A Novel Nuclear Export Signal Sensitive to Oxidative Stress in the Fission Yeast Transcription Factor Pap1*

Nobuaki KudoDagger , Hiroshi TaokaDagger , Takashi Toda§, Minoru YoshidaDagger , and Sueharu HorinouchiDagger

From the Dagger  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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha  (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 beta  that recruits the complex of importin alpha  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.

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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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 Delta 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 (Delta 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-Delta 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 Delta 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-Delta 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 (Delta 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.

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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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-Delta 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-Delta 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-Delta 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-Delta 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-Delta 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-Delta 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).

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.

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 (Delta 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 Delta 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.

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-Delta CRD-Rev NES (F-Delta CRD-NESRev) in response to DEM treatment.

To verify this hypothesis, we expressed the CRD-deleted Pap1 fusion containing the Rev NES (F-Delta 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-Delta 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-Delta CRD-NESRev) was insensitive to DEM treatment (Fig. 6B, F-Delta 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-Delta 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-Delta CRD fusion (F-Delta 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Ikappa 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 alpha -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 alpha  (PKIalpha ) (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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