivo experiments indicate that HeLa cells expressing GFP-NFAT are
appropriate for use in an in vitro nuclear export assay.
Nuclear Export of GFP-NFAT In Vitro
After the expression of GFP-NFAT was induced in stably transfected HeLa cells, the nuclear import of the reporter was triggered by treatment with ionomycin. Next, the cells were removed from the dish by trypsinization, permeabilized with digitonin, and then washed to remove the endogenous cytosol. In our standard export assay, the permeabilized cells were first preincubated for 15 min at 30°C in the absence of cytosol and in the presence of ATP. To retain most of the GFP-NFAT in the nucleus during the preincubation step, we included lithium in the preincubation buffer to inhibit GSK-3 (refer to Introduction and Materials and Methods). Typically, between 80 and 90% of the GFP-NFAT remained in the nucleus after the preincubation (data not shown). Subsequently, the cells were incubated under various export conditions in the absence of lithium and were analyzed by flow cytometry. As shown below, the 30°C preincubation step depletes rate-limiting export factors from the permeabilized cells, thereby allowing the detection of these activities in the subsequent export assay.
A double-stranded oligonucleotide corresponding to an
NFAT DNA-binding site (Northrop et al., 1994) was included in all export reactions. This oligonucleotide stimulated in vitro ATP-dependent nuclear export of GFP-
NFAT approximately twofold at a concentration of 1 µM
(data not shown), apparently by promoting release of the protein from chromatin (see below). Export reactions in
the absence of the oligonucleotide gave qualitatively similar results, although the absolute levels of export were
lower. Interestingly, the addition of lithium to export reactions containing oligonucleotide did not significantly decrease the level of GFP-NFAT export (data not shown).
This indicates that the inclusion of the oligonucleotide abrogates the requirement for phosphorylation by GSK-3 for
efficient export. Thus, the lithium-sensitive phosphorylation of NFAT appears to stimulate export by releasing
NFAT from chromatin, not by activating an NES on
NFAT per se. Decreased DNA binding activity of NFAT upon phosphorylation has been described previously (Park
et al., 1995
).
Fluorescence micrographs of permeabilized cells that
had been incubated in the standard export assay are
shown in Fig. 2 b. When cells were incubated at 0°C, the
amount of GFP-NFAT retained in the nucleus (Fig. 2 b;
0°C) was essentially identical to the amount retained in the
nuclei of cells examined immediately after permeabilization (data not shown). In contrast, incubation at 30°C in
the presence of cytosol and an ATP-regenerating system led to the loss of most nuclear fluorescence (Fig. 2 b;
30°C). The export of GFP-NFAT, as seen by the loss of
fluorescence, was largely prevented if the reaction included an ATP-depleting system (Fig. 2 b; ATP) or
wheat germ agglutinin (WGA; Fig. 2 b; +WGA), which
binds to a group of O-glycosylated proteins of the NPC
(Hanover et al., 1987
) and inhibits mediated nuclear import and export in vivo (Yoneda et al., 1987
; Dargemont
and Kühn, 1992
). As shown below, nuclear export of
GFP-NFAT was largely dependent on the addition of cytosol (see Figs. 4 b and 5 b).
|
Flow cytometry provided a rapid and quantitative method to analyze nuclear export in a large number of cells. Fig. 3 shows the result of a standard nuclear export assay carried out in the presence of cytosol that was analyzed by this method. The original flow cytometry data used to generate the graph in Fig. 3 a is shown in Fig. 3 b. It should be noted that in assays measuring nuclear export, low values of nuclear fluorescence indicate high levels of transport, whereas in assays measuring nuclear import (see below), high fluorescence values represent high levels of transport. Incubation at 30°C resulted in the retention of only 18.2 units of nuclear fluorescence as compared with 100 units retained in the 0°C control (Fig. 3 a). Export was strongly inhibited by depleting ATP or by including the lectin WGA, both of which gave export values similar to the 0°C control (Fig. 3 a).
Several controls indicated that the loss of fluorescence
during the export incubations was due to physiologically
relevant export of GFP-NFAT from intact nuclei. When
we included rhodamine-labeled dextrans of different molecular weights in the export reactions (Melchior et al.,
1993), we found that a small dextran (4.5 kD), but not a
large one (155 kD) was able to enter the nucleus (data not
shown). This demonstrated that the permeability barrier
of the nuclear envelope remained normal. Additional validation for the integrity of the nuclear envelope was obtained by incubating permeabilized cells with Cy5-labeled
NLS-BSA and cytosol in the standard export assay. In this
case, efficient nuclear accumulation of the NLS-BSA occurred at the same time as export of the GFP-NFAT (see
Fig. 4), and both the import and export were inhibited by WGA (data not shown). A further control involved centrifuging the assay mixture after the export reaction and examining the resulting supernatants and pellets by Western
blotting to detect GFP-NFAT (refer to Fig. 1). The total
amount of GFP-NFAT in a reaction incubated at 30°C
was similar to that in a reaction incubated at 0°C. However, in the 0°C incubation, most of the GFP-NFAT was found in the permeabilized cell pellet, whereas in the 30°C
incubation, most of the GFP-NFAT was found in the supernatant. Thus, the loss of GFP fluorescence at 30°C does
not result from degradation of GFP-NFAT, but instead
reflects its release from the permeabilized cells into the supernatant. It should be noted that after incubation at 30°C,
the electophoretic mobility of GFP-NFAT decreases and resembles that of the protein before nuclear import in vivo
(refer to Fig. 1, compare in vivo with in vitro panels). This
apparently reflects rephosphorylation of the NFAT, which
activates the export of NFAT and inactivates its NLS in vivo
(Beals et al., 1997). Since calcineurin-mediated dephosphorylation of GFP-NFAT cannot occur in vitro due to the presence of EGTA in our assay buffer, the exported GFP-NFAT
is trapped outside the nucleus and cannot be reimported.
Further Characterization of the Standard Nuclear Export Assay
To further characterize nuclear transport in the standard export assay, we simultaneously measured export of GFP- NFAT and import of Cy5-NLS-BSA in the same cells (Fig. 4). Fig. 4 a shows a concurrent time course of nuclear import and export over a range of 30 min. Nuclear import of Cy5-NLS-BSA was linear for at least 30 min, yielding a final value that was approximately sixfold greater than the 0°C background (100 versus 16.4 units). Examination of nuclear export of GFP-NFAT in the same cells showed that the fluorescent signal of GFP-NFAT was reduced from 100 units at the beginning of the reaction to a final typical value of 24.5 units after a 30-min incubation. Nuclear export was roughly linear for 15-20 min and then started to level off, which was probably due to a limitation of the export substrate after this time. In cells expressing much lower levels of GFP-NFAT, export was linear for only ~10 min (data not shown).
Fig. 4 b shows a comparison of the cytosol requirement of nuclear import and nuclear export. The reactions were carried out in the presence of excess recombinant Ran so that it would not be rate limiting. We found that 1.25 mg/ml cytosol stimulated nuclear export of GFP-NFAT to almost the maximal level (from 72.9 to 19 units) and also gave maximal stimulation of import of Cy5-NLS-BSA into the nucleus (from 21.7 to 100 units). Thus, the cytosol requirement for nuclear import and export is quantitatively very similar under these conditions.
Under steady-state conditions, nuclear export factors
are expected to be somewhat concentrated in the nucleus.
Therefore, we tested the ability of a HeLa cell nuclear extract to stimulate nuclear export. Fig. 4 c shows that the
nuclear extract stimulated export of GFP-NFAT to
roughly the same extent as did cytosol when used at the
same protein concentration (from 100 units at 0°C to 28.6 units for nuclear export, compared with 23.6 units for cytosol). In contrast, the nuclear extract had only a minor stimulatory effect on protein import into the nucleus as compared with cytosol (from 14 units at 0°C, to 22.6 and 100 units at 30°C for the nuclear extract and cytosol, respectively). A nuclear extract prepared with a low-salt extraction
buffer (50 mM NaCl) was able to stimulate nuclear export
at least as well as the nuclear extract prepared with a high-salt buffer (500 mM NaCl, refer to Materials and Methods), suggesting that the extracted factors are not tightly bound
to NPCs and are probably intranuclear (data not shown).
These results suggest that factors distinct from those required for the nuclear import of NLS-containing substrates
are involved in the export of GFP-NFAT from the nucleus. The recombinant factors other than Ran that support
the nuclear import of cargoes with classical NLS in permeabilized cells (NTF2, importin , and importin
) strongly
stimulated import of Cy5-NLS-BSA into the nucleus without affecting export of GFP-NFAT (data not shown).
Thus, whereas some or all of these import factors are rate-limiting for the nuclear import of a protein containing a
classical NLS in our system, none of these components are
rate-limiting in the assay measuring export of GFP-NFAT.
Ran and Other Nucleocytoplasmic Shuttling Factors Are Involved in Nuclear Export of NFAT
Ran has a well-established role in nuclear import and recent studies have suggested a direct role for Ran in nuclear
export as well (refer to Introduction). To investigate a possible involvement of Ran and other nucleocytoplasmic
shuttling factors in nuclear export in our assay, we analyzed the stimulation of nuclear export of GFP-NFAT by
Ran alone or by cytosol, after preincubation of the permeabilized cells either at 0°C or at 30°C in the presence of
ATP and absence of cytosol (Fig. 5, a and b). The preincubation at 30°C, as used in our standard assay, might be expected to deplete rate-limiting export factors whose exit
from the nucleus is ATP- and temperature-dependent,
whereas the preincubation at 0°C would deplete only factors that are small enough to exit the nucleus by passive
diffusion. Previous work has shown that Ran is almost completely depleted from the nucleus of permeabilized
cells during digitonin treatment and washing at 0°C (Melchior et al., 1995).
|
In permeabilized cells that had been preincubated at 0°C, Ran alone strongly stimulated export to a degree that was similar to the export stimulation obtained with complete cytosol (Fig. 5, compare a with b): the fluorescence decreased from 81.2 units after incubation with buffer to 34.3 units with Ran (100 µg/ml) or to 37.6 units with cytosol (5 mg/ml). By contrast, in cells that had been preincubated at 30°C, Ran by itself only weakly stimulated nuclear export (reducing the fluorescence from 87.2 to 67.4 units), whereas cytosol still stimulated export to the same extent as in cells preincubated at 0°C (reducing fluorescence from 87.2 to 28.9 U; Fig. 5, a and b). These results indicate that Ran is a rate-limiting export factor that is depleted from cells that have been preincubated at 0°C, whereas additional rate-limiting factors are depleted from the nucleus by preincubating the permeabilized cells at 30°C.
We next investigated the effect of the RanQ69L and
RanT24N mutants on nuclear export in cells that had been
preincubated at 0°C to deplete Ran only but not other
rate-limiting export factors. RanQ69L cannot hydrolyze
GTP and thus, is predominantly in the GTP-bound form
(Klebe et al., 1995). RanT24N is either in the nucleotide-free state or bound to GDP (Klebe et al., 1995
). These two
mutants are well-characterized inhibitors of nuclear protein import, both in vivo (Dickmanns et al., 1996
) and in
permeabilized cells (Palacios et al., 1996
). Interestingly, increasing concentrations of RanQ69L progressively stimulated nuclear export of GFP-NFAT in the absence of cytosol (Fig. 5 c; from 83.9 units without addition to 67.2 units with 40 µg/ml of RanQ69L), albeit to a lower extent than the stimulation obtained with cytosol (from 83.9 to
41.9 units) or with wild-type Ran (data not shown; also compare Fig. 5, a with b). However, in the presence of cytosol,
RanQ69L inhibited export from 41.9 to 68.2 units, a value
that is very similar to that observed with RanQ69L alone
(67.2 units). In cells that had been preincubated at 30°C,
RanQ69L had no effect on nuclear export in the absence
of cytosol and had an inhibitory effect in the presence of
cytosol (data not shown). RanT24N had no effect on nuclear export in the absence of cytosol and inhibited export in the presence of cytosol in cells that had been preincubated at 0°C (data not shown).
These data indicate that Ran has a dual effect on nuclear
export of GFP-NFAT in vitro. One effect reflects the ability
of GTP-bound Ran to stimulate nuclear export in the absence of nuclear protein import and of RanGTP hydrolysis,
resulting in a moderate increase in export when RanQ69L
is added to the export assay. This effect is consistent with
recent results indicating that RanGTP, but not GTP hydrolysis, is required for nuclear export of several substrates
(Izaurralde et al., 1997; Richards et al., 1997
). A second effect involves the additional stimulation of export by wild-type Ran, which allows GTP hydrolysis. We suggest that this
stimulatory effect reflects the RanGTP hydrolysis-dependent import of rate-limiting export factors into the nucleus.
This Ran-dependent import appears to be inhibited by either RanQ69L or RanT24N, resulting in an inhibition of
export by these mutants in the presence of cytosol.
Well-characterized Pathways for Mediated Nuclear Protein Import Are Not Required for Nuclear Export of NFAT In Vitro
Since our data indicate that our standard nuclear export
assay requires the nuclear import of export factors by a
Ran-dependent-mediated pathway, we analyzed whether
the well-characterized pathways for import of proteins
containing either a basic amino acid-rich NLS or the M9-type NLS are involved. To address this we performed kinetic competition experiments with either classical NLS-BSA or GST-M9 transport substrates, and also carried out
antibody inhibition experiments to inactivate importin ,
which is involved in import of proteins containing basic
amino acid-type NLSs (refer to Introduction).
Fig. 6 a shows that a high concentration of NLS-BSA inhibited the nuclear import of Cy5-NLS-BSA (from 100 fluorescent to 18 units, with a 0°C background of 14 units).
In contrast, nuclear export was not significantly affected
by NLS-BSA (resulting in 27 units in the presence of
NLS-BSA, as compared with 23.6 units without NLS-BSA).
We next examined the effects of a monoclonal antibody
against importin that inhibits nuclear import of NLS-containing substrates (Chi et al., 1995
). In the presence of
cytosol, this antibody reduced the level of import of Cy5-
NLS-BSA into the nucleus approximately fourfold without affecting export of GFP-NFAT (data not shown). In
the presence of a nuclear extract, which supports import
to a much lower extent than cytosol (see above), the anti-importin
antibody completely blocked import (Fig. 6 b):
fluorescence was reduced from 41.6 units with buffer to
16.1 units with antibody; a 0°C control resulted in 16.3 units.
Again, the antibody had only a minor effect on the nuclear
export of GFP-NFAT (30.2 units with antibody compared
with 25.4 units with buffer).
|
Similar to the results with NLS-BSA, unlabeled GST-M9 competed for the import of Cy5-GST-M9 (Fig. 6 c). The fluorescence was reduced from 100 to 45.6 units with a 0°C background of 23.2 units (in this case the applied concentration of GST-M9 was not high enough to achieve complete inhibition). In contrast, the export of GFP-NFAT remained essentially unchanged under these conditions (resulting in a fluorescence of 17.8 units with competitor, compared with 16.9 units without competitor). These results indicate that the nuclear protein import pathways specified by the two well-characterized NLSs are not involved in nuclear export of GFP-NFAT. Thus, the nuclear export factors provided by exogenously added cytosol or nuclear extract appear to reenter the nucleus by a nonconventional-mediated import pathway that is dependent on Ran.
CRM1 Is a Major Cytosolic Export Factor In Vitro
Since Ran alone was unable to support export after preincubation at 30°C to the same extent as cytosol (refer to
Fig. 5, a and b), we carried out biochemical fractionation
of cytosol to isolate a major activity other than Ran that
promotes nuclear export in our assay. As an initial purification step, we enriched approximately half of the export
activity by a selective ammonium sulfate precipitation step
(data not shown; refer to Materials and Methods). The
sample from ammonium sulfate precipitation then was
loaded onto a Mono Q ion exchange column and then individual fractions were tested for their ability to stimulate
nuclear export in our standard assay in the presence of an
excess of exogenous Ran (Fig. 7 a). A single strong peak of
export-stimulating activity was obtained from this column.
Fraction 22, which had the highest activity, reduced the
nuclear fluorescence from a background of 69.2 to 39.2 units. A saturating amount of total cytosol supported nuclear export to the same extent as fraction 22 (data not
shown). Since CRM1 has been implicated as an export receptor for certain leucine-rich NESs (refer to Introduction),
we also analyzed the column fractions for the presence of
CRM1 using immunoblotting (Fig. 7 a, inset). Interestingly, the peak of export activity in this column precisely
coincided with the elution profile of CRM1. The peak fraction from the Mono Q column was subsequently chromatographed onto a gel filtration column, fractions were tested for their ability to stimulate nuclear export under standard
conditions in the presence of excess Ran, and were also immunoblotted to detect CRM1 (Fig. 7 b). A single peak of export activity was detected in this column profile, and again,
the peak coincided with the elution of CRM1. The export
activity peak and CRM1 eluted at the position of a monodisperse protein at ~120 kD (Fig. 7 b, inset), a size very similar to the calculated molecular mass (123 kD) of CRM1
(Fornerod et al., 1997a). Fraction 13, which had the highest activity, stimulated nuclear export of NFAT from 63.8 to 47.3 units. In comparison, cytosol reduced the nuclear
fluorescence to 23.4 units in this assay. As shown by silver
staining, CRM1 appeared to be the only protein in the
most active fractions (Fig. 7 c). In other experiments, we were
able to recover larger amounts of export activity (and also of
CRM1 antigen) by including 100 µg/ml BSA in the gel filtration column buffer. With Ran and the CRM1 obtained in
this preparation, we obtained a level of nuclear export that
was comparable to that seen with cytosol (data not shown).
|
Fig. 7 d depicts the export activity of a partially purified CRM1 fraction in the absence and presence of exogenous Ran. Whereas little export was obtained with CRM1 alone or with Ran alone, strong stimulation was obtained with a combination of CRM1 and Ran (from 79.3 units of nuclear fluorescence in the absence of both proteins to 36.6 units). This demonstrates that Ran is required together with CRM1 to reconstitute in vitro export of NFAT in our standard export assay.
To further evaluate the role of CRM1 as an export factor in our assay, we analyzed the distribution of endogenous CRM1 during cell permeabilization and preincubation to determine whether its fractionation behavior
paralleled the presence of nuclear export activity (Fig. 7 e).
After digitonin permeabilization, the majority of CRM1
was recovered in the cytosolic fraction (Fig. 7 e, lane 2), although a significant amount of the protein remained in the permeabilized cells (Fig. 7 e, lane 1). Some additional
CRM1 protein was removed from permeabilized cells by a
0°C incubation (Fig. 7 e, lanes 3 and 4). However, preincubation of cells at 30°C in the absence of cytosol led to the
loss of the great majority of the remaining CRM1 from the
permeabilized cells (Fig. 7 e, lane 6), leaving only a small
amount behind (Fig. 7 e, lane 5). Thus, most CRM1 is released from the permeabilized cells during the 30°C preincubation step when the loss of export activity occurs, whereas substantial CRM1 remains after a 0°C preincubation step when the permeabilized cells still support strong
export. In further experiments, we found that leptomycin
B, a drug that inhibits the formation of a complex between
CRM1 and NES substrates and thus blocks nuclear export
in vivo (Fornerod et al., 1997b, Fukuda et al., 1997
, Ossareh-Nazari et al., 1997
), inhibited nuclear export in our in
vitro assay at submicromolar concentrations (data not
shown). Moreover, when we immunodepleted CRM1 from
cytosol, we found that the antibody-depleted cytosol had
substantially reduced nuclear export activity when compared with cytosol that had been treated with a nonspecific
IgG (data not shown). Considering these observations together with the results of our biochemical fractionation
and reconstitution studies, we conclude that CRM1 and
Ran are nucleocytoplasmic shuttling factors that are required for the nuclear export of NFAT.
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Discussion |
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An In Vitro Assay for Nuclear Export of NFAT
We have used the shuttling transcription factor NFAT as a
transport substrate to develop a rapid, quantitative in vitro
assay to study nuclear protein export. In our standard export assay, the permeabilized cells are subjected to a preincubation step at 30°C in the absence of cytosol. This step
depletes shuttling export factors from the nucleus under
conditions in which the nuclear export of GFP-NFAT is
reversibly arrested. During a subsequent incubation, efficient export of GFP-NFAT can be achieved if nuclear or
cytosolic extracts are added to the permeabilized cells. Numerous criteria demonstrate that the assay reflects physiologically relevant in vitro export of GFP-NFAT. Nuclear
export occurs in a time-, temperature-, and ATP-dependent manner, and can be blocked by WGA, a reagent that
is known to inhibit signal-mediated import and export
through NPCs (Yoneda et al., 1987; Dargemont and Kühn, 1992
). Moreover, the nuclei retain their normal diffusional
permeability barrier and can simultaneously import proteins containing NLSs.
Reimport of the GFP-NFAT into the nucleus during
the export assay, which would substantially hinder quantitative analysis of nuclear export, does not seem to occur in
our system to any significant level. This is because GFP-
NFAT becomes partially rephosphorylated during the export assay in a manner that apparently inactivates its NLSs
and traps it outside the nucleus. The activity of calcineurin, the calcium-dependent protein phosphatase that is responsible for NFAT dephosphorylation, is likely to be
inhibited by the EGTA present in our assay buffer. Consistent with this possibility is our observation that cyclosporin A, which inhibits the activity of calcineurin in
vivo (Flanagan et al., 1991; Liu et al., 1991
), has no further
stimulatory effect on nuclear export of GFP-NFAT in vitro.
Effects of the GTPase Ran on Nuclear Export
Moroianu and Blobel (1995) previously found that a protein containing a classic NLS was released from the nucleus of permeabilized cells in a Ran-stimulated fashion.
The meaning of these results is not entirely clear, because
the presence of a basic type NLS does not appear to specify shuttling between the nucleus and cytoplasm in vivo after nuclear import (Michael et al., 1995
). The GFP-NFAT that we have used as a nuclear export substrate in these
studies is a nucleocytoplasmic shuttling protein with a
well-characterized NES, and thus serves as a good model
for in vitro nuclear export studies.
Our results clearly demonstrate that the GTPase Ran is required for nuclear export of GFP-NFAT in vitro. When in vitro nuclear export of GFP-NFAT is analyzed in permeabilized cells that have not been subjected to a 30°C preincubation step, Ran alone stimulates the nuclear export of GFP-NFAT to the level achieved with cytosol. Analysis of the effects of Ran mutants in this assay suggest that Ran stimulates nuclear export by two different mechanisms that have additive effects. In the first of these mechanisms, nuclear export is stimulated by RanQ69L, which cannot hydrolyze its bound GTP, but is unaffected by RanT24N, which is either free of nucleotides or in a GDP-bound state. Thus, this mechanism appears to require GTP-bound Ran but not RanGTP hydrolysis. A second mechanism through which Ran stimulates nuclear export is seen with wild-type Ran and appears to require RanGTP hydrolysis, since it is inhibited by RanQ69L. We suggest that this requirement reflects an involvement of Ran in the reimport of CRM1 (see below) and possibly other export factors that are released from the nucleus during the preincubataion. This dual effect of Ran can explain the higher stimulation of nuclear export in the absence of cytosol by wild-type Ran versus RanQ69L.
Our observation that GTP-bound Ran but not RanGTP
hydrolysis is required for (an intermediate level) of nuclear export of NFAT is consistent to the results of in vivo
studies indicating that nuclear export of a leucine-rich
NES substrate (Richards et al., 1997) and of various RNAs
(Izaurralde et al., 1997
) requires GTP-bound Ran. This requirement for RanGTP probably relates to the cooperative binding of RanGTP with export substrate to export receptors to form putative export complexes (Fornerod et al., 1997b
; Kutay et al., 1997
).
CRM1 Is a Nucleocytoplasmic Shuttling Factor Involved in NFAT Export
With the exception of Ran, all rate-limiting factors required for efficient nuclear export of NFAT are retained
in the permeabilized cells after digitonin treatment. However, by preincubating cells at 30°C in the absence of cytosol, additional rate-limiting factor(s) are released, thereby
making the assay dependent on exogenous cytosol or nuclear extract, rather than on Ran alone. This provides the
basis for isolating these activities by biochemical fractionation. We have purified a major cytosolic activity involved
in the stimulation of nuclear export in our standard export
assay, and have found that this activity corresponds to
CRM1 (Fornerod et al., 1997b; Fukuda et al., 1997
; Ossareh-Nazari et al., 1997
; Stade et al., 1997
). Stimulation of
nuclear export by purified CRM1 depends on the presence
of Ran. This requirement probably reflects a need for Ran
for the import of CRM1 into the nucleus, as well as the involvement of Ran in the formation of a CRM1-RanGTP-NFAT complex (Fornerod et al., 1997b
) that can be exported from the nucleus (see above). Considered together,
our results involving fractionation and reconstitution contribute strong direct evidence that CRM1 functions as a
nucleocytoplasmic shuttling receptor for nuclear export of
NFAT, and complements the results of earlier in vivo studies involving temperature-sensitive mutants (Fukuda
et al., 1997
; Stade et al., 1997
) and overexpression of
CRM1 (Fornerod et al., 1997b
). The ability to reconstitute
nuclear export in this assay with CRM1 and Ran will allow
a detailed structure-function analysis of these components.
We believe that it will be possible to identify additional factors that regulate NFAT export using this assay. Indeed, we have partially purified a potent activity from cytosol that inhibits NFAT export in permeabilized cells (our unpublished observations). Additional positively acting export factors may be revealed under different preincubation and assay conditions.
Nuclear Export Factors for NFAT Enter the Nucleus by a Nonconventional Import Pathway
Shuttling nuclear export factors like CRM1 have to reenter the nucleus after one round of export. The strong inhibition of nuclear export by RanQ69L in our standard, cytosol-dependent export assay indicates that these factors
are imported into the nucleus by a Ran-dependent transport pathway. Whereas importin and transportin can be
imported into the nucleus when added to permeabilized
cells in the absence of Ran (Kose et al., 1997
; Nakielny and
Dreyfuss, 1997
), we consider it very likely that the nuclear
import of these proteins under more physiological conditions (i.e., in the presence of cytosol and Ran) involves
RanGTP hydrolysis, since they both strongly bind RanGTP
and their import (and that of their cargos) is efficiently inhibited by RanQ69L and nonhydrolyzable GTP analogues (Melchior and Gerace, 1995
; Bonifaci et al., 1997
; Izarraulde et al., 1997).
We determined that neither the importin nor transportin pathways are involved in the import of export factors (including CRM1) for NFAT in our assay because
inhibiting these pathways with an excess of competing import substrates or with a monoclonal antibody against importin
does not diminish nuclear export of GFP-NFAT.
Taken together, our results indicate that the recycling of
nuclear export factors for NFAT involves a nonconventional nuclear import pathway. If this pathway were selective for shuttling export factors, it would provide a means
to regulate nuclear export in a manner that is independent
of the nuclear protein import pathways used to transport
the bulk of nuclear proteins. Furthermore, this would allow the recycling of nuclear export factors without competition by other major import substrates. Understanding the ability of the NPC to accommodate multiple different signaling pathways for import as well as for export represents
an important future challenge.
![]() |
Footnotes |
---|
Received for publication 19 February 1998 and in revised form 3 April 1998.
Address all correspondence to Larry Gerace, Department of Cell Biology and Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. Tel.: (619) 784-8514. Fax: (619) 784-9132. E-mail: lgerace{at}scripps.eduWe wish to thank G. Crabtree for providing the cDNA clone for NFAT,
the anti-NFAT antibody, and for helpful advice, G. Grosveld for the gift
of anti-CRM1 antibody, G. Dreyfuss for providing a cDNA clone for
GST-M9, J. Becker for the RanQ69L and RanT24N proteins, C. Delphin
(Scripps Research Institute, La Jolla, CA) for importin and
, and D. Sweet (Trends in Cell Biology, Cambridge, UK) for the preparation of
NTF2. The manuscript was improved by critical comments from S. Lyman, F. Melchior, A. Kehlenbach, and C. Fritze (all four from The Scripps
Research Institute). We also gratefully acknowledge J. Hauber (Institute
for Clinical and Molecular Virology, University Erlangen, Nürnberg, Germany) for his support of this project.
This work was supported by a grant from Novartis Pharmaceuticals to L. Gerace and by fellowships from the Human Frontier Science Program to R.H. Kehlenbach (LT-400/96) and the Deutsche Forschungsgemeinschaft to A. Dickmanns (Di 676/1-1).
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Abbreviations used in this paper |
---|
FACS®, fluorescence activated cell sort(er)ing; GFP, green fluorescent protein; GSK-3, glycogen synthase kinase-3; NES, nuclear export sequence; NFAT, nuclear factor of activated T cells; NLS, nuclear localization sequence; NPC, nuclear pore complex; WGA, wheat germ agglutinin.
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