Pse1p Mediates the Nuclear Import of the Iron-responsive Transcription Factor Aft1p in Saccharomyces cerevisiae*

In Saccharomyces cerevisiae, the iron-responsive transcription factor Aft1p plays a critical role in maintaining iron homeostasis. The activity of Aft1p is induced in response to iron starvation and as a consequence the expression of the iron-regulon is increased. We have shown previously that Aft1p is localized to the cytoplasm under iron-replete conditions but that it is localized to the nucleus under iron-depleted conditions. In this study, we identified the transport receptor that mediates the import of Aft1p into the nucleus, located the nuclear localization signal (NLS) sequences of Aft1p, and examined whether the nuclear import of Aft1p is affected by iron status. In pse1-1 cells, which bear a temperature-sensitive mutation of PSE1, Aft1p was misdirected to the cytoplasm during iron starvation at the restrictive temperature. Aft1p could also directly bind to Pse1p and was dissociated from the complex by Ran-GTP in vitro. These results indicate that Aft1p is imported into the nucleus by Pse1p. Supporting this is that the induction of an Aft1p target gene, FTR1, in response to iron starvation was greatly reduced in pse1-1 cells. Furthermore, we demonstrated that the nuclear localization of a mutant Aft1 protein that contains an NLS derived from SV40 was regulated by iron status regardless of whether Pse1p could interact with Aft1p. This suggests that the interaction between Aft1p and Pse1p is not a critical step that controls the iron-regulated nucleo-cytoplasmic transport of Aft1p.

Regulation of the nucleo-cytoplasmic localization of transcription factors is a strategy by which eukaryotic cells respond to environmental changes. The nuclear translocation of transcription factors occurs commonly in response to extracellular signals and appears to be a key mechanism by which these factors alter the expression of their target genes (1,2).
The nucleus and the cytoplasm are separated by the nuclear envelope (NE). Molecules are exchanged through the nuclear pore complex (NPC), which is embedded in the NE. Macromolecules, such as proteins with molecular masses exceeding 40 -60 kDa, are imported to or exported from the nucleus via sequence-mediated processes. Transport receptors called karyopherins (also called importins or exportins) recognize spe-cific nuclear import and export sequences (called nuclear localization signals (NLS) 1 and nuclear export signals (NES), respectively) within their cargo proteins and mediate their translocation across the NPC. The small GTPase Ran plays an essential role in this transport process since it, along with its effector molecules, establishes the directionality of transport. A large family of transport receptors that contain the conserved region required for binding to Ran-GTP have been identified and shown to operate within the multiple pathways of nucleocytoplasmic transport (3)(4)(5). Fourteen transport receptors that are members of the karyopherin ␤ family have been identified in yeast genome databases (6). To date, eight of these have been designated as import receptors, five as export receptors, and one as an import and export receptor. In metazoans, the existence of more than 25 karyopherin ␤ members has been demonstrated (7,8). These include importin ␤, the best studied karyopherin ␤, which often functions in association with an adaptor protein, importin ␣, to bind cargo proteins (9,10). Importin ␣ recognizes cargo proteins containing one or two stretches of basic amino acid residues termed the classical NLS (11). The sequences recognized by other receptors identified so far are not similar to the classical NLS (2), and the mechanisms by which these receptors recognize their cargo remain to be elucidated.
Fluctuation in environmental nutrient concentrations elicits signals that modulate gene expression. Iron is an essential nutrient, but it can be toxic in excess. Therefore, cells have acquired mechanisms through which they maintain iron homeostasis (12,13). The major iron-responsive transcriptional activator Aft1p plays a critical role in iron homeostasis in budding yeast as its activity is induced in response to iron starvation and as a consequence the expression of the ironregulon is up-regulated (14,15). Aft2p, a paralog of Aft1p, shares 39% homology with Aft1p (16) and plays overlapping but non-redundant roles with Aft1p in the transcriptional regulation of the iron regulon (17). We have shown previously that Aft1p is localized to the cytoplasm in Saccharomyces cerevisiae under iron-replete conditions, but that it is localized to the nucleus under iron-depleted conditions (18).
Here we demonstrate that Aft1p is imported into the nucleus of S. cerevisiae by the nonclassical import receptor Pse1p/ Kap121p. Consistent with the importance of PSE1 in the nuclear localization of Aft1p is that the iron-regulon was poorly expressed in a mutant strain deficient in the Pse1p activity and had been subjected to iron-impoverished conditions. We also identified two NLSs of Aft1p and demonstrate that each is sufficient for its PSE1-dependent nuclear import in vivo, which suggests that these two regions are independently recognized by Pse1p. In addition, we demonstrate that a mutant Aft1p containing a classical NLS responds to iron status independently of Pse1p, which suggests that the recognition of Aft1p by Pse1p is not important for the iron-regulated nuclear localization of Aft1p.

EXPERIMENTAL PROCEDURES
Yeast Strains and Media-The yeast strains used in this study are listed in Table I. Strains expressing hemagglutinin (HA)-tagged Aft1p (Aft1p-HA) or its derivatives were created by transforming the strain indicated with pRS416-AFT1-HA or its derivatives. Cells were grown routinely in YPD or synthetic dextrose (SD) medium supplemented with amino acids. To create iron-deprived conditions, cells were grown in SD-Fe media consisting of yeast nitrogen base lacking iron, 2% glucose, amino acid supplements, and 50 mM MES buffer (pH 6.1). To create iron-replete conditions, ferrous sulfate and ferrozine were added to final concentrations of 200 and 500 M, respectively.
Plasmids-pRS416-AFT1-HA has been described previously (18). To create pRS416-AFT1-SV40-HA, the DNA fragment containing SV40 NLS was excised from pPS1372 (19) and introduced into the BglII site of pRS416-AFT1-HA. As a template for histidine-tagged Aft1 protein (Aft1p-His) synthesis in vitro, pEU-AFT1-Hisϫ6 was constructed by inserting the SmaI-HindIII fragment encoding the region encompassing amino acids 1-473 of Aft1p and the HindIII-BamHI fragment encoding the histidine tag into the EcoRV and BamHI sites of pEU-NII (Invitrotech). pGEX-PSE1 (20) and pGEX-KAP95 (21) were used to express glutathione S-transferase (GST)-fused Pse1 protein (GST-Pse1p) and GST-fused Kap95 protein (GST-Kap95p), respectively. pET-GSP1 G21V (20) and pET-GSP1 T26N were used to express mutant histidine-tagged Gsp1 proteins. pET-GSP1 T26N was constructed by inserting the NdeI-XhoI fragment encoding Gsp1p T26N into NdeI and XhoI sites of pET15b (Novagen). Expression plasmids for the green fluorescent protein (GFP)-fused proteins were constructed as follows: Within pPS 1372, the ADH promoter was replaced with the MET25 promoter, and the NES derived from the protein kinase inhibitor (PKI) was mutated to create pMET25-SV40 NLS-GFP. The DNA fragments encoding various regions of Aft1p with a ClaI site and SmaI site at each end were then inserted between ClaI and SmaI sites of pMET25-SV40 NLS-GFP.
Indirect Immunofluorescence and GFP Microscopy-For indirect immunofluorescence, cells expressing HA-tagged proteins were fixed by adding formaldehyde to a final concentration of 4% for 30 min, followed by the addition of buffered formaldehyde (4% formaldehyde, 50 mM potassium phosphate buffer, pH 6.5, 0.5 mM MgCl 2 ) for 90 min. Cell walls were digested with 300 units of zymolyase (Seikagaku Kogyo) in SPM (1.2 M sorbitol, 50 mM potassium phosphate buffer, pH 6.5, 0.5 mM MgCl 2 ) for 1 h at 30°C, followed by the addition of 2% SDS for 2 min. Spheroplasts were fixed on polylysine-coated cover glass using phosphate-buffered saline (PBS) containing 4% formaldehyde, permeabilized with 0.05% saponin for 10 min, and then incubated with an anti-HA antibody (Roche Diagnostics). Signals were amplified by Alexa Fluor 594 Signal-Amplification Kit (Molecular Probes) and visualized by fluorescent microscopy.
For GFP microscopy, cells were cultured in medium lacking methionine to induce the expression of the GFP-fused proteins. Shifting the temperature from the permissive temperature to the restrictive temperature was performed before the proteins were induced. GFP-fused proteins were visualized directly by fluorescent microscopy. The expression of the tagged proteins was measured by Western blot and found to be similar in each of the strains under the different iron conditions employed.
Recombinant Protein Expression and Purification-The expression and purification of recombinant GST-Pse1p and GST-Kap95p were performed as follows: Escherichia coli BL21(DE3) cells transformed with pGEX-PSE1 or pGEX-KAP95 were grown in LB at 37°C. At an OD 600 nm of 0.5, the incubation temperature was shifted to 20°C and then isopropyl-␤-D-thiogalactoside was added to a final concentration of 1 mM. After another 2 h at 20°C, the cells were harvested and resuspended in PBS. The cells were then lysed using 1 mg/ml lysozyme for 1 h and nucleic acids were digested with 5 g/ml DNaseI and 5 g/ml RNaseA. The recombinant proteins were purified using glutathione-Sepharose 4B (Amersham Biosciences). The expression and extraction of His-Gsp1p G21V and His-Gsp1p T26N were performed in the same way as for the GST-fused proteins except that 5 M GTP or GDP was added to all buffers. After purification of His-Gsp1p G21V and His-Gsp1p T26N by Ni-NTA-agarose (Qiagen) chromatography, the buffer was exchanged with Ran buffer (50 mM Tris-Cl, pH 7.5, 300 mM NaCl) using HiTrap Desalting (Amersham Biosciences). Aft1p-His was synthesized using a PROTEIOS kit according to the manufacturer's instructions (Invitrotech).
Dissociation of Aft1p-Pse1p Complexes by Ran-GTP-His-Gsp1p G21V and His-Gsp1p T26N were loaded with GTP or GDP as follows: His-Gsp1p G21V and His-Gsp1p T26N dissolved in Ran buffer were incubated in the presence of 20 mM dithiothreitol, 20 mM EDTA, pH 7.5, and 4 mM GTP or GDP for 2 h at room temperature. After the addition of MgCl 2 to a final concentration of 50 mM, the mixture was further incubated for 20 min on ice, and then the buffer was exchanged for Ran-G buffer (50 mM Tris-Cl, pH 7.5, 300 mM NaCl, 2 mM MgCl 2 , 2 mM ␤-mercaptoethanol). GST-Pse1p and Aft1p-His were bound to glutathione-Sepharose beads as above. The beads were divided into two equal parts and incubated with the GTP-loaded form of His-Gsp1p G21V (His-Gsp1p G21V -GTP) or the GDP-loaded form of His-Gsp1p T26N (His-Gsp1p T26N -GDP) for 2 h at room temperature. The supernatant, which contained the unbound fraction, was collected and concentrated by acetone precipitation. The proteins bound to the beads were eluted as the bound fraction. These samples were fractionated by SDS-PAGE and the proteins were detected by immunoblotting.
Total Yeast RNA Isolation and Northern Blotting-Cells cultured to mid-log phase were harvested and subjected to total RNA preparation as described (22). The RNA was separated on a 1% agarose gel containing formaldehyde, transferred to a Biodyne B membrane (Pall corporation) and hybridized with 32 P-labeled probes for FTR1 (nucleotides from 1 to 649 of the FTR1 open reading frame) and ACT1 (nucleotides from 37 to 1070 of the ACT1 open reading frame). The hybridized membrane was analyzed by BAS-2000 (Fuji Film).

Pse1p Is Required for the Nuclear Localization of Aft1p-We
have previously shown that Aft1p stays in the cytoplasm under iron-replete conditions (Fig. 1A, panel 2) but that it localizes to the nucleus under iron-depleted conditions (Fig. 1A, panel 1) (18). To determine whether the nuclear import of Aft1p is regulated by iron, we first tried to identify the import receptor for Aft1p. The Aft1p sequence contains several short stretches of basic amino acids that could potentially act as classical NLSs (although nuclear proteins that are imported via the non-classical pathway are also rich in basic amino acids). As described above, proteins containing classical NLSs are imported into the nucleus by the transport receptor complex that consists of Importin ␣ and Importin ␤. The yeast homologs of these proteins are Srp1p/Kap60p and Rsl1p/Kap95p, respectively. Thus, we examined the nuclear localization of Aft1p in temperaturesensitive mutants of SRP1 (srp1-31) or RSL1 (rsl1-4) ( Table I) expressing Aft1p-HA by indirect immunofluorescence microscopy. As shown in Fig. 1A (panels 3 and 5), Aft1p localized to the nucleus under iron-depleted conditions in both strains even at the restrictive temperature (37°C), as was also observed for the wild-type strain (Fig. 1A, panel 1). However, as expected, GFP fused with a classical NLS derived from SV40 (SV40 NLS-GFP) driven by the MET 25 promoter as a control could not accumulate in the nucleus in these mutant strains at the restrictive temperature (37°C) whereas it did localize in the nucleus in the wild-type strain (compare Fig. 1B, panel 1 with  panels 2 and 3). This indicates that Aft1p is not imported into the nucleus by the classical NLS pathway.
We next studied the localization of Aft1p in other strains that harbor mutations in karyopherin ␤ family members. In strains carrying mutations in KAP104, KAP123, SXM1, NMD5, and PDR6, no defect in the nuclear accumulation of Aft1p was observed (data not shown). However, we found that when the strain carrying a temperature-sensitive mutation in PSE1 (pse1-1) was cultured at the restrictive temperature (37°C), Aft1p-HA does not accumulate in the nucleus during iron starvation (Fig. 1A, panel 7), which suggests that the nuclear localization of Aft1p requires Pse1p. However, the import of proteins with classical NLSs was not perturbed in  1, 3, 5, and 7) or iron-replete (panels 2, 4, 6, and 8) medium at 25°C to mid-log phase growth and then were shifted to 37°C for 3 h. After fixation, the subcellular localization of Aft1p-HA was examined by indirect immunofluorescence microscopy using an anti-HA antibody. Transmitted and fluorescent images are shown. B, the subcellular localization of the SV40 NLS-GFP fusion protein was observed in wild-type (panel 1), rsl1-4 (panel 2), srp1-31 (panel 3), or pse1-1 (panel 4) cells that had been cultured at 25°C to mid-log phase growth and then shifted to 37°C for 3 h. pse1-1 cells because SV40 NLS-GFP accumulated in the nucleus (Fig. 1B, panel 4). Thus, not all nuclear proteins are mislocalized in pse1-1 cells under the conditions used in these experiments.
Pse1p Directly Binds to Aft1p and Is Dissociated from Aft1p by Ran-GTP-To confirm that Pse1p is the transport receptor for Aft1p, we examined the interaction between Aft1p and Pse1p in vitro. GST-fused Pse1 protein (GST-Pse1p) and histidine-tagged Aft1 protein (Aft1p-His) were synthesized and subjected to a GST pull-down assay. Thus, GST-Pse1p was immobilized on glutathione-Sepharose and incubated with Aft1p-His. GST-fused Rsl1p/Kap95p (GST-Kap95p) was used as a negative control. After washing, the bound proteins were eluted and subjected to SDS-PAGE and analyzed by immunoblotting with an anti-His antibody and by protein staining. As shown in Fig. 2A, Aft1p-His was retained by GST-Pse1p but not by GST-Kap95p, which indicates that Pse1p directly interacts with Aft1p.
In general, stable complexes of the import receptors and cargos that are formed in the cytoplasm are dissociated in the nucleus by binding to the GTP-bound form of Ran (3,4). To further investigate the specificity of the interaction between Pse1p and Aft1p, the disassembly of this interaction by the GTP-bound form of Gsp1p, a yeast homolog of Ran, was examined using histidine-tagged mutant Gsp1 proteins (His-Gsp1p G21V and His-Gsp1p T26N ) that had been purified from E. coli. Gsp1p G21V harbors a mutation that causes the loss of the GTPase activity of Gsp1p (23), while Gsp1p T26N is known to bind only GDP (24). Thus, GTP-loaded His-Gsp1p G21V (His-Gsp1p G21V -GTP) or GDP-loaded His-Gsp1p T26N (His-Gsp1p T26N -GDP) was added to glutathione-Sepharose beadimmobilized GST-Pse1p to which Aft1p-His was bound. In the presence of His-Gsp1p G21V -GTP, Aft1p-His was dissociated from GST-Pse1p (Fig. 2B, lanes 1 and 2). However, unlike His-Gsp1p G21V -GTP, Aft1p was still associated with Pse1p in the presence of His-Gsp1p T26N -GDP (Fig. 2B, compare lanes 1  and 2 with lanes 3 and 4). Together, these results suggest that Pse1p mediates the nuclear import of Aft1p.
pse1-1 Cells Have a Defect in Aft1p-induced Transcription-As revealed above, Pse1p mediates the nuclear import of Aft1p. Consequently, we hypothesized that the impaired nuclear import of Aft1p may cause the pse1-1 cells to be defective in the induction of the Aft1p regulon, thereby leading to a defect in iron homeostasis. To address this, we analyzed the expression in wild-type and pse1-1 cells at 37°C of an Aft1p target gene, FTR1, which encodes the iron permease that is required for high affinity iron uptake in response to iron starvation. The mRNA levels of FTR1 in pse1-1 cells were indeed much lower than in wild-type cells in iron-starved conditions (Fig. 3, lanes 2 and 4), although the induction of FTR1 was not completely lost in pse1-1 cells (Fig. 3, lanes 3 and 4). This implies that pse1-1 cells have a defect in iron homeostasis.
Identification of Two Independent NLSs of Aft1p-To iden-  1 and 2) or His-Gsp1p T26N -GDP (lanes 3 and 4). The proteins in the supernatant (lanes 1 and 3) and bound to beads (lanes 2 and 4) were fractionated by SDS-PAGE and detected by immunoblotting using an anti-His antibody.

FIG. 3. The induction of FTR1 expression is impaired in pse1-1 cells.
Wild-type or pse1-1 cells grown to mid-log phase in iron-replete medium were further cultured at 37°C for 3 h. Cells were then transferred to iron-depleted medium. Total RNA was then prepared from cells cultured in iron-replete medium (Feϩ) and 30 min after transferring to iron-depleted medium (FeϪ). The FTR1 mRNA levels were then analyzed by Northern blotting. MATa ura3-52 leu2⌬1 trp1 his3⌬200 lys2 pdr6⌬::HIS3 P.A. Silver tify the region in Aft1p required for its nuclear import, we constructed a series of vectors that express various regions of Aft1p fused in-frame with GFP under the control of the MET25 promoter. The fusion proteins were then expressed in the wildtype strain cultured under iron-depleted conditions, and their localization was analyzed. As shown in Fig. 4, A and B (panels  1 and 2), fusion proteins containing amino acids 198 -225 or 332-365 of Aft1p (Aft1p-(198 -225)-GFP and Aft1p-(332-365)-GFP) both localized to the nucleus, which suggests that each of these two regions mediates the nuclear import of Aft1p. Each of these two regions contains a stretch of basic amino acid residues, namely, KPKKKR (residues 202-207) and RKPK (residues 352-355), respectively. This is similar to what is seen in classical NLSs and consequently we wondered if these regions could be recognized by the classical nuclear import receptor complex, even though whole Aft1p is imported into the nucleus by Pse1p. However, when Aft1p-(198 -225)-GFP and Aft1p-(332-365)-GFP were expressed in the srp1-31 and rsl1-4 strains, both regions were able to localize to the nucleus at the restrictive temperature (data not shown). In contrast, the mislocalization of these fusion proteins was evident in pse1-1 cells cultured at the restrictive temperature (Fig. 4B, panels 3 and  4). This implies that each region in Aft1p (namely, amino acids 198 -225 or 332-365) functions independently as an NLS for Pse1p. Further deletion of each region led to the loss of nuclear localization (data not shown), as has been previously reported for the NLSs in other cargo proteins that are imported by Pse1p (20,25,26).
Some of the sequences that have been reported to date as being NLSs recognized by Pse1p are not homologous to each other except in that they contain basic amino acids (20,(25)(26)(27). As described above, many basic amino acids are located in the Aft1p NLSs. Thus, we assessed the contribution of these residues to NLS function. The residues within or proximal to the basic amino acid stretch were mutated, and the subcellular localization of the resulting mutant Aft1p-GFPs in wild-type cells under iron-depleted conditions was then determined. Aft1p-(198 -225)-GFP that contains six alanine substitutions in amino acids 202-207 (m(198 -225)-GFP) was misdirected (Fig. 5, A and B, panel 1). Mutation of amino acids 332-335 (m1(332-365)-GFP) or 352-356 (m2(332-365)-GFP) to alanine residues also partially impaired the NLS function of the 332-365 region, and the double mutation (m3(332-365)-GFP) completely abolished the nuclear localization of Aft1p-(332-365)-GFP (Fig. 5, A and B, panel 2).
In addition, we tested whether the mutations introduced in these two regions abolish the nuclear localization of Aft1p. Thus, we introduced the same alanine substitutions in m(198 -225)-GFP and m3(332-365)-GFP into the full-length Aft1p fused with GFP (Aft1p(mut-nls)-GFP) (Fig. 5C), and observed its subcellular localization. As shown in Fig. 5D (panel 2), GFP-fused Aft1p(mut-nls) no longer accumulates in the nucleus, even under iron-starved conditions. However, when the alanine substitutions in either m(198 -225)-GFP or m3(332- C, schematic depiction of wild-type (Aft1p-(1-690)-GFP) and mutant Aft1p containing alanine substitutions in both NLSs (Aft1p(mut-nls)-GFP). In Aft1p(mut-nls)-GFP, the same amino acids residues that were 365)-GFP were singly introduced into the full-length Aft1p so that one of the NLSs would remain intact, the mutant Aft1p-GFPs accumulated in the nucleus, albeit more weakly than the wild-type Aft1p-GFP (data not shown). These results demonstrate that both of the Aft1p NLS regions, namely, amino acids 198 -225 and 332-365, are necessary and sufficient for the nuclear import of intact Aft1p.
Pse1p Does Not Play a Key Role in the Iron-regulated Subcellular Localization of Aft1p-We examined the effect of iron concentration on the nuclear import of Aft1p and thus assayed the subcellular localization of Aft1p-(198 -225)-GFP and Aft1p-(332-365)-GFP in iron-rich medium. Both Aft1p-(198 -225)-GFP and Aft1p-(332-365)-GFP were localized in the nucleus under iron-replete conditions (Fig. 4A), which indicates that the direct interaction between Pse1p and NLSs of Aft1p is not affected by iron levels.
Although the NLSs of Aft1p are imported to the nucleus irrespective of the iron levels, it is still possible that under iron-replete conditions the Aft1p NLSs are masked either by a conformational change of Aft1p itself or by an unknown partner. To examine this possibility, we constructed two Aft1p mutants, Aft1p-SV40 and Aft1p(mut-nls)-SV40. In these mutants, the C terminus of either the wild-type or the NLSmutated Aft1p was fused to the NLS of SV40, which is recognized by the classical import receptor complex (Fig. 6A). It is unlikely that an exogenous NLS located at the extreme C terminus of Aft1p would be routinely masked if an NLS-masking mechanism is involved in the regulation of Aft1p localization. As shown in Fig. 1, wild-type Aft1p cannot localize to the nucleus during iron starvation in pse1-1 cells. In contrast, Aft1p-SV40, which is imported into the nucleus via the classical NLS pathway, localized to the nucleus under iron-depleted conditions and stayed in the cytoplasm under iron-replete conditions in pse1-1 cells at the restrictive temperature (Fig. 6B,  panel 1 and 2). Alternatively, Aft1p(mut-nls)-SV40, which is no longer imported to the nucleus by Pse1p (see Fig. 5A), localized to the nucleus and the subcellular localization of Aft1p(mutnls)-SV40 expressed in ⌬aft1 cells was regulated by iron status similar to wild-type Aft1p expressed in ⌬aft1 cells (Fig. 6C,  panels 1 and 2). These results indicate that the accessibility and subsequent interaction of Pse1p with Aft1p are not critical events that control the iron-regulated subcellular localization of Aft1p. DISCUSSION We have previously reported that the subcellular localization of Aft1p is regulated by iron status. To understand the mechanisms regulating Aft1p activity, the molecular basis of its nucleo-cytoplasmic transport needs to be elucidated.
In this report, in vivo genetic evidence and in vitro biochemical analyses demonstrated that Pse1p is an import receptor for Aft1p. Moreover, the pse1-1 mutant strain, which is impaired in the nuclear import of Aft1p, showed a defect in iron homeostasis since FTR1, a target gene of Aft1p, is expressed at lower levels in these cells upon iron starvation.
Pse1p was first reported as an auxiliary import receptor of ribosomal protein L25, since the defective import of L25 observed in the kap123⌬ strain was reversed by the overexpression of Pse1p (28). Recently, Pse1p was also shown to be a transporter for several transcription factors (20,25,27,29) and a regulatory protein for sporulation (26). The NLS-containing regions identified within these proteins to date do not share any distinct homology. However, it appears that the regions required for NLS function within these proteins are relatively large compared with classical NLSs and that they all contain a short stretch of basic amino acids, including serine and proline amino acids. In this regard, the Aft1p NLSs that we identified here are similar to those observed in other proteins transported by Pse1p. We found that multiple successive mutations in the basic amino acid stretches were needed to impair the NLS activity of these regions, since single alanine substitutions within these residues was not effective (data not shown). This suggests that proper folding may be needed before these NLSs in the cargo proteins can be recognized by Pse1p.
It has been reported that Pse1p preferentially binds to unphosphorylated Pho4p (25). As Aft1p has been reported to be a phosphoprotein (30), it would be of interest to determine if the phosphorylation of Aft1p affects its interaction with Pse1p.
The data reported here suggest that the interaction of Aft1p and Pse1p is not regulated by iron status, as we found that the iron concentration did not affect the nuclear localization of the two Aft1p NLS-GFP fusion proteins. This indicates that the binding of Aft1p NLSs to Pse1p occurs with similar affinity in iron-replete and iron-depleted conditions, although we did not extensively compare the rate of import. It has been reported that the nuclear localization of a protein can be regulated by the intra-or intermolecular masking of the NLS that inhibits the interaction between the NLS and its import receptor (31,32). However, this is unlikely to be a mechanism affecting the recognition of Aft1p by Pse1p since SV40 NLS fused to the C terminus of Aft1p can substitute for the Aft1p NLSs. Nevertheless, these findings do not completely exclude the possibility that the import of Aft1p may play some role in regulating its activity. It may be that this protein is anchored in the cytoplasm, which overrides the presence of any NLS, even though the NLS of the cargo is accessible to the import receptor. Supporting this notion is a recent report that demonstrated that the transcription factor Nrf2, which is essential for the antioxidant responsive element (ARE)-mediated induction of phase II detoxifying and oxidative stress enzymes, is bound in the cytoplasm to Keap1. Keap1 is closely related to the Drosophila actin-binding protein Kelch, which is anchored to the actin cytoskeleton. Upon exposure to electrophiles, the Keap1-Nrf2 complex is disrupted and Nrf2 migrates to the nucleus (33). Thus, it is possible that Aft1p interacts with such an anchoring protein and is located to the cytoplasm in the presence of iron but that when iron is scarce, it is released and results in its translocation into the nucleus. It is also possible that the subcellular localization of Aft1p might be regulated solely at the export step, as is the case for Yap1p (20). The recognition of Yap1 by the export receptor, Crm1p/Xpo1p is regulated in a redox-sensitive manner (34,35). Further investigation is required to determine which of these possibilities are true and to fully understand the mechanisms that regulate Aft1p localization in response to iron status.