Subcellular Localization of Aft1 Transcription Factor Responds to Iron Status in Saccharomyces cerevisiae *

The Aft1 transcription factor regulates the iron regulon in response to iron availability in Saccharomyces cerevisiae . Aft1 activates a battery of genes required for iron uptake under iron-starved conditions, whereas Aft1 function is inactivated under iron-replete conditions. Previously, we have shown that iron-regulated DNA binding by Aft1 is responsible for the controlled expression of target genes. Here we show that this iron-regu-lated DNA binding by Aft1 is not due to the change in the total expression level of Aft1 or alteration of DNA binding activity. Rather, nuclear localization of Aft1 responds to iron status, leading to iron-regulated expression of the target genes. We identified the nuclear export signal (NES)-like sequence in the AFT1 open reading frame. Mutation of the NES-like sequence causes nuclear retention of Aft1 and the constitutive activation of Aft1 function independent of the iron status of the cells. These results suggest that the nuclear export of Aft1 is critical for ensuring iron-responsive transcriptional activation of the Aft1 regulon and that the nuclear import/ export systems are involved in iron sensing by Aft1 in S. cerevisiae . Plasmid Construction— Plasmids pRS416-AFT1-HA and pRS416- AFT1–1 up -HA carry the AFT1 or AFT1–1 up gene, respectively, modified by insertion of the HA 12 tag at the C terminus under the control of its native promoter in the centromere vector pRS416 (Stratagene). A PCR-based strategy was performed to introduce alanine substitution to hy- drophobic residues in Aft1. DNA domain-Aft1 pGBD-Aft1(1– pGBD-Aft1(1– Detection by West Pico) was performed after incubation with horse-radish peroxidase-conjugated second antibody (Amersham Biosciences) according to the instructions of manufacturer (Pierce). (cid:2) -Galactosidase activity in yeast transformants was assayed as described (19). Immunofluorescence Microscopy and Northern Blot Analysis— Indirect immunofluorescence microscope was performed as de- scribed (20). cells grown iron-starved iron-replete potassium Cell wall digestion of fixed cells sorbitol, M Spheroplasts treated 2% SDS/1.2 M sorbitol for 1–2 min, SPM, applied polylysine- coated coverslips. Cells were permeabilized with 0.05% saponin, incubated with anti-HA antibody (Roche Molecular Diagnostics). After washing with phosphate-buffered saline, Alexa Fluor 594-conjugated antibodies (signal-amplification kit, Molecular Probe) were used to am- plify and visualize the protein. DNA was counterstained with 4 (cid:3) , 6-dia-midino-2-phenylindole, dihydrochloride (DAPI). Cells with SlowFade solution (Molecular Probe) and visualized by microscopy. Total RNA was extracted cells by hot phenol and Northern blot analysis was performed as described previously (12).

Iron is an essential nutrient for virtually all organisms, working as a cofactor for critical proteins that mediate diverse processes such as cellular respiration and synthesis of metabolic intermediates (1). On the other hand, excess iron is extremely toxic, being capable of generating free radicals that damage macromolecules such as proteins, lipids, and DNA (2). The uptake and metabolism of iron must therefore be strictly regulated. In all organisms studied, the rates of iron uptake are tightly coupled to the levels of available iron and the needs of cells (3). In metazoans, the mechanisms that underlie regulation of iron metabolism including uptake, sequestration, and utilization have been well characterized. Two RNA-binding proteins called "iron regulatory protein," IRP1 1 and IRP2, are responsible for this regulation by controlling mRNA translation or mRNA stability by iron regulated mRNA-protein interactions (4). Although these proteins are closely related, IRP1 is a relatively stable protein (5,6), and its RNA binding activity is inactivated by iron-sulfur cluster formation in iron-replete cells (7). By contrast, IRP2 is degraded by the ubiquitin-proteasome system in iron-replete cells (8).
In Saccharomyces cerevisiae, iron deprivation induces activities of a high affinity iron uptake system (9) and siderophoremediated iron uptake system (10,11). A transcription factor, Aft1, plays a critical role in this process (12). Aft1 protein binds to a regulatory region in the DNA of the genes required for iron uptake and induces the transcription of these genes in ironstarved cells (13). Conversely, Aft1 is not bound to its regulatory target element in iron-replete cells, and the expression of its target genes is not induced. An increasing number of Aft1 target genes have been implicated in iron metabolism, including a putative transporter complex located in the vacuole as well as plasma membrane and siderophore transporters (11,(13)(14)(15), indicating that Aft1 is a key regulator of cellular iron homeostasis.
Many transcription factors respond to external stimuli by modulating the expression of target genes (16). One way to modulate the activity of transcription factors is to allow them to traverse from the cytoplasm to the nucleus by means of cargo receptors such as importins and exportins (17,18).
In this report, we show that Aft1 responds to iron by regulated changes in the Aft1 subcellular localization; Aft1 localized to the nucleus under iron-depleted conditions, whereas Aft1 localized to the cytoplasm under iron-replete conditions. Introducing mutation in AFT1 open reading flame reveals hydrophobic residues important for nuclear export of Aft1, which resembles NES sequences identified previously (18). Inhibition of nuclear export of Aft1 results in constitutive activation of Aft1 function and thereby causes activated expression of target genes regardless the changing iron status in the cells. These results suggest that the nuclear import/export systems are involved in iron sensing by Aft1 and that the nuclear retention of Aft1 in response to iron starvation activates the iron regulon in S. cerevisiae.
Plasmid Construction-Plasmids pRS416-AFT1-HA and pRS416-AFT1-1 up -HA carry the AFT1 or AFT1-1 up gene, respectively, modified by insertion of the HA 12 tag at the C terminus under the control of its native promoter in the centromere vector pRS416 (Stratagene). A PCRbased strategy was performed to introduce alanine substitution to hydrophobic residues in Aft1.
Immunoblot Analysis and ␤-Galactosidase Assay-Cells grown in the iron-starved or iron-replete medium were harvested by centrifugation and resuspended in 150 l of 1.85 M NaOH/1% ␤-mercaptoethanol and incubated on ice for 10 min. An equal volume of 50% trichloroacetic acid was added and incubated on ice for at least 30 min. Trichloroacetic acid precipitates were collected by centrifugation and resuspended in 50 l of 2 ϫ SDS sample buffer supplemented with 0.1 M Tris base to neutralize the trichloroacetic acid. The sample was subjected to SDSpolyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with use of anti-HA monoclonal antibody (Roche Molecular Diagnostics) or anti-Fet3 polyclonal antibody (a gift from Andy Dancis (University of Pennsylvania)). Detection by chemiluminescence (SuperSignal, West Pico) was performed after incubation with horseradish peroxidase-conjugated second antibody (Amersham Biosciences) according to the instructions of manufacturer (Pierce). ␤-Galactosidase activity in yeast transformants was assayed as described (19).
Immunofluorescence Microscopy and Northern Blot Analysis-Indirect immunofluorescence microscope was performed essentially as described (20). Briefly, cells grown in iron-starved or iron-replete medium were fixed by direct addition of formaldehyde (final concentration 4%) and followed by buffered formaldehyde (4% formaldehyde, 50 mM potassium phosphate, pH 6.5, 0.5 mM MgCl 2 ). Cell wall digestion of fixed cells was carried out with 300 units of zymolyase (Seikagaku Kougyo) for 1 h at 30°C in SPM (1.2 M sorbitol, 50 mM potassium phosphate, pH 6.5, 0.5 mM MgCl 2 ). Spheroplasts were then treated with 2% SDS/1.2 M sorbitol for 1-2 min, washed with SPM, and applied to polylysinecoated coverslips. Cells were permeabilized with 0.05% saponin, incubated with anti-HA antibody (Roche Molecular Diagnostics). After washing with phosphate-buffered saline, Alexa Fluor 594-conjugated antibodies (signal-amplification kit, Molecular Probe) were used to amplify and visualize the protein. DNA was counterstained with 4Ј, 6-diamidino-2-phenylindole, dihydrochloride (DAPI). Cells were mounted with SlowFade solution (Molecular Probe) and visualized by microscopy. Total RNA was extracted from cells by the hot phenol method (21), and Northern blot analysis was performed as described previously (12).

The Amount of Aft1 Protein Is Not Affected by Iron Status-
We have demonstrated previously that the iron-regulated gene expression mediated by Aft1 occurs though the iron-regulated occupancy of the Aft1 binding site on its target sequences (13). The AFT1 transcript has a long 5Ј UTR that contained two small open reading frames preceding the AFT1 open reading frame, suggesting the possibility that expression of the Aft1 protein might be translationally regulated (12). Moreover, IRP2, which is one of the regulators of iron homeostasis in metazoans is degraded by an iron-dependent mechanism (8,22). Therefore we considered the possibility that the amount of Aft1 protein might be affected by the cellular iron status. The diminished Aft1 binding to the target sequences in iron-replete cells might be a consequence of decreased protein expression. The AFT1-disrupted strain (Y21) was transformed with a centromere-based plasmid expressing HA-tagged Aft1 under its own promoter. The transformants with the plasmid expressed and regulated Aft1 target genes in a manner identical to the cells expressing the wild type Aft1 (data not shown). The total amount of Aft1-HA protein in the cells grown in iron-depleted or iron-replete medium was measured by immunoblotting with anti-HA antibody. The iron concentration in the medium had little effect, if any, on the total amount of Aft1 protein. In the same experiment, the expression of one of the Aft1 targets, Fet3 protein was well regulated, being induced by iron starvation and repressed by iron availability (Fig. 1). These results suggest that the amount of Aft1 protein is not responsible for its iron-regulated function.
The Activity of Aft1 Is Not Regulated through Activation Domain-Although Aft1 binds to its target DNA in an ironregulated manner in vivo, Aft1 might have other regulatory mechanisms that respond to iron. Thus, we first mapped the transcriptional activation domain of Aft1. To identify the activation domain of Aft1, we fused the Gal4 DNA binding domain (GBD) to the full-length or truncated Aft1 ( Fig. 2A). The ability of transcriptional activation of each plasmid was assayed for ␤-galactosidase activity in the cells possessing GAL1-lacZ reporter cultured in the iron-depleted medium. Expression of each GBD-Aft1 fusion protein was confirmed by immunoblotting (data not shown). The Gal4 binding domain alone did not give rise to any ␤-galactosidase activity in the cells. As shown in Fig. 2B, the full-length fusion, GBD-Aft1(1-690) conferred high level expression of GAL1-lacZ in the iron-depleted cells. The N-terminal half, GBD-Aft1(1-412) failed to activate GAL1-lacZ, whereas the GBD-Aft1(413-690), which contains the C-terminal half rich in glutamine and acidic (aspartate and glutamate) residues, potential domains for transcriptional activation induced strong expression of GAL1-lacZ. Further mapping of the transcriptional activation domain with shorter constructs, GBD-Aft1(413-572) and GBD-Aft1(573-690) revealed that the main activation domain of Aft1 resides within the region from 413 to 572 amino acid residues. We then examined the iron responsiveness of these GBD fusion proteins. The fusion proteins without an activation domain, GBD-Aft1(1-412) and GBD-Aft1(573-690), failed to confer expression on GAL1-lacZ in iron-replete cells as in iron-depleted cells. The fusion proteins containing an activation domain, GBD-Aft1(413-690) and GBD-Aft1(413-572), induced strong activation of GAL1-lacZ in the presence of iron, indicating that activity of the Aft1 activation domain is not regulated by iron status in the cells. Interestingly, the full-length fusion, GBD-Aft1(1-690) showed iron-regulated activation of ␤-galactosidase activity (Fig. 2B). This suggests that the DNA binding activity of Aft1 through its own DNA binding domain may not be essential for its iron-regulated activation function. However, we can not rule out the involvement of cooperative regulation of DNA-binding and activation domains as described for several transcription factors (23)(24)(25).
Subcellular Localization of Aft1 Responds to Iron Status-Alternatively, the iron-regulated activity of the Aft1 might be explained by the iron effects on nuclear localization of Aft1. Therefore, we examined the subcellular localization of Aft1 by indirect immunofluorescence microscopy. As mentioned before, an HA tag introduced at the C terminus of Aft1 does not affect its function. Cells expressing the wild type Aft1-HA and the gain-of-function mutant, Aft1-1 up -HA, were used for this assay. As we described previously, mutant strains carrying the AFT1-1 up allele exhibit a phenotype in which the expression of the Aft1 target genes cannot be repressed by available iron in the environment (12,13). The Aft1-HA in the cells grown in media containing different concentrations of iron was visualized by anti-HA antibody in combination with Alexa Fluor 594-conjugated secondary antibodies. Due to the low expression level of Aft1 protein, the intensity of fluorescence needed to be amplified for the detection of Aft1 as described under "Experimental Procedures." We observed that the Aft1-1 up -HA protein localized to the nucleus even in the presence of iron in the medium (Fig. 3). On the contrary, the wild type Aft1-HA was localized to the cytoplasm in iron-replete cells and redirected to the nucleus in iron-depleted cells. These results suggest that the subcellular partitioning of Aft1 in response to iron availability in the medium causes the iron-regulated DNA occupancy by Aft1in vivo.
Nuclear Retention of Aft1 Results in Activation of Aft1 Regulon-There is accumulating evidence that NES are involved in regulating the functions of various nuclear proteins (17,18,26,27). A canonical NES, often called the leucine-rich NES, was first found in the HIV Rev protein (28) and the cellular protein kinase inhibitor (29). These proteins are known to be substrates for a nuclear export receptor, CRM1/expotin 1 (30 -33). Thus we searched for a candidate leucine-rich NES in the Aft1 open reading flame. As shown in Fig. 4A, the NES-like sequence was found around leucine residues at position 99 and 102 and could be aligned with other well characterized leucinerich NES sequences. These leucine-rich NES sequences may diverge from the consensus (LX 2-3 (F/I/L/V/M)X 2-3 LX(L/I)) (18,34) so that this alignment is also somewhat imprecise. To determine whether this NES-like sequence regulates the subcellular localization of Aft1 function, we introduced single amino acid substitutions to hydrophobic residues between position 63 and 105 in the context of Aft1-HA protein (Fig. 4B) and examined the subcellular localization of each Aft1 mutant in the cells cultured in iron-replete medium. The mutants, Aft1L63A, Aft1L69A, Aft1I88A, and Aft1L94A, which harbor an alanine substitution outside the NES-like sequence showed cytoplasmic localization as did the wild type Aft1 (Fig. 4, B and  C). In contrast, The Aft1L99A and Aft1L102A constitutively localized in the nucleus even in the iron-replete cells, suggesting that these leucine residues found in the NES-like sequence are critical for nuclear export of Aft1. Unexpectedly, a valine residue at position 105 within the NES-like sequence was dispensable for export of Aft1 from the nucleus.
Then, the effect of mutation in the NES-like sequence on expression of the Aft1 target gene, FTR1, was evaluated by Northern blot. When expressed in aft1 cells, both wild type Aft1 and the mutant Aft1L99A induced FTR1 transcript expression in the cells grown in iron-depleted medium (Fig. 5). The FTR1 transcript was repressed in the Aft1-expressing cells 1 h after addition of iron to the medium, whereas the induced expression of FTR1 was still observed in the Aft1L99A-expressing cells at least 4 h after addition of iron to the medium. These results suggested that the mutation in the NES-like sequence results in the constitutive activation of Aft1 function. Taken together, nuclear retention of Aft1 protein is sufficient to ensure transcriptional activation of target genes. DISCUSSION In S. cerevisiae, three metal-responsive transcription factors have been known to modulate the expression of genes involved in metal homeostasis in similar fashion: Mac1 for copper (35), Zap1 for zinc (36), and Aft1 for iron (12). Under conditions of deprivation of each metal, the respective metal-responsive transcription factors induce expression of a battery of genes required for the specific metal uptake system. Under metalreplete conditions, the function of these respective transcription factors are inhibited; as a consequence, expression of genes required for specific metal uptake is repressed (37). The mechanisms regulating Mac1 and Zap1 function have been well characterized. Each one localizes in the nucleus in any concentration of the respective metals (25,38). It has been proposed that Mac1 function is inhibited by a copper-induced intramolecular interaction that attenuates the DNA binding activity and transactivation activity (25,39,40). Similarly, it has been reported that Zap1 is regulated through its zinc fingers prob- ably by DNA binding activity and/or transactivation activity (38). However, regulation of Aft1 function is different from Mac1 and Zap1. Based on the results presented here, we conclude that the regulation of Aft1 function occurs at the level of its subcellular localization. Aft1 localizes in the nucleus only when cells are iron-depleted, and the expression of target genes is required.
There are a growing number of transcription factors that are regulated by nuclear localization. Regulated nuclear localization provides a mechanism by which cells can rapidly respond to changing environmental conditions (41,42). Besides metals, yeast must be able to sense nutrient availability. Nutrients such as phosphate and glucose regulate the subcellular localization of responsive transcription factors, Pho4 and Mig1, respectively. Pho4 up-regulates the expression of PHO5, which encodes a secreted acid phosphatase, upon the phosphate starvation. Pho4 is predominantly in the cytoplasm when cells are grown in phosphate-rich medium, whereas Pho4 is concentrated in the nucleus when yeast are starved for phosphate (43). The Mig1 glucose repressor localizes to the cytoplasm in FIG. 4. The effect of a mutation in a putative leucine-rich NES for Aft1 function. A, a putative NES sequence locates at residues 97-107 in Aft1. The NES sequence from yeast proteins Yap1 (49,50) and Pap1 (51), viral protein HIV-1 rev (28), and metazoan proteins PKI (29), MAPKK (32), IB (57), and Dsk-1 (58) are aligned. Important hydrophobic residues in the sequences are boxed. B, various hydrophobic amino acid residues were substituted to alanine residues at the indicated sites between residues 63 and 105 in the Aft1 protein. The localization of the wild type and mutant proteins in the cells grown in the iron-replete condition is summarized on the right. C, subcellular localization of wild type and mutant Aft1-HA proteins. Aft1-HA derivatives indicated were visualized by indirect immunofluorescence microscopy as described in Fig. 3. Aft1L99A (Y24) (right panel) were grown in the iron-starved medium, and total RNA was isolated after the addition of iron at the indicated times. RNA was separated on a 1% agarose gel containing formaldehyde and subjected to Northern blot analysis. The region from nt 1 to 649 (relative to the ATG start codon) of FTR1 was used for the probe. glucose-free medium and to the nucleus in glucose medium (44). In addition to nutrient availability, regulated nuclear transport has also been implicated in the response to environmental stress. Yap1 in S. cerevisiae and its homologue in Schizosaccharomyces pombe, Pap1, are also controlled by their nuclear localization in response to oxidative stress (45,46).
Many important questions concerning how iron regulates Aft1 nuclear localization remain to be elucidated. The questions include whether the iron status in cells regulates either import or export of Aft1. One well studied example for regulated nuclear import is the NF-B protein, a ubiquitous regulator of a variety of genes essential for cellular immune responses, inflammation, cell growth and development in mammalian cells. In uninduced cells, the nuclear localization signal (NLS) of NF-B is masked by interaction with the inhibitor IB. Upon stimulation, the NLS of NF-B becomes available for the import factor by disrupting this interaction (47,48). A well studied example of regulated nuclear export is Yap1. Recognition of the NES in Yap1 by the nuclear exporter, Crm1, is inhibited by oxidation of a cysteine-rich domain containing its NES (49 -51). A binary switch model, which implies both regulated import and export, has also been proposed for phosphate regulation. Phosphorylation of Pho4 abolishes binding to the nuclear import factor, Pse1 (52) and concomitantly leads to binding to the nuclear export factor, Msn5 (53) under conditions of high phosphate.
Another important question is whether iron directly regulates the nuclear localization of Aft1 or not. Based on features of the amino acid sequence of Aft1, iron may interact directly with Aft1 and inhibit nuclear localization of Aft1. Alternatively, the posttranslational modification of Aft1 may be involved in regulation of Aft1 nuclear localization in response to cellular iron status. Aft1 is reported to be phosphorylated (54). Iron status in the cell may change the phosphorylation/dephosphorylation status of Aft1 protein, thereby changing the recognition by import/export receptor(s), which mediate nucleo-cytoplasmic trafficking. Moreover, like IB, the interacting protein may be responsible for mediating the signal from iron status.
Interestingly, in metazoans, RNA binding proteins, IRP1 and IRP2, are responsible for sensing iron status in the cells and transducing this signal into regulated gene expression (4,55,56). This sensing is supposed to occur in the cytoplasm although a possible role of mitochondria in this process should not be neglected. The mechanism by which Aft1 senses iron in cells might be conserved even though the regulation occurs at the level of transcription in S. cerevisiae and at a posttranscriptional level in metazoans.