A Novel Nuclear Export Signal Sensitive to Oxidative Stress in
the Fission Yeast Transcription Factor Pap1*
Nobuaki
Kudo
,
Hiroshi
Taoka
,
Takashi
Toda§,
Minoru
Yoshida
¶, and
Sueharu
Horinouchi
From the
Department of Biotechnology, Graduate School
of Agriculture and Life Sciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113, Japan and the § Laboratory of Cell
Regulation, Imperial Cancer Research Fund,
London WC2A 3PX, United Kingdom
 |
ABSTRACT |
Pap1, a fission yeast AP-1-like transcription
factor, is negatively regulated by CRM1/exportin 1, the nuclear export
factor. Pap1 was localized normally in the cytoplasm but was
accumulated in the nucleus when Crm1 was inactivated by a
temperature-sensitive mutation or by treatment with leptomycin B, a
specific export inhibitor. Deletion of the C-terminal cysteine-rich
domain (CRD) resulted in nuclear accumulation of Pap1, while a
glutathione S-transferase-green fluorescent protein-CRD
fusion protein was localized in the cytoplasm in a
Crm1-dependent manner. Deletion and mutational analyses
identified several important amino acids in a 19-amino acid region in
the CRD as a nuclear export signal (NES). Strikingly, a cysteine
residue (Cys-532), in addition to two leucines and an isoleucine, was
important for the NES function and the presence of at least one of the
two cysteine residues was essential. Unlike classical NESs such as the
human immunodeficiency virus Rev NES, the Pap1 NES lost the function
upon treatment with oxidants such as diethyl maleate. The oxidative
stress response is conserved through evolution, as green fluorescent
protein-fused proteins bearing the Pap1 NES expressed in mammalian
cells responded to diethyl maleate. These results show that the
hydrophobic amino acid-rich region containing two important cysteines
in Pap1 serves as a novel NES, which is sensitive to oxidative stress.
 |
INTRODUCTION |
A nuclear export signal
(NES),1 a short leucine-rich
sequence, is a transport signal that is necessary and sufficient to
mediate nuclear export of proteins (1, 2). Many proteins including human immunodeficiency virus (HIV) Rev (3), protein kinase inhibitor
(4), and MAP kinase kinase (5) have been reported to be spatially
controlled by their NESs. Recently, CRM1/exportin 1 was shown to be a
receptor for the NES in both lower and higher eukaryotes (6-9).
CRM1/exportin 1 shares low but distinct homology with importin
that
recruits the complex of importin
and the nuclear localization
signal (NLS)-containing proteins to the nuclear pore complex (NPC) by
directly interacting with the NPC (10, 11). CRM1/exportin 1 conveys
NES-bearing proteins through the NPC in a Ran-GTP-dependent
manner. Genetic alterations in the CRM1 locus caused a defect in
nuclear export of NES-bearing proteins in yeast (6, 8, 12). Nuclear
microinjection of a specific antibody to CRM1 that prevents the
in vitro NES binding inhibited in vivo protein
nuclear export in mammalian cells (13). Thus, the NES-mediated nuclear
export of proteins is a universal and conserved mechanism by which
subcellular localization of proteins is controlled in cells.
CRM1/exportin 1 was originally identified as an essential protein
controlling chromosome structure in fission yeast
Schizosaccharomyces pombe (14). Temperature- or
cold-sensitive mutations of S. pombe Crm1 caused abnormal
chromosome morphology and other pleiotropic phenotypes such as
resistance to several structurally unrelated antifungal agents and
overproduction of p25Apt1, a functionally unknown
flavoprotein. At least some of these phenotypes are mediated by
activation of Pap1, an S. pombe bZip (basic leucine-zipper)
DNA binding motif-containing protein that has homology with and binding
specificity similar to the mammalian c-Jun, a component of the AP-1
transcription factor (15). Pap1 is involved in oxidative stress
response, heavy metal detoxication, and multidrug resistance. It
controls the expression of the following stress-induced genes:
ctt1+ for catalase (16),
trx2+ for thioredoxin (17),
trr1+ for thioredoxin reductase (18),
pgr1+ for glutathione reductase (19), and
hba2+/bfr1+ and
pmd1+ for ABC transporters responsible for
multidrug resistance (20-22). Chromosomal deletion of
pap1+ resulted in increased sensitivity to a
variety of toxic compounds and loss of p25Apt1 production
(23, 24). We previously showed that a mutation of S. pombe
crm1+ conferred resistance to leptomycin B (LMB) (25),
which had been discovered as a potent antifungal antibiotic blocking
the eukaryotic cell cycle (26, 27). We proposed that S. pombe Crm1 is the cellular target of LMB, which was evidenced by
the identical phenotypes between crm1 mutant cells and
LMB-treated wild-type cells (25). Recently, Wolff et al.
(28) rediscovered LMB as an agent that inhibits the nuclear export of
HIV-1 Rev. We and others found that LMB abolishes association of
CRM1/exportin 1 with the NES by directly binding to CRM1/exportin 1, thereby inhibiting nuclear export of proteins (7-9, 13). These
results, together with the finding that LMB causes p25Apt1
overproduction (25), raise a possibility that CRM1/exportin 1 negatively regulates Pap1 activity by exporting it from the nucleus to
the cytoplasm.
Budding yeast Saccharomyces cerevisiae contains two AP-1
homologs, Yap1 and Yap2 (29-33). Yap1 acts as a major transcription factor that induces expression of various genes required for stress response or drug resistance in response to oxidative stress generated by hydrogen peroxide, diamide, and diethyl maleate (DEM) (34-37). The
Yap2 function may be redundant to that of Yap1 at least in some
response pathways, although YAP2 deletion had no effect on some of the Yap1 target genes (31, 32, 34). These transcription factors
show significant homology with Pap1 in the two distinct regions, the
bZip domain and the C-terminal cysteine-rich domain (CRD) (15).
Recently, Kuge et al. (38, 39) showed that the Yap1 CRD was
responsible for the CRM1/exportin 1-dependent cytoplasmic localization of Yap1, which may be important for sensing the redox state of the cell. These observations led us to assume that the Pap1
CRD also regulates the subcellular localization in response to
oxidative stress.
Most recently, Toone et al. (17) reported that Pap1
relocalizes from the cytoplasm to the nucleus in a process that is
dependent on Sty1, a fission yeast stress-activated MAP kinase
(40-42). This relocalization appears to result from regulated protein
export by CRM1/exportin 1 and a Ran nucleotide exchange factor. In this report, we demonstrate that the CRD of Pap1 contains an NES.
Interestingly, the Pap1 NES, an atypical NES consisting of a
leucine-rich sequence with an important cysteine residue, is functional
in not only yeast but also animal cells and is sensitive to oxidative
stress. The novel NES of Pap1 highlights a mechanism of stress response in eukaryotes.
 |
MATERIALS AND METHODS |
Yeast Strains, Media, and Chemicals--
S. pombe
strains used in this study were 972 (h
), JY266
(h+ leu1-32), TP17-13C
(h
leu1-32 crm1-1R), and
TP108-3C (h
leu1-32 ura4
pap1::ura4+). The S. pombe strains were grown in complete YPD medium (1% yeast
extract, 2% polypeptone, and 2% glucose) or minimal medium (43),
which was used for plasmid selection and induction of the
nmt1 promoters. For suppression of the nmt1
promoters of pREP plasmids, thiamine hydrochloride was added to minimal
medium at the final concentration of 50 µg/ml. LMB was prepared as
described (26). Diethyl maleate was purchased from Sigma.
Plasmid Constructions--
pREP1, pREP41, and pREP81 were used
for controlled expression of various proteins in S. pombe
cells. pREP1 contains the wild-type nmt1 promoter, which can
strongly direct transcription when thiamine is absent in the medium
(44). pREP41 and pREP81 possess mutations in their promoter sequences,
which result in the moderate and weak promoter activities, respectively
(45). Plasmids for expressing various GFP-fused proteins in S. pombe were constructed on the basis of pR1GF1 (13) and pR1F1. A
DNA fragment encoding EGFP ORF was amplified by PCR using
synthetic oligonucleotides
(5'-GGCAGATCTCTCATATGGCTAGCATGGTGAGCAAGGGCGAGGAG-3' and
5'-GCCCCCGGGTCAATCTCGAGGGATCCCCTTGTACAGCTCGTCCATGCC-3') as the primers
and pEGFP-N1 (CLONTECH) as a template. pR1F1 was
constructed by inserting the fragment between NdeI and
SmaI sites on pREP1. pR1FPA1 and pR1FPA2 for GFP fused with
full-length Pap1 and CRD-deleted Pap1 (represented as
CRD, residues
1-483), respectively, were constructed by inserting the PCR-amplified
fragments obtained using pST1 (15) as a template into the
BamHI-XhoI site on pR1F1, resulting in the
connection at the 3'-end of EGFP ORF. Synthetic oligonucleotides used
for pR1FPA1 were 5'-CGGCTCGAGGGGATCCAGATGTCCGGACAAACTGAGACGTTG-3' (pap1-1) and 5'-CGGCTCGAGCCCGGGTCAGCTAGCACATCGTCTTTCATCGAGTAATAC-3' (pap1-a) whereas those for pR1FPA2 were pap1-1 and
5'-CGGCTCGAGCCCGGGTCAGCTAGCTTGAGAACTAGTATCATGTTTACC-3' (pap1-b). In
pR1FPA1, pR1FPA2, and pR1GFPA4 (below), an NheI site was
created at the end of the coding sequences, resulting in the addition of residues of an alanine and a serine at the C-terminal ends
of the fusion proteins. To construct pR1GFPA4, -5, and -6 for the
expression of GST-GFP fused with the CRD and its subdomains, we
inserted fragments amplified by PCR into the
BamHI-XhoI site on pR1GF1. The primers used were
5'-CCTGGATCCCTAATGAAATCGTTCCGGCCAAGGAACG-3' (pap1-3) and pap1-a for
pR1GFPA4; pap1-3 and 5'-CCGCTCGAGTCAATCATCAATGTCGAAACTCTC-3' (pap1-k)
for pR1GFPA5 and 5'-CCTGGATCCCTGCATACCTCAGCTGCCCCAAGG-3' (pap1-10) and
5'-CCGCTCGAGTCATACACCCGAAGAAGAACATTTAGC-3' (pap1-j) for pR1GFPA6.
pR1GFPA7, -8, -9, -10, and -11 were constructed by inserting DNA
fragments composed of annealed synthetic oligonucleotides into the same
site on pR1GF1. Oligonucleotides used were
5'-GATCCCTTCAAAGATTATCAATCACCCTCGATTTGAGAGTTTC-3' (pap1-8),
5'-GACATTGATGATTTGTGTAGCAAGTTGAAGAATTGAC-3' (pap1-9), 5'-CGAGTCAATTCTTCAACTTGCTACA-3' (pap1-g),
5'-CAAATCATCAATGTCGAAACTCTCAAATCG-3' (pap1-h) and
5'-AGGGTGATTGATAATCTTTGAAGG-3' (pap1-i) for pR1GFPA7; 5'-GATCCCTGAGAGTTTCGACATTGATGATTTGTGTAGCAAGTTGAAG-3' (pap1-6), 5'-AATAAAGCTAAATGTTCTTCTTCGGGTGTATGAC-3' (pap1-7),
5'-TCGAGTCATACACCCGAAGAAGA-3' (pap1-d),
5'-ACATTTAGCTTTATTCTTCAACTTGCTACA-3' (pap1-e) and
5'-CAAATCATCAATGTCGAAACTCTCAGG-3' (pap1-f) for pR1GFPA8;
5'-GATCCCTGAGAGTTTCGACATTGATGATTTGTGTAGCAAGTTGAAGAATTGAC-3' (pap1-4)
and 5'-TCGAGTCAATTCTTCAACTTGCTACACAAATCATCAATGTCGAAACTCTCAGG-3' (pap1-c) for pR1GFPA9;
5'-GATCCCTCACCCTCGATTTGAGAGTTTCGACATTGATGATTTGTGTAGC-3' (pap1-11),
5'-AAGTTGAAGAATAAAGCTAAATGTTCTTGAC-3' (pap1-12),
5'-TCGAGTCAAGAACATTTAGC-3' (pap1-m),
5'-TTTATTCTTCAACTTGCTACACAAATCATC-3' (pap1-n) and
5'-AATGTCGAAACTCTCAAATCGAGGGTGAGG-3' (pap1-o) for pR1GFPA10 and
5'-GATCCCTGAGAGTTTCGACATTGATGATTTGTGTAGC-3' (pap1-12),
5'-GATCCCTGAGAGTTTCGACATTGATGATTTGTGTAGC-3' (pap1-13), pap1-m, pap1-n
and 5'-AATGTCGAAACTCTCAGG-3' (pap1-p) for pR1GFPA11. To construct the
plasmids for expressing GST-GFP-fused PA11 with single or double
mutation(s), the same strategy for constructing pR1GFPA11 was
employed using pap1-12, pap1-13, pap1-m, pap1-n, and pap1-p
containing base exchanges that cause amino acid substitutions. For
creating mutations into alanine, serine, and leucine, the corresponding
codons were exchanged by GCT, TCT, and TTA, respectively. The annealed
synthetic DNA fragments encoding PA11 and its mutants were also
inserted into the BamHI-XhoI site on pR1GsvNLSF1
(12), for constructing the expression plasmids for GST-SV40 T-antigen NLS-GFP-fused PA11 and its derivatives. pR1GrevNESF1 for GST-Rev NES-GFP was described previously (12). pR1FPA2NES1 for expressing GFP-fused CRD-deleted Pap1 containing the Rev NES was constructed by
inserting annealed synthetic DNAs,
5'-CTAGCTTGCCTCCCTTAGAGCGTTTAACTCTAGACTGCTGAC-3' and
5'-TCGAGTCAGCAGTCTAGAGTTAAACGCTCTAAGGGAGGCAAG-3', into the NheI-XhoI site of pR1FPA2. To express Pap1 or
CRD-deleted Pap1 (
CRD)without GFP in S. pombe, we
constructed pR1PA1, pR41PA1, pR81PA1, pR1PA2, pR41PA2, and pR81PA2 by
inserting the DNA fragments corresponding to Pap1 ORF excised from
pR1FPA1 and pR1FPA2 into the BamHI-SmaI site of
pREP1, pREP41, and pREP81, respectively. pE16GPA4F1 for expressing
GST-CRD-GFP by T7 promoter in Escherichia coli was
constructed as follows: Multiple cloning sites were created between
NdeI and BamHI sites of pET16b (Novagen) using
annealed synthetic DNAs
(5'-TATCGAGCTCCATATGGGGATCCAGACTAGTGCTCGAGCCCGGGTGACTGACTGAC-3' and
5'-GATCGTCAGTCAGTCACCCGGGCTCGAGCACTAGTCTGGATCCCCATATGGAGCTCGA-3'). DNA
fragments encoding GST and GFP amplified by PCR using pGEX-5X (Amersham
Pharmacia Biotech) as the template and primers
(5'-GGCGAGCTCATGTCCCCTATACTAGGTTATTGG-3' and
5'-GCCCCATATGACGACCTTCGATCAGATCCGATTT-3') for GST, and pS65T (CLONTECH) as the template and primers
(5'-GCGCTCGAGCCCGGGATATGGGTAAAGGAGAAGAACTTTTC-3' and
5'-CTACTTGTATAGTTCATCCATGCCATG-3') for GFP were inserted into the
SacI-NdeI and XhoI-SmaI
sites, respectively. The plasmid thus obtained was digested by
BamHI and SpeI and ligated with the DNA fragment
encoding CRD obtained from digestion of pR1FPA4 by BamHI and
NheI, resulting in the construction of pE16GPA4F1. The
mammalian expression plasmids for various GFP-fused proteins were
constructed as follows. We inserted the NheI-XhoI
fragments excised from pR1FPA1, pR1FPA2, pR1GFPA4, and pR1GFPA11 into
the NheI-XhoI site of pEGFP-C1 (CLONTECH) to construct pFPA1 for GFP-Pap1, pFPA2
for GFP-
CRD, pFPA4 for GFP-CRD, and pFPA11 for GFP-PA11. pFRevNES1
for GFP-Rev NES was constructed by inserting the annealed synthetic
DNAs (5'-TCGAGGCTTGCCTCCCTTAGAGCGTTTAACTCTAGACTGCTGAG-3' and
5'-GATCCTCAGCAGTCTAGAGTTAAACGCTCTAAGGGAGGCAAGCC-3') into the XhoI-BamHI site of pEGFP-C1. Nucleotide
sequences of all the cloned DNA fragments mediated by PCR and by
annealing of synthetic DNAs were confirmed.
Observation of Subcellular Localization of GFP-fused Proteins in
S. pombe--
Various plasmids for expression of GFP-fused proteins
were introduced into S. pombe TP108-3C
(pap1
strain) or TP17-13C
(crm1ts strain). The fused proteins were
expressed by cultivating the transformants in the absence of thiamine
for about 12-15 h at 30 °C (pap1
) or
26.5 °C (crm1ts), and then subcellular
distributions of the proteins were observed under a Zeiss Axiophot-2
fluorescent microscope. The effects of LMB and DEM were analyzed by
adding the drugs to the above cultures at a final concentration of 50 ng/ml for LMB and 2 mM for DEM, respectively, and
cultivating for an additional 0.5-6 h. Periods for drug treatment
depended on the proteins, since the proteins containing potential NLS
such as the full-length Pap1 or the
CRD fusion rapidly relocalized
into the nucleus (0.5 h for LMB and 1 h for DEM), while other
proteins without NLS showed relatively slow nuclear translocation (3 h
for LMB and 4-6 h for DEM), probably due to the passive diffusion. To
see the effect of the crm1 mutation, we kept the above
culture of crm1ts at a restrictive temperature
(37 °C) for 2-6 h. Location of the nucleus was determined by
staining the cells with DAPI.
Mammalian Cell Culture, Microinjection, and Transfection--
A
human carcinoma cell line (HeLa) and a murine fibroblast cell line
(NIH3T3) were cultured at 37 °C under a humidified atmosphere of 5%
CO2 in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum. GST-CRD-GFP fusion protein was expressed in
E. coli BL21 (DE3) harboring pE16GPA4F1 and purified by
glutathione-Sepharose 4B (Amersham Pharmacia Biotech). HeLa cells grown
on glass coverslips were pretreated with or without 10 ng/ml LMB for
1.5 h, and were then injected with the purified GST-CRD-GFP
protein using a Micromanipulator 5171 and a Transjector 5246 (Eppendorf) with glass capillary needles under a Zeiss Axiovert 135 microscope. Successful injection was monitored by co-injection with
tetramethylrhodamine isothiocyanate (TRITC)-labeled dextran
(Mr 70,000, Molecular Probes). After
microinjection, the cells were incubated for 1 h at 37 °C or
4 °C, and then fixed with 3.7% paraformaldehyde in
phosphate-buffered saline for 10 min. Fluorescence of GFP and TRITC was
observed under a Zeiss Axiophot-2 fluorescent microscope. Transfection
of NIH3T3 cells with pFPA1 for expression of GFP-fused Pap1, pFPA2 for
GFP-
CRD, pFPA4 for GFP-CRD, pFPA11 for GFP-PA11, and pFRevNES1 for
GFP-Rev NES was performed with LipofectAMINE (Life Technologies, Inc.) according to the furnished instructions. The cellular distribution of
GFP-fused proteins was observed under a Zeiss Axiovert 135 fluorescent microscope.
Preparation of S. pombe Cell Extracts and Western
Blotting--
S. pombe TP108-3C
(pap1
) was transformed with pR1PA1,
pR41PA1, and pR81PA1 for expression of full-length Pap1 or pR1PA2,
pR41PA2, and pR81PA2 for CRD-deleted Pap1 (
CRD). For empty vector
control, pREP1 was used. The transformants were cultured overnight at
30 °C in minimal medium supplemented with 50 µg/ml thiamine and
transferred to the medium without thiamine for expression from the
nmt1 and its derivative promoters. After cultivation for
14 h at 30 °C, cells grown exponentially were cultured for an
additional 2 h in the presence or absence of 50 ng/ml LMB. For
identification of the stimuli that activate Pap1-dependent
gene expression, exponentially growing S. pombe 972 (wild-type) cells cultured for 10 h in minimal medium were
subjected to the following stress conditions for 6 h: 2 mM DEM, 2 mM hydrogen peroxide, 0.4 mM menadione, 1.5 mM diamide, 2 µg/ml
staurosporine, 50 µg/ml cycloheximide, 5 µg/ml brefeldin A, 10 mM caffeine, 0.5 M KCl, and 50 ng/ml LMB. These cells were harvested, and whole cell extracts were prepared as follows.
The cells (about 30 µl wet volume) were freeze-thawed, suspended with
150 µl of extraction buffer (20 mM Tris-Cl, pH 7.5, 1 mM EDTA, 5 mM dithiothreitol, 5 mM
MgCl2, 50 mM KCl, 5% glycerol, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, and 0.6 µg/ml pepstatin
A), and disrupted with glass beads at 4 °C. Supernatants obtained by
centrifugation at 15,000 × g for 10 min at 4 °C
were used as whole cell extracts. The protein concentrations of the extracts were measured by Bradford's method. Proteins (0.5 µg of
total protein) of the whole cell extracts were separated on a 12.5%
SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride
membrane. The blot was incubated overnight with a diluted (1:500)
anti-p25Apt1 rabbit antiserum (23) at 4 °C.
p25Apt1 was detected with an ECL system (Amersham Pharmacia
Biotech) using horseradish peroxidase-conjugated anti-rabbit Ig.
 |
RESULTS |
The C-terminal CRD Is Responsible for Nuclear Export of
Pap1--
Pap1 has significant homology with Yap1 and Yap2 in two
distinct domains, bZip in the N-terminal region and the cysteine-rich domain (CRD) at the C terminus (Fig.
1A). The Yap1 CRD has been shown to be responsible for its cytoplasmic localization and
autonomously negative regulation (38, 46). To test whether the Pap1 CRD (residues 488-544) is also important for its subcellular localization, we expressed three GFP fusion proteins, GFP-Pap1 (F-Pap1), GFP-Pap1 lacking the CRD (F-
CRD), and GST-GFP-CRD (G-F-CRD) in
crm1+ cells in the absence of endogenous Pap1
(pap1-). The full-length Pap1 localized in the
cytoplasm (Fig. 1B) but was not detected in the nucleus. On
the other hand, CRD-deleted Pap1 was observed as single spots that
colocalized with DAPI-stained nuclei. GST-GFP-CRD was present in the
cytoplasm, indicating that the CRD is sufficient for the Pap1
cytoplasmic localization. If the cytoplasmic localization is due to the
presence of an NES but not a cytoplasmic retention signal, then LMB, a
specific nuclear export inhibitor, would disrupt the cytoplasmic
localization of Pap1. When the cells expressing GFP-Pap1 were exposed
to 50 ng/ml LMB for 3 h, Pap1 relocalized to and accumulated in
the nucleus (F-Pap1), although the nuclear localization of CRD-deleted
Pap1 was unaffected (F-
CRD). Furthermore, GST-GFP-CRD was
redistributed in both the cytoplasm and the nucleus upon LMB treatment.
Since GST-GFP without any signal expressed in S. pombe was
diffused throughout the cell probably due to passive diffusion through the NPC (12, 13), it is suggested that the CRD contains an NES.
Essentially the same results were obtained with
pap1+ cells (JY266; data not shown).

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Fig. 1.
Requirement of Crm1 for regulated subcellular
localization of Pap1. A, schematic representation of
GFP-fused Pap1 and its derivatives that are expressed under the control
of the wild-type nmt1 promoter. B, subcellular
localization of Pap1 (F-Pap1), CRD-truncated Pap1 (F- CRD), and CRD
(G-F-CRD). S. pombe TP108-3C
(pap1 ) and S. pombe TP17-13C
(crm1ts) were transformed with pR1FPA1 for
F-Pap1, pR1FPA2 for F- CRD, and pR1GFPA4 for G-F-CRD. The fusion
proteins were expressed by transferring to thiamine-depleted medium and
culturing for 12-15 h at 30 °C for pap1 or
26.5 °C for crm1ts transformants. Changes in
the localization of the fusion proteins in response to LMB were
observed by fluorescence microscopy (GFP) after treating the
pap1 transformants with 50 ng/ml LMB for
0.5 h (F-Pap1 and F- CRD) or 3 h (G-F-CRD). The effect of
crm1 mutation was also analyzed by culturing the
crm1ts transformants at 37 °C for 2 h
(F-Pap1 and F- CRD) or 6 h (G-F-CRD). Differences in time for
the treatment depends on the different import rates of each protein.
Nuclear DNA in the same microscopic fields was visualized by staining
with DAPI (DNA). Abnormality in the nuclear morphology was observed
when crm1+ cells were treated with LMB or
crm1ts cells were cultured at the nonpermissive
temperature for longer than 3 h, as described previously (14,
25).
|
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To verify the involvement of CRM1/exportin 1 in the Pap1 cytoplasmic
localization further, we expressed the same fusion proteins in
ts crm1 mutant cells (crm1-1R) (24), and
analyzed the effect of the deficiency in Crm1 at a nonpermissive
temperature (37 °C) on their subcellular localization. Again, Pap1
was present only in the nucleus as was CRD-truncated Pap1, and
GST-GFP-CRD became present in both the cytoplasm and the nucleus. These
results indicate that the Pap1 CRD contains an NES that is exported by
a mechanism dependent on CRM1/exportin 1. Constitutive nuclear
localization of CRD-deleted Pap1 suggests that Pap1 contains a nuclear
localization signal in the region other than the CRD. These
observations suggest that Pap1 normally shuttles between the nucleus
and the cytoplasm by its NLS and NES.
The Pap1 CRD Can Be Exported in Mammalian Cells--
Mammalian or
viral NES can work in yeast (6, 12). CRM1 and Ran are required for
nuclear export in both yeast and mammalian cells, indicating that the
mechanism for protein nuclear export is highly conserved in eukaryotes
(47, 48). To test whether the Pap1 CRD is also exported in mammalian
cells, we injected the bacterially produced GST-CRD-GFP (G-CRD-F)
fusion protein into the HeLa cell nuclei and observed its location
(Fig. 2). GST-CRD-GFP was rapidly
exported from the nucleus to the cytoplasm. This transport was
completely blocked by pretreatment with 10 ng/ml LMB for 1.5 h.
The active nuclear export is dependent on energy and is inhibited by
cold treatment (3). When the cells were incubated at 4 °C after
injection, GST-CRD-GFP mostly remained in the nucleus. These results
indicate that the Pap1 CRD, like other NESs, is functional in both a
CRM1-dependent manner and an energy-dependent
manner in mammalian cells.

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Fig. 2.
Nuclear export of CRD in mammalian
cells. A fusion protein, GST-CRD-GFP (G-CRD-F) was produced by
expressing in E. coli harboring pE16GPA4F1, and was
affinity-purified. The purified protein was injected together with
TRITC-labeled dextran into the nuclei of HeLa cells preincubated with
(+LMB) or without ( ) 10 ng/ml LMB for 1.5 h. The
localization of injected protein was observed by fluorescence
microscopy (GFP panels) after 1 h incubation at
37 °C or 4 °C following injection. The TRITC
panels indicate the injection sites.
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Effect of Pap1 Nuclear Translocation on Target Gene
Expression--
To determine the role of the CRD NES in regulation of
Pap1-directed gene expression, we expressed full-length or CRD-deleted Pap1 using three thiamine-repressive promoters with different promoter
activities in the pap1
background. The
transcriptional activity directed by Pap1 was examined by monitoring
the amount of p25Apt1, one of the Pap1 target gene products
(23). As we reported previously, LMB treatment caused increased
expression of p25Apt1 in wild-type cells (Fig.
3, lanes 1 and
2). On the other hand, LMB did not induce
p25Apt1 expression in the absence of Pap1, suggesting that
Pap1 is essential for p25Apt1 expression (lanes
3 and 4). p25Apt1 became detectable
when full-length Pap1 was expressed from the moderate or strong
promoter on the expression vector (lanes 5-7). When these cells were treated with LMB for 3 h,
p25Apt1 was detectable, even in the presence of a small
amount of Pap1 expressed from the weak promoter, and this increased in
a Pap1 dose-dependent manner (lanes
8-10). On the other hand, cells expressing CRD-deleted Pap1
always produced a plateau level of p25Apt1 regardless of
the differences in the promoter activity or LMB treatment
(lanes 11-16). These results imply that the Pap1
transcriptional activity is negatively regulated by the NES present in
the CRD and that nuclear translocation is sufficient to induce the
target gene expression.

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Fig. 3.
Effects of Pap1 and CRD-truncated Pap1
expressed by nmt1 promoters on the production of
p25Apt1, a Pap1-target gene product. The expression
level of Pap1 and CRD-truncated Pap1 ( CRD) was controlled by a
series of plasmids containing nmt1 promoters, pREP81
(lanes 5, 8, 11, and
16), pREP41 (lanes 6, 9,
12, and 15), and pREP1 (lanes
7, 10, 13, and 16).
S. pombe TP108-3C was transformed with the following
plasmids: pREP1 for empty vector control (lanes 3 and 4), pR81PA1 (lanes 5 and
8), pR41PA1 (lanes 6 and
9), and pR1PA1 (lanes 7 and
10) for Pap1, and pR81PA2 (lanes 11 and 14), pR41PA2 (lanes 12 and
15) and pR1PA2 (lanes 13 and
16) for CRD. Total proteins isolated from each of the
indicated strains treated with or without LMB (50 ng/ml) were separated
on SDS-polyacrylamide gels and analyzed with Western blotting using the
anti-p25Apt1 antibody. The amounts of p25Apt1
in pap1+ cells (WT) cultured in the
absence or presence of LMB were also shown (lanes
1 and 2).
|
|
Identification of an NES in the Pap1 CRD--
To determine the
region sufficient for the NES activity in the CRD, we fused various
lengths of the CRD sequence to GST-GFP and expressed them in S. pombe. Localization of these fusion proteins were examined in the
presence or absence of LMB (Fig. 4). The protein containing a 19-amino acid region named PA11 was localized in
the cytoplasm. LMB treatment caused diffused distribution of this
protein in both the cytoplasm and the nucleus (Figs. 4 and 5B). These results indicate
that the Pap1 NES is present within PA11.

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Fig. 4.
Schematic representation of alignment of the
CRD sequences of Yap1, Yap2, and Pap1; structures of the deletion
mutants of the Pap1 CRD; and the subcellular localization of the
mutants fused with GST-GFP, which are expressed in S. pombe. Localization of the proteins was determined by
fluorescence microscopy. C and N represent the
cytoplasm and the nucleus, respectively. Asterisks indicate
the C termini of proteins.
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Fig. 5.
Identification of an NES in the Pap1
CRD. A, alignment of PA11 and the corresponding regions
of the Yap1 and Yap2 CRD and site-directed mutagenesis of PA11. The
identical amino acids are emphasized by shaded
boxes. Amino acids conserved among three proteins are marked
by asterisks. Amino acids conserved between two of three
proteins are marked by closed circles. In
particular, they are marked by double closed
circles when those of two of three proteins are identical and the other
one is also similar. Amino acids not identical among the three but all
hydrophobic are indicated by an open circle. The
amino acids substituted in various mutants are indicated below the
sequences. The ability of nuclear export of a variety of PA11 mutants
was analyzed by examining the localization of both GST-GFP-fused PA11
derivatives (G-F-PA11 mutants) and GST-T NLS-GFP-fused derivatives
(G-L-F-PA11 mutants) containing an NLS of SV40 large T-antigen (13),
which were expressed in the pap1 disruptant.
Crm1-dependent nuclear export was confirmed by detecting
nuclear translocation upon LMB treatment. DEM response was also
analyzed by observing the localization of these mutant fusion proteins
after treatment with 2 mM DEM for 6 h. Response (yes)
indicates the DEM-dependent nuclear translocation.
nd, not determined. B, subcellular localization
of PA11 and its mutants. The localization in S. pombe of the
GST-GFP-PA11 and its derivatives, L522A, C523S, C532S, and C523S/C532S,
was shown. The cytoplasmic localization of G-F-PA11 was completely
disrupted by the L522A and C523S/C532S mutations, fairly impaired by
the C532S mutation, and unaffected by the C523S mutation.
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|
The amino acid sequence of PA11 is very similar to the corresponding
regions of Yap1 and Yap2 (Fig. 4). To determine the amino acid residues
responsible for the nuclear export, we introduced single or double
amino acid substitutions into 14 conserved amino acids, and examined
the effects on its NES activity of PA11. As shown in Fig.
5A, the nuclear export of PA11 was impaired by either L522A
or L526A mutation, and the mutant proteins were relocalized in both the
cytoplasm and the nucleus (Fig. 5B, G-F-PA11-L522A; and not
shown for L526A). In addition, the export activity of I519A- or
C532S-containing protein was greatly reduced. However, a single
mutation at other conserved amino acid residues did not affect the
nuclear export of PA11 (Fig. 5A). These results indicate that three hydrophobic amino acid residues (Ile-519, Leu-522, and
Leu-526) and Cys-532 are important in the Pap1 NES. To characterize the
Pap1 NES further, we tested the export activity of several double
mutants of PA11, namely E515A/S516A, F517A/I519A, F517L/I519L, D518A/D520A, C523S/C532S, and K529A/K531A. Among these, the cytoplasmic localization was completely disrupted by F517A/I519A and C523S/C532S. Since the additional mutation at Phe-517 resulted in the complete loss
of the NES activity of the I519A mutant, it is likely that Phe-517 may
have a role in the NES activity as a hydrophobic residue in addition to
Ile-519, Leu-522, and Leu-526. It is not surprising that a
phenylalanine serves as one of the hydrophobic residues in NESs, since
the cyclin B1 NES contains a phenylalanine residue (49-51). It is,
however, striking that the double mutations at the two cysteine
residues completely abolished the export activity (Fig. 5B).
This finding implies that the presence of at least one of the two
cysteines is required for the CRD NES function. Since deletion of five
amino acids containing Cys-532 resulted in the loss of activity (Fig.
4, PA7 and PA9), and the single C532S mutation by
itself but not C523S showed markedly decreased export activity (Fig.
5B), Cys-532 is more significantly involved in the function
of the Pap1 NES.
DEM Inhibits Nuclear Export of Pap1--
Pap1 has been shown to
play a major role in the expression of multiple genes involved in the
oxidative stress response (17). We then analyzed the Pap1 activity in
response to various stressors by monitoring the p25Apt1
protein level, a Pap1-target gene product. Just like LMB in wild-type cells, expression of p25Apt1 was strongly induced by DEM
and hydrogen peroxide, while these agents failed to enhance
p25Apt1 expression in the pap1 disruptant (Fig.
6A). Other agents caused no
significant increase in the amount of p25Apt1 in the
pap1+ background. Effects of oxidative stress on
the subcellular localization of various GFP fusion proteins expressed
in S. pombe were examined in the presence of DEM as an
inducer of the oxidative stress response (Fig. 6B).
GFP-Pap1, which is normally cytoplasmic (Fig. 1B), was
highly accumulated in the nucleus in response to DEM (Fig. 6B, F-Pap1). Similarly, the cytoplasmic
localization of GST-GFP-PA11 (Fig. 5B) was abolished by DEM
and the protein uniformly distributed throughout the cell (Fig.
6B, G-F-PA11). This finding indicates that PA11
is sufficient for both the NES activity and the DEM response. On the
other hand, the localization of GST-Rev NES-GFP, which had been shown
to localize in the cytoplasm in S. pombe (12, 13), was
unaffected by DEM (Fig. 6B,
G-NESRev-F). These results suggest that the Pap1
NES is different in response to oxidative stress from the HIV-1 Rev
NES.

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Fig. 6.
Inhibition of Pap1 NES function by a subset
of oxidants. A, activation of Pap1 under the various
stress conditions was monitored by detecting the amounts of
p25Apt1. The Pap1-dependent expression of
p25Apt1 upon treatment with the indicated agents was
determined by Western blotting and compared between the cells in the
pap1+ and pap1
backgrounds as described under "Materials and Methods."
B, changes in the subcellular localization of GFP-Pap1
(F-Pap1), GST-GFP-PA11 (G-F-PA11), GST-Rev NES-GFP
(G-NESRev-F), and GFP- CRD-Rev NES
(F- CRD-NESRev) in response to DEM treatment.
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|
To verify this hypothesis, we expressed the CRD-deleted Pap1 fusion
containing the Rev NES (F-
CRD-NESRev) and observed the
effect of DEM on the subcellular localization. Although CRD-deleted
Pap1 was present only in the nucleus (Fig. 1B,
F-
CRD), the addition of the Rev NES caused exclusion of
the fusion protein from the nucleus, indicating that the Rev NES could fully work as an NES instead of the CRD (Fig. 6B). In
parallel with Rev NES alone, the Pap1 fusion containing the Rev NES
(F-
CRD-NESRev) was insensitive to DEM treatment (Fig.
6B, F-
CRD-NESRev+DEM).
Importantly, this protein localized in the nucleus upon LMB treatment.
These results indicate that the Pap1 NES is unique in that it is
sensitive to the oxidative stress generated by DEM.
To identify the amino acid residues involved in the oxidative stress
response of PA11, we examined the location of point-mutated PA11 in the
presence of DEM. As shown in Fig. 5A, all GST-GFP-PA11 derivatives that undergo nuclear export did respond to DEM and relocalized into the nucleus. The conserved amino acids with negative charges (Asp-518 and Asp-520), positive charges (Lys-529 and Lys-531), and hydroxy groups (Ser-524 and Ser-533) appeared not to be involved in
the DEM response (Fig. 5A). These results strongly suggest that the NES function of PA11 cannot be separated from its ability to
respond to DEM.
The Stress Response of the Pap1 NES Is Conserved in Mammalian
Cells--
The CRD containing the Pap1 NES can be exported in
mammalian cells (Fig. 2). To test whether the nuclear export of the
Pap1 NES is also sensitive to DEM in mammalian cells, we analyzed the effect of DEM on the localization of full-length Pap1, CRD-deleted Pap1, CRD, PA11, and the Rev NES. These proteins were transiently expressed as GFP-fused proteins in NIH3T3 cells under the control of
the cytomegalovirus promoter. As shown in Fig.
7, while CRD-deleted Pap1 (F-
CRD) was
highly accumulated in the nucleus, full-length Pap1 (F-Pap1), CRD (data
not shown), and PA11 (F-PA11) were present in the cytoplasm as in
S. pombe. The cytoplasmic localization of these proteins was
disrupted by treatment with LMB (5 ng/ml) within 1 h, confirming
that the Pap1 NES works in mammalian cells. Upon DEM treatment (0.8 mM), Pap1 relocalized to and accumulated in the nucleus
within 2 h, and PA11 was redistributed in both the cytoplasm and
the nucleus, indicating that the Pap1 NES function was abolished by
oxidative stress. On the other hand, the localization of the protein
containing the Rev NES (F-NESRev) was not affected by DEM.
These observations indicate that the Pap1 NES can fully respond to DEM
in not only yeast but also mammalian cells.

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Fig. 7.
Effect of DEM on the localization of the
transiently expressed proteins containing the Pap1 NES in mammalian
cells. NIH3T3 cells were transfected with pFPA1 for GFP-Pap1
fusion (F-Pap1), pFPA2 for GFP- CRD fusion (F- CRD), pFPA11 for
GFP-PA11 fusion (F-PA11), and pFRevNES1 for GFP fusion containing the
Rev NES (F-NESRev). Cells expressing these fusion proteins
were treated with 5 ng/ml LMB for 1 h or 0.8 mM DEM
for 2 h. Localization of the fusion proteins expressed in the
cells was observed by fluorescence microscopy.
|
|
 |
DISCUSSION |
We demonstrated that Pap1, a fission yeast bZip transcription
factor, contained an NES by which its subcellular localization and
transcriptional activity were regulated. The Pap1 NES was mapped into
the CRD at the C terminus. Truncation or mutations of the CRD caused
constitutive nuclear localization of Pap1, thereby activating the
target gene expression. Thus, transcriptional activity of Pap1 depends
on its localization that is mediated by CRM1-Pap1 NES. This is
consistent with the previous observation that Pap1 activity is
down-regulated by Crm1 (23). Although an NES in mammalian AP-1 has not
yet been identified, the active nuclear export mediated by the NES has
been shown with IRF3 (52) and I
B (53), indicating that nuclear
export is one of the general mechanisms by which activity of
transcription factors is spatially regulated.
We mapped the Pap1 NES into the 19-amino acid sequence (PA11) in the
CRD. Although the sequence requirements for NES function are only
loosely defined, PA11 does contain a sequence FDIDDLCSKL (residues
517-526) that is similar to the consensus sequence for the functional
Rev/Rex NES
(L-X2-3-(L/I/F/V/M)-X2-3-L-X-(L/I)) (54). In fact, mutational analyses revealed that four hydrophobic residues in PA11 were important for the NES function. In particular, both of the two leucine residues Leu-522 and Leu-526 were essential. These results suggest that the Pap1 NES belongs to the leucine-rich NESs that are recognized by CRM1/exportin 1 (Fig.
8). It should be noted that, when
compared with the consensus sequence, the Pap1 NES is inverse in the
spacing of hydrophobic amino acids. The Pap1 NES may be similar in
topology to the side chain of the Rev/Rex NES if their main chains form
an
-helix. Another characteristic feature of the Pap1 NES is its
unique requirement of at least one of the two cysteine residues.
Site-directed mutagenesis together with deletion analysis of the CRD
suggest that Cys-532 is particularly important for the NES activity.
Thus, Pap1 nuclear export is mediated by an atypical hydrophobic NES
with cysteine residues in the CRD. The Pap1 NES apparently required the
NES receptor, CRM1/exportin 1 for nuclear export, since the
inactivation of Crm1 by the treatment with LMB or by the mutation in
crm1+ completely abolished the export activity
(Fig. 1). Although it is likely that Pap1 is exported directly by
CRM1/exportin 1, there may be an intermediary protein between them.

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Fig. 8.
Comparison of a novel NES of Pap1 with the
classical NESs. The important amino acids for the Pap1 NES and the
NESs of mouse protein kinase inhibitor (PKI ) (4) and HIV-1 Rev
(61) are emphasized by open letters and
filled boxes. The amino acids partially involved
in NES function are marked by shaded boxes. These
sequences were compared with the proposed consensus sequence
(54).
|
|
Pap1 has been shown to activate the expression of multiple genes that
are induced by oxidative stress. Pap1 is thought to be activated by
oxidants through nuclear translocation due to decreased nuclear export
upon oxidative stress (17). We demonstrated that the Pap1 NES was fully
functional and responsive to DEM in mammalian cells (Figs. 2 and 7).
This finding suggests that the mechanism by which Pap1 relocalizes in
response to oxidative stress is conserved in higher eukaryotes. How
does oxidative stress reduce the export rate of Pap1? One possibility
is that oxidative stress suppresses CRM1/exportin 1 function. The
addition of the Rev NES to Pap1 instead of the CRD resulted in the
constitutive cytoplasmic localization of Pap1 irrespective of oxidative
stress (Fig. 6B). It is therefore unlikely that oxidative
stress reduces protein nuclear export in a general manner. These
results also rule out the possibility that DEM enhances the potential
NLS activity in Pap1. A more likely possibility is that Pap1 contains
such a novel NES that is responsive to oxidative stress and is unable
to interact with CRM1/exportin 1 under the stress conditions.
Consistent with this idea, the nuclear export by the Pap1 NES sequence
alone (G-F-PA11) was sensitive to DEM. This result ruled out the
possibility that the intramolecular interaction between the CRD and
another portion of Pap1 modulates the NES activity upon oxidative
stress. We thus conclude that PA11 is sufficient for the DEM response.
We first considered that some additional residue(s) might confer the
ability to respond to oxidative stress onto the classical hydrophobic amino acid-rich NES. However, the site-directed mutagenesis of PA11
does not predict such residues. Recently, modulation of the NES
function by protein phosphorylation (51, 55) and masking by the
adjacent protein sequence (56) has been suggested. Double mutations in
the highly conserved amino acids, which might be involved in the
intramolecular interaction within PA11 or undergo possible
modifications, such as aspartates, lysines, or serines did not affect
the DEM response. These results imply that the amino acid residue(s)
that is responsible for the oxidative stress response is present within
the NES sequence. The most probable ones are two cysteine residues
(Cys-523 and Cys-532), since cysteine residues have been implicated in
oxidative stress-induced modulation of function of c-Jun (57, 58), SoxR
(59), and OxyR (60). It seems possible that the thiol group may be a
direct sensor for the redox conditions; oxidation of the cysteine
residues modulates the activity of the Pap1 NES. Alternatively, and
perhaps more likely, upon oxidative stress the cysteine residues can be
associated with some other protein(s), thereby inhibiting the nuclear
export. Since Sty1/Spc1 MAP kinase is involved in the stress-mediated activation of Pap1 (17), it is conceivable that protein phosphorylation induces the potential Pap1-binding protein to be able to interact with
the Pap1 NES. Due to the high similarity in the CRD sequence between
Pap1 and Yap1, Yap1 may also be exported with the similar NES sequence.
Most recently, Kuge et al. (39) reported that the Yap1 CRD
was also exported by the CRM1/exportin 1-dependent mechanism. However, the requirements of cysteine residues for the Pap1
CRD were different from those for the Yap1 CRD (38), and the Pap1 CRD
was not functional in S. cerevisiae (39). Furthermore, there
is no evidence for the involvement of a stress-activated MAP kinase in
the oxidative stress response of Yap1. These observations suggest that
the Pap1 and Yap1 CRDs are similar in their CRM1/exportin 1-dependent nuclear export but not identical in the
molecular mechanisms of the stress-sensitive nuclear export. We show
here a novel example of the NES containing important cysteine residues, the function of which is inhibited by oxidative stress probably through
an evolutionarily conserved mechanism. It is obviously important to
elucidate how oxidative stress prevents the Pap1 NES from being
exported by the export machinery.
 |
ACKNOWLEDGEMENTS |
We thank Dr. M. Yanagida, Graduate School of
Science, Kyoto University, for strains and anti-p25Apt1
antibody. We are also grateful to Dr. M. Nishiyama, Biotechnology Research Center, The University of Tokyo, for discussion. We also thank
Dr. S. Y. Shinozaki, Graduate School of Science, The University of
Tokyo, for technical advice.
 |
FOOTNOTES |
*
This work was supported in part by a special grant for
Advanced Research on Cancer from the Ministry of Education, Culture and
Science of Japan, Takeda Science Foundation (to M. Y.), and by
Research Fellowships of the Japan Society for the Promotion of Science
for Young Scientists (to N. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence and reprint requests should be
addressed. Fax: 81-3-3812-0544, E-mail:
ayoshida{at}hongo.ecc.u-tokyo.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
NES, nuclear export
signal;
DAPI, 4', 6-diamidino-2-phenylindole;
DEM, diethyl maleate;
NLS, nuclear localization signal;
GST, glutathione
S-transferase;
LMB, leptomycin B;
CRD, cysteine-rich domain;
GFP, green fluorescent protein;
EGFP, enhanced GFP;
HIV, human
immunodeficiency virus;
TRITC, tetramethylrhodamine isothiocyanate;
MAP, mitogen-activated protein;
NPC, nuclear pore complex;
bZip, basic
leucine-zipper;
ORF, open reading frame;
PCR, polymerase chain
reaction.
 |
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