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J. Biol. Chem., Vol. 279, Issue 10, 9462-9474, March 5, 2004

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The Schizosaccharomyces pombe Corepressor Tup11 Interacts with the Iron-responsive Transcription Factor Fep1^*

Sadri Znaidi, Benoit Pelletier¶, Yukio Mukai||, and Simon Labbé**

From the Département de Biochimie, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada and the ^||Department of Biotechnology, Osaka University, Osaka 565-0871, Japan

Received for publication, November 24, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The Schizosaccharomyces pombe fep1⁺ gene encodes a GATA transcription factor that represses the expression of iron transport genes in response to elevated iron concentrations. This transcriptional response is altered only in strains harboring a combined deletion of both tup11⁺ and tup12⁺ genes. This suggests that Tup11 is capable of negatively regulating iron transport gene expression in the absence of Tup12 and vice versa. The tup11⁺- and tup12⁺-encoded proteins resemble the Saccharomyces cerevisiae Tup1 corepressor. Using yeast two-hybrid analysis we show that Tup11 and Fep1 physically interact with each other. The C-terminal region from amino acids 242 to 564 of Fep1 is required for interaction with Tup11. Within this region, a minimal domain encompassing amino acids 405-541 was sufficient for Tup11-Fep1 association. Deletion mapping analysis revealed that the WD40-repeat sequence motifs of Tup11 are necessary for its interaction with Fep1. Analysis of Tup11 mutants with single amino acid substitutions in the WD40 repeats suggested that the Fep1 transcription factor interacts with a putative flat upper surface on the predicted -propeller structure of this motif. Further analysis by in vivo coimmunoprecipitation showed that Tup11 and Fep1 are physically associated. In vitro pull-down experiments further verified a direct interaction between the Fep1 C terminus and the Tup11 C-terminal WD40 repeat domain. Taken together, these results describe the first example of a physical interaction between a corepressor and an iron-sensing factor controlling the expression of iron uptake genes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Iron is required for a number of biological functions in most life forms, from microbes to mammals (1, 2). Because its chemistry allows a reversible one-electron oxidation-reduction reaction, iron plays a crucial role in electron-transfer reactions (3). On the other hand, the redox active nature of iron can make it toxic due to its ability to unleash highly reactive species in the presence of oxygen that can damage biological cellular components (4). Because of its essential but toxic nature, highly regulated homeostatic controls have evolved across different species in order to maintain intracellular concentration of iron at levels needed for essential biochemical processes while preventing its accumulation to toxic levels (5).

Although iron is an abundant transition metal on earth, under aerobic conditions, it is usually present as insoluble ferric hydroxides (6). Because of its low bioavailability, organisms have developed a variety of mechanisms for iron acquisition including secretion and utilization of siderophores, ferrireductase activity at the cell surface, and heme-iron introduction inside the cell (7-10). In the model organism Schizosaccharomyces pombe, two pathways for iron uptake have been identified (11-13). The first one consists of a siderophore-iron transport system. Although S. pombe does not produce siderophores, the fission yeast can utilize siderophore-bound iron complexes produced by other microorganisms using three transmembrane proteins, Str1, Str2, and potentially Str3 (11). Of these three siderophore transporters, Str1 exhibits specificity for ferrichrome-iron, while Str2 is specific for ferroxiamine B-iron and to a lesser extent ferrichrome-iron. Although Str3 may participate in the mobilization of iron bound to siderophores, its substrate specificity has not been determined (11). The second pathway for iron assimilation in S. pombe involves three components, Frp1, Fio1, and Fip1 (12, 13). The Frp1 protein reduces Fe³⁺ to Fe²⁺ at the cell surface, rendering iron accessible to iron-binding extracellular ligands found in the iron-transport proteins in the plasma membrane (13, 14). Once reduced, iron is transported by two high affinity iron uptake proteins encoded by the fio1⁺ and fip1⁺ genes (12). The Fio1 protein is a multicopper ferroxidase that converts Fe²⁺ to Fe³⁺, which is then transported by Fip1, a transmembrane iron permease (12). The frp1⁺, fio1⁺, and fip1⁺ genes are regulated by the cellular need for iron. When cells are grown under iron-deficient conditions, frp1⁺, fio1⁺, and fip1⁺ mRNA levels are induced. In contrast, iron-replete conditions repress the expression of these genes (12, 13, 15). As expected, str1⁺, str2⁺, and str3⁺ gene expression is up-regulated under conditions of iron starvation and down-regulated under conditions of iron repletion (11). It was recently shown that the transcription factor Fep1¹ mediates the iron-dependent repression of str1⁺, str2⁺, str3⁺, frp1⁺, fio1⁺, and fip1⁺ transcription (11, 16). The N-terminal region of Fep1 is highly similar to the N-terminal regions of the Urbs1, SREA, and SRE proteins that have been shown to negatively regulate, in an iron-dependent manner the siderophore biosynthesis pathways of Ustilago maydis, Aspergillus nidulans, and Neurospora crassa, respectively (17-22). This region of Fep1 harbors two consensus GATA-type zinc finger motifs that has been shown to be required for DNA binding to the consensus sequence 5'-(A/T)GATAA-3' (16). Thus, Fep1 is related to a family of GATA-type transcription factors (23).

Interestingly, a mutant fission yeast strain with deletions in both tup11⁺ and tup12⁺ genes exhibited a fio1⁺ gene expression that was highly derepressed and unresponsive to repression by iron (16). Elimination of either Tup11 or Tup12 alone was not sufficient to annihilate the iron-mediated repression of fio1⁺ (16). These observations suggest that, tup11⁺ and tup12⁺, known to encode transcriptional corepressors, are functionally redundant in down-regulating the expression of the iron transport genes. Recent work has demonstrated that Tup11 assembles with itself or the Tup12 protein (24). The S. pombe Tup11 and Tup12 proteins exhibit 39.9 and 43.5% identity to Saccharomyces cerevisiae Tup1, respectively (25). Based on the extended homology of Tup11 and Tup12 to Tup1, putative functional domains have been designated for the Tup1 homologues in S. pombe (26). The N-terminal 70 and 87 amino acids of the S. pombe Tup11 and Tup12 proteins, respectively, bear homology to a similar region in Tup1 that is known to interact with Ssn6. Like Tup1, both Tup11 and Tup12 contain a homologous region located in the middle part of their N-terminal halves that is required for interaction with histones H3 and H4. Consistent with this observation, it has been shown that Tup11 associates with histones H3 and H4 of S. cerevisiae in vitro (26). Within their C termini, Tup11 and Tup12 contain seven copies of a repeated amino acid motif, named WD40 repeat (27), found in Tup1 and other Tup1-like corepressors as well as in other proteins that are unlinked to transcription like the -subunit of the trimeric G-proteins (28). Based on the x-ray crystal structures of three proteins with seven WD40 repeats, including the Tup1 corepressor, each repeat folds into four antiparallel strands, forming a blade structure (29-31). Each WD40 repeat unit is interconnected to the other by a loop to form a seven-bladed -propeller, structure that is highly symmetrical and assumes an overall donut shape (29, 31). Although biochemical data has shown that the sides of the -propeller are capable of establishing protein-protein contacts, the flat upper surface has been shown to bind to some proteins (30, 32).

Previous studies have shown that S. cerevisiae Tup1 and Ssn6 proteins physically interact in vivo to form a corepressor complex (33). Furthermore, it has been demonstrated that Tup1 forms a tetramer with one Ssn6 molecule (34). Although the Ssn6-Tup1 corepressor complex is incapable of binding to DNA, it is attracted to the regulatory regions of different genes by interacting with transcription factors that function in specific metabolic pathways (28). A number of potential mechanisms by which the Ssn6-Tup1 complex can repress gene expression have been described (28). First, upon location to the target promoter, the corepressor complex may inhibit the activities of the basal transcription factors that activate gene expression through the RNA polymerase II holoenzyme (35). Second, the Ssn6-Tup1 may promote the formation of a repressive chromatin structure (36, 37). Third, the formation of a complex between Tup1 and the mRNA 5'-triphosphatase Cet1 may abrogate mRNA stability and translation through the association of Tup1 with the mRNA capping enzyme (38).

Based on our previous findings that the S. pombe Tup11 protein has the potential to regulate the expression of the fio1⁺ iron transport gene (16), we sought to determine its ability to interact with the iron-sensing DNA-binding repressor Fep1. Using two-hybrid analysis, we demonstrate that Tup11 interacts with the C-terminal region of Fep1, but not with the two adjacent zinc fingers in the N-terminal region. The WD40-repeat domain in Tup11 is necessary for its interaction with Fep1. In addition, we showed that the amino acid residues Tyr³⁶² and Leu⁵⁴², located on the same surface of the predicted -propeller structure of the Tup11 WD40 repeats, are critical for the Tup11-Fep1 interaction. When coexpressed in fission yeast, the Tup11 and Fep1 proteins were detected in a heteroprotein complex by coimmunoprecipitation experiments. Furthermore, in vitro protein binding analysis using fusion proteins that were expressed in and purified from Escherichia coli revealed that the Tup11 and Fep1 C-terminal regions directly interact with each other. Taken together, our findings indicate that Tup11 and Fep1 are components of a heteromeric complex that is required for transcriptional down-regulation of genes that are critical for iron acquisition in fission yeast.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Yeast Strains and Media—The S. cerevisiae strain L40 (Mata his3200 trp1-901 leu2-3, 112 ade2 LYS2::(lexAop)₄-HIS3 URA3::(lexAop)₈-lacZ) (39) was used for two-hybrid analysis. When plasmid selection was required, the L40 strain was grown in synthetic complete (SC) medium minus the indicated nutrients (40). For routine growth, yeast extract (1%), bactopeptone (2%), dextrose (2%) (YPD) medium was used (41). The wild-type S. pombe strain used in this study was JY741 (h- leu1-32 ura4-18 ade6-M210) (16). The fission yeast strain was maintained on yeast extract plus supplement (YES) medium (42, 43). Under selective conditions, S. pombe cells were grown on Edinburgh minimal medium lacking specific nutrients required for plasmid selection (43). Iron starvation or iron repletion was performed by adding the indicated amount of bathophenanthrolinedisulfonic acid (BPS) or FeCl₃ to cells grown to mid-logarithmic phase (OD_{600 nm} 1.0). At this mid-logarithmic phase, cells were treated for 90 min at 30 °C. Subsequent to treatment, 20-ml samples were withdrawn from the cultures for steady-state mRNA or protein analyses (16, 44).

Plasmids—To generate the LexA-tup11⁺codons1-614 plasmid, the full-length tup11⁺ gene was isolated by PCR using primers that corresponded to the start and stop regions. The tup11⁺ gene was amplified from the plasmid pBTM-tup11 (26, 38). The PCR product obtained was digested with BamHI and PstI and cloned into the corresponding sites of pBluescript SK. Once sequenced to verify that no DNA sequence alterations were present, the tup11⁺ gene was cloned into the BamHI and PstI sites of pLexN-a (45) to produce pLexA-tup11⁺codons1-614. Plasmids pLexA-tup11⁺codons1-92, pLexA-tup11⁺codons1-301, and pLexA-tup11⁺codons1-356 were created by a similar strategy, except that only the first 92, 301, and 356 codons of tup11⁺ were isolated by PCR. Likewise, the wild-type tup11⁺ codons 69-301, 69-356, 69-614, and 301-356 were isolated by PCR and cloned downstream of and in-frame to the lexA gene, creating the pLexA-tup11⁺codons69-301, pLexA-tup11⁺codons69-356, pLexA-tup11⁺codons69-614, and pLexA-tup11⁺codons301-356 plasmids. Chimeric plasmids containing the first 211 codons of lexA fused to the tup11⁺codons 134 through 614, tup11⁺codons 192 through 614, tup11⁺codons 245 through 614, tup11⁺codons 267 through 614, or tup11⁺codons 284 through 614 were also created and designated pLexA-tup11⁺codons134-614, pLexA-tup11⁺codons192-614, pLexA-tup11⁺codons245-614, pLexA-tup11⁺-codons267-614, and pLexA-tup11⁺codons284-614, respectively. Using pLexA-tup11⁺codons1-614, the mutations Tyr362 Cys and Leu⁵⁴² Ser were created by the overlap extension method (46). The DNA sequence of the 1845-bp BamHI-PstI fragment from each respective PCR-amplified fragment was used to replace the equivalent fragment from plasmid pLexA-tup11⁺codons1-614 to produce the pLexA-Tup11 Tyr³⁶² Cys and pLexA-Tup11 Leu⁵⁴² Ser mutant plasmids. The DNA sequence of the BamHI-PstI fragment from each respective mutant was confirmed by dideoxy sequencing. To create the prey plasmids, pVP16-fep1⁺codons2-564, pVP16-fep1⁺codons242-564, pVP16fep1⁺-codons319-564, pVP16-fep1⁺codons360-564, pVP16-fep1⁺codons390-564, pVP16-fep1⁺codons405-564, and pVP16-fep1⁺codons432-564, BamHI-NotI fragments of the fep1⁺ gene containing different 5'-termini relative to the start codon of the gene but all extending through the stop codon were inserted into pVP16. Likewise, the fep1⁺codons 2-281 were amplified by PCR, purified, and inserted in-frame into pVP16, producing the pVP16-fep1⁺codons2-281 plasmid. Plasmids pVP16-fep1⁺codons242-457, pVP16-fep1⁺codons319-457, pVP16-fep1⁺-codons360-457, pVP16-fep1⁺codons360-491, pVP16-fep1⁺codons360-541, and pVP16-fep1⁺codons405-541 contained the wild-type fep1⁺ C-terminal codons 242-457, 319-457, 360-457, 360-491, 360-541, and 405-541, respectively. All six plasmids contained these fep1⁺ sequences cloned into the BamHI and NotI sites of pVP16.

Two-hybrid Analysis—To study the association between Tup11 and Fep1, the complete or truncated versions of the tup11⁺ open reading frame were inserted downstream of and in-frame to the E. coli lexA gene as bait. The prey plasmid, pVP16 (45, 47), contains the VP16 acidic activation domain followed by a polylinker in which different fragments of the fep1⁺ gene were introduced. Each L40 transformant strain harboring the indicated bait and prey plasmids was tested for the interaction of the two fusion proteins using previously described standard procedures (48). For quantitative measurements, -galactosidase activity was determined using early logarithmic phase cultures (OD_{600 nm} of 0.5) of yeast cells transformed with the indicated plasmids. 4-ml samples were withdrawn from the cultures, and the cells were harvested, washed with sterile water, and resuspended in 700 µl of Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, pH 7.0, 10 mM KCl, 1 mM MgSO₄, 50 mM -mercaptoethanol). The cells were permeabilized by adding 50 µl of chloroform and 50 µl of 0.1% SDS. After the cell suspension was vortex-mixed for 10 s, 200 µl of 4 mg/ml o-nitrophenyl--D-galactopyranoside was added to each sample. Following a 10-min incubation at 30 °C, 350 µl of 1 M Na₂CO₃ was added to stop the reactions. After clarification by centrifugation at 4 °C, the A_{420 nm} was measured within the linear response range and expressed in standard units (49). Values shown are the average of triplicate assays of three independent transformants. In addition to liquid -galactosidase assays, a riboprobe derived from the plasmid pKSlacZ (50) was used to monitor the steady-state levels of lacZ mRNAs from the integrated (lexAop)₈-lacZ reporter construct in the L40 strain. Total RNA was extracted by the hot phenol method as described previously (51). RNase protection analyses were carried out as described previously (52) using the plasmid pKSACT1 (50) to probe ACT1 mRNA as an internal control. For protein expression analysis of the LexA-Tup11 and VP16-Fep1 fusion protein derivatives, and PGK, the following antisera were used for immunodetection: monoclonal anti-LexA antibody 2-12; monoclonal anti-VP16 antibody 1-21 (both from Santa Cruz Biotechnology, Santa Cruz, CA); and monoclonal anti-PGK antibody (Molecular Probes, Eugene, OR).

Protein Coimmunoprecipitation Experiments—To determine whether Fep1 interacts with Tup11 in fission yeast cells, plasmid pNTAP-fep1⁺codons2-564 was constructed as follows. The fragment containing the fep1⁺ gene (codons 2-564) was isolated by PCR using Pfu Turbo polymerase (Stratagene, La Jolla, CA), purified, and cloned into the BamHI and NotI sites of pREP1-NTAP (53). Plasmid pNTAP-fep1⁺codons319-564 was generated by using a similar approach, except that the DNA fragment harboring the fep1⁺ gene corresponded to codons 319 through 564. Plasmid pGST-tup11⁺ containing the full-length S. pombe tup11⁺ gene fused downstream of and in-frame to the GST coding region was created by inserting a BamHI-SacI fragment encompassing the entire coding sequence of the tup11⁺ gene into the corresponding sites of pAAUGST (54). The coding region of the tup11⁺ gene that contains either the mutation Tyr³⁶² Cys or Leu⁵⁴² Ser was also swapped for an identical DNA region into the pGST-tup11⁺ plasmid, creating pGST-tup11Tyr³⁶² Cys and pGST-tup11Leu⁵⁴² Ser. For co-immunoprecipitation experiments, JY741 cells were co-transformed with pGST-tup11⁺ or mutant derivatives or pAAUGST, and pNTAP-fep1⁺codons2-564, or pNTAP-fep1⁺codons319-564. Cells were grown to OD_{600 nm} of 1.0 in the absence of thiamine after 18-20 h in culture. Cells were broken with glass beads in buffer G (30 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA, pH 8.0, 5 mM dithiothreitol, 10 mM -mercaptoethanol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride) in the presence of a complete protease inhibitor mixture (P8340, from Sigma) followed by a centrifugation at 3500 rpm at 4 °C for 5 min. Protein extracts (3 mg of total protein) were incubated for 16 h with 50 µl of glutathione-Sepharose 4B beads (Amersham Biosciences, Uppsala, Sweden). Beads were washed five times with 1.5 ml of buffer G, with the beads completely transferred to a fresh microtube before the last wash. The immunoprecipitates were resuspended in 50 µl of SDS loading buffer, incubated for 5 min at 95 °C, and resolved by electrophoresis on a 9% SDS-polyacrylamide gel. Proteins were electrophoretically transferred to nitrocellulose membranes. Blots were incubated with primary antibody for 2 h at room temperature. For protein analysis of GST-Tup11, NTAP-Fep1, and PCNA, the following primary antisera were used: polyclonal anti-GST antibody Z-5 (Santa Cruz Biotechnology); polyclonal anti-mouse IgG antibody (ICN Biomedicals, Aurora, OH); and monoclonal anti-PCNA antibody PC10 (Sigma). After immunoblots were washed in TBS (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% bovine serum albumin), membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) for 30 min, washed, and developed with enhanced chemiluminescent detection reagents.

Expression and Purification of the MBP-Fep1 and GST-Tup11 Fusion Proteins in E. coli—The plasmid pMAL-fep1⁺(codons 319-564) was constructed by cloning a 738-bp BamHI-PstI DNA fragment containing the C-terminal 319-564 codons of Fep1 into the same sites of pMAL-c2X (New England BioLabs, Beverly, MA). The expression and purification of MBP-³¹⁹Fep1⁵⁶⁴ fusion protein from E. coli were carried out essentially as described previously (16). Plasmids pGEX-tup11N (codons 1-298) and pGEX-tup11C (codons 297-614) were described previously (38). These fusion plasmids were transformed into E. coli TB1 cells. Fresh transformants of TB1 cells were grown to early growth phase (OD_{600 nm} of 0.5) in Luria-Bertani (LB) medium supplemented with 0.2% glucose and 100 µg/ml ampicillin. At this early logarithmic growth phase, the cells were induced with 0.2 mM isopropyl--D-thiogalactopyranoside for 2 h at 25 °C. Purification of the GST-Tup11 fusion proteins from E. coli cells was performed using glutathione-Sepharose 4B beads following the manufacturer's protocol, with minor modifications. Cells were broken with glass beads in buffer G as described above. Protein extracts (1.4 mg of total protein) were incubated for 30 min at 4 °C with 50 µl of glutathione-Sepharose 4B using siliconized microtubes (Fisher). Beads were washed extensively with buffer G as described above. The different GST fusion proteins bound to the beads were eluted with 2-bed volumes of 5 mM glutathione in 30 mM Tris-HCl (pH 8.0), separated by SDS-PAGE, and visualized by Coomassie Brilliant Blue stain, or used for Western blot analysis. Purified fusion proteins produced in E. coli were used for GST pull-down experiments. The MBP and MBP-Fep1 fusion proteins (3.2 µg) were incubated with equivalent amount of GST-Tup11N, GST-Tup11C, or GST proteins bound to glutathione-Sepharose 4B beads. After 16 h at 4 °C, the resin was sedimented, and the supernatant removed. The resin was washed five times with 1.5 ml of buffer G. The bound protein complexes were resuspended in Laemmli buffer and heated at 95 °C for 5 min prior to SDS-polyacrylamide gel electrophoresis and immunoblot analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Tup11 Associates with the S. pombe Fep1 Iron-sensing Transcription Factor—We have previously shown by genetic analysis that Fep1 requires the presence of the cofactor Tup11 or Tup12 to repress the expression of the iron transport genes under conditions of iron repletion (16). Based on this observation, we examined the possibility that Tup11 physically interacts with Fep1. We performed two-hybrid analyses using the full-length tup11⁺ gene fused to the LexA coding region as bait and different coding regions of the fep1⁺ gene fused to the coding region of the VP16 acidic activation domain as prey. Interestingly, coexpression of the full-length Fep1 fused to VP16 with the LexA-Tup11 fusion protein, produced high levels of -galactosidase activity (448 Miller units) (Fig. 1), indicating a strong interaction between these proteins. To gain insight into the specific region of Fep1 that interacts with Tup11, we first tested an N-terminal segment of Fep1 (residues 2-281) that contains its DNA-binding domain (16). This region, which bears two Cys₂/Cys₂-type zinc finger motifs failed to interact with the LexA-Tup11 fusion protein. In contrast, the C-terminal portion of Fep1 (residues 242-564) interacted with the LexA-Tup11 fusion protein. Although the induction of lacZ was lower by 30% compared with the full-length Fep1, a clear transactivation of the reporter gene expression was observed (Fig. 1). Further removal of the amino-terminal residues 242-318 of Fep1 had little effect (8% decrease) on the activity of the reporter gene. Based on this observation, we used the chimeric protein that contains the N-terminal 78 amino acids of VP16 and the C-terminal 245 amino acids of Fep1 (residues 319-564) in subsequent analyses. To ensure that the fusion proteins were expressed in the transformed cells, immunoblot analyses of protein extracts were performed using anti-LexA and anti-VP16 antibodies (Fig. 1C). Although all of the VP16-Fep1 fusion proteins used in this study were consistently detected by immunoblotting, we were unable to detect the VP16 polypeptide alone, perhaps, owing to its low predicted molecular weight of 8 kDa. As shown in Fig. 1D, the association between the LexA-Tup11 and VP16-Fep1 fusion proteins in bakers' yeast cells using a two-hybrid system was not modulated by cellular iron status. Indeed, the LexA-Tup11 and VP16-Fep1 fusion proteins that specifically associate with each other gave a constitutive steady-state level of lacZ mRNA as assayed by RNase protection experiments under both iron-starvation and iron-replete conditions.

View larger version (30K): [in this window] [in a new window]   FIG. 1. Tup11 interacts with the Fep1 C-terminal region. A, schematic illustration of the LexA DNA-binding domain (DBD) used without or with the full-length Tup11 protein. The indicated bait molecule was coexpressed with the VP16 activation domain (AD) or different VP16AD-Fep1 fusion derivatives. The amino acid sequences of the Tup11 and Fep1 proteins are numbered relative to their first initiator codons, respectively. B, the constructs shown in A were coexpressed in the S. cerevisiae strain L40 grown under basal conditions. Positive interactions between the proteins were detected by liquid -galactosidase assays. The values are the averages of triplicate determinations ± S.D. C, whole cell extracts were prepared from aliquots of cultures used in B and analyzed by immunoblotting. D, total RNA from 100 µM BPS (B), basal (-), and 100 µM FeCl3 (Fe) cultures was isolated and analyzed. Shown is a representative RNase protection assay of lacZ and ACT1 mRNA steady-state levels.

View larger version (29K): [in this window] [in a new window]   FIG. 2. Minimal C-terminal region of Fep1 required for interaction with LexA-Tup11 fusion protein by two-hybrid assay. A, LexA-Tup11 fusion protein was co-expressed with a series of VP16-Fep1 truncations in the L40 strain. Positive interactions between the proteins were detected by liquid -galactosidase assays. B, the LexA-Tup11 fusion protein and truncated versions of VP16-Fep1 fusion proteins were detected by immunoblotting using either anti-LexA or anti-VP16 antibody. As a control, total extract preparations were probed with anti-PGK antibody.

View larger version (32K): [in this window] [in a new window]   FIG. 3. C terminus of Tup11 encompassing the WD repeats is required for interaction with the C-terminal region of Fep1. A, schematic illustration of truncated versions of the LexA-Tup11 fusion protein (left side). The N-terminal 69 amino acid residues of Tup11 represent a putative Ssn6-binding region (Ssn6). Likewise, residues 69-301 of Tup11 are potentially required for interaction with histones H3 and H4. The C terminus of Tup11, from residues 301 to 614, bears seven putative WD40 repeats. Right side shows the fusion of VP16 activation domain (AD) to the last 246 amino acids of the Fep1 protein used as prey in these assays. B, as a measure of protein-protein interactions, liquid -galactosidase assays were carried out. Error bars indicate the S.D. of samples analyzed in triplicate. C, protein extracts were prepared from aliquots of cultures used in B, and then analyzed by immunoblotting using either anti-LexA, anti-VP16, or anti-PGK (as an internal control) antibody. D, cells were incubated in the absence (-) or presence of 100 µM BPS (B) or 100 µM FeCl3 (Fe). After total RNA extraction, the lacZ steady-state mRNA levels were analyzed by RNase protection assay with actin (ACT1) as an internal control. Results shown are representative of three independent experiments. lacZ and ACT1 are indicated by arrowheads.

View larger version (30K): [in this window] [in a new window]   FIG. 4. The C-terminal 331 amino acid residues of Tup11 containing the WD40 motifs are sufficient for interaction with the C terminus of Fep1. A, anatomy of the LexA-Tup11 fusion proteins (left side). Chimeric VP16-Fep1 molecule used as a prey is shown (right side). B, the -galactosidase activity of a (lexAop)8-lacZ reporter was measured. Each set of constructs was assayed in triplicate. C, total cell extracts from cultures used in B were analyzed by immunoblotting using anti-LexA, anti-VP16, or anti-PGK antibody. D, total RNA was extracted from cultures incubated under conditions of iron starvation (B, 100 µM BPS) versus basal (-) or iron-repleted (Fe, 100 µM FeCl3) conditions. The lacZ and ACT1 (as a control) mRNA steady-state levels are indicated with arrowheads.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Substitutions of the Tyr362 and Leu542 amino acid residues within the C-terminal region of Tup11 block the interaction with Fep1. A, schematic diagram of the LexA-Tup11 fusion derivatives. The point mutations are marked with an X. Chimeric VP16-Fep1 molecule composed of the indicated region of Fep1 was co-expressed with the LexA-Tup11 fusion proteins. B, liquid -galactosidase activities were assayed in the L40 strain using a (lexAop)8-lacZ reporter. Each sample was assayed in triplicate. C, whole cell extracts from cultures used in B were analyzed for LexA-Tup11, LexA-Tup11 Tyr362 Cys, LexA-Tup11 Leu542 Ser, VP16-Fep1, or PGK levels by immunoblotting. D, cells transformed with the indicated constructs, were grown under iron-starvation conditions (B), untreated (-), or treated with FeCl3 (Fe) at final concentration of 100 µM for 90 min. After total RNA extraction, the lacZ and ACT1 steady-state mRNA levels (indicated by arrowheads) were analyzed by RNase protection assay. Results shown are representative of three independent experiments.

View larger version (38K): [in this window] [in a new window]   FIG. 6. Three-dimensional model of the C-terminal WD40 repeat domain of Tup11. A, amino acid sequence of Tup11 WD40 repeats aligned with the C-terminal WD40 repeat domain of the S. cerevisiae Tup1 corepressor. Amino acids that are identical in both proteins are indicated with an asterisk, whereas dots represent amino acid similarity between Tup11 and Tup1. The Tyr362 and Leu542 residues are underlined. The sequence of the WD40 regions in Tup1 that were used to model the structure of the Tup11 WD40 repeats contains internal deletions (indicated by a triangle below) because these regions were not structurally interpretable (31). One deletion corresponding to residues 514-528 was made in

View larger version (21K): [in this window] [in a new window]   FIG. 7. Tup11 and Fep1 physically interact in S. pombe. Strain JY741 was transformed with TAP-tagged full-length Fep1 or TAP-tagged 319Fep1564 (retaining amino acids 319-564) containing plasmids expressing either GST-tagged Tup11 or GST alone. Extracts (Total) were subjected to GST pull-down using glutathione-Sepharose. The proteins bound to the beads were heated, loaded, and separated on 9% SDS-polyacrylamide gels, blotted to nitrocellulose, and visualized with the anti-mouse IgG antibody. TAP-2Fep1564 and TAP-319Fep1564 are indicated with arrowheads. As specific controls, aliquots of whole cell extracts and bound fractions were probed with anti-PCNA antibody and anti-GST antibody (shown with arrowheads). Extracts (Total) loaded on gels represent 2% of the whole extracts used in the binding reactions. WB, Western blot.

View larger version (19K): [in this window] [in a new window]   FIG. 8. In vivo association between Tup11 and Fep1 requires the conserved Tyr362 and Leu542 residues within the WD40 repeat domain of Tup11. Total cell extracts (T) were prepared from JY741 cells harboring TAP-319Fep1564 fusion protein and the indicated GST-tagged-1Tup11614 derivatives or GST alone. Lysates were incubated with a glutathione affinity resin and washed, and the bound fraction (B) was analyzed by immunoblot assay using anti-mouse IgG antibody. A portion of the total cell extracts (2%) was included to verify the presence of the immunoblotted proteins prior to chromatography. Furthermore, aliquots of all samples (extracts and bound fractions) were analyzed by immunoblotting using anti-GST antibody. WB, Western blot.

View larger version (20K): [in this window] [in a new window]   FIG. 9. The C-terminal region of Fep1 interacts directly with the Tup11 protein through its WD40 repeat domain. Purified MBP or MBP-319Fep1564 (Input) was incubated with a suspension of glutathione Sepharose resin carrying GST-1Tup11N298, GST-297Tup11C614, or GST. Bound fractions (B) were analyzed by SDS-polyacrylamide gel electrophoresis and subjected to Western blotting (WB) using MBP monoclonal antibody (top panel) and GST polyclonal antibody (bottom panel).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Deletion mapping studies of VP16-²Fep1⁵⁶⁴ fusion protein have shown that the C-terminal amino acids from residues 242 to 564 are required for physical interaction of Fep1 with the corepressor Tup11. Within this region of Fep1 lies a domain from residues 405 to 541 that constitutes the minimal module for this interaction. Interestingly, this minimal module contains several leucine-proline dipeptide repeats. One of these repeats, ⁴¹⁴Leu-Pro-Pro-Ile-Leu-Pro⁴¹⁹, is highly conserved in other iron responsive transcriptional repressors including Urbs1 from U. maydis (22), Srea from A. nidulans (18), and Srep from P. chrysogenum (67). Furthermore, in S. cerevisiae, this dipeptide repeat is also found in the Mig1 and Rox1 sequence-specific DNA binding transcription factors necessary for glucose and oxygen repression, respectively (68, 69). In fact, it has been suggested that this di-leucine-proline motif may play a role in protein-protein interactions with the Tup1 corepressor complex (68). The contribution of these residues or other residues in the C-terminal region encompassing amino acids 405-541 of Fep1 to the interaction between Fep1 and Tup11 must await a comprehensive dissection of that minimal module.

A hallmark of the fungal GATA factors Fep1 (16), Urbs1 (70), Srea (18), Sre (71), and Srep (67) is that, like the vertebrate GATA transcription factors, two Cys₂-Cys₂-type zinc finger motifs are found in their DNA-binding domain. In Fep1, these motifs are within the N-terminal portion of the protein (residues 12-220). In the mammalian GATA-1 transcription factor, the C-terminal zinc finger has been shown to be necessary and sufficient for DNA binding (72, 73). Although the N-terminal zinc finger has been shown to increase stability and influence specificity of the binding of GATA-1-DNA complexes, this zinc finger has also been demonstrated to mediate specific protein-protein interactions (73-77). Using two-hybrid analyses and in vitro protein binding experiments, we showed that Tup11 interacts with the C-terminal portion of Fep1, which does not contain the two adjacent zinc finger motifs. This suggests that the N-terminal zinc finger is not required for association between Fep1-Tup11. Further studies will be needed to assess the nature of the function of the zinc fingers in Fep1 with respect to its DNA binding activity and ability to mediate interactions with other proteins.

In this study, we have identified and demonstrated a role for the C-terminal WD40 repeat domain of the S. pombe Tup11 protein. This domain consists of seven WD40 repeats. The model predicts that these repeats clasp together into a circular, seven-bladed propeller with a relatively flat top and bottom. Deletion mapping analyses revealed that the WD40 repeat sequence motifs of Tup11 are requisite for the Fep1-Tup11 interaction. The first Tup11 WD40 repeat failed to interact with Fep1 when fused to the LexA domain by two-hybrid assay, suggesting that amino acids from more than one WD repeat are required for binding Fep1. Analysis of the WD40 repeat structure using computer algorithms predicted that the Tup11 Tyr³⁶² and Leu⁵⁴² residues in the WD40 repeats 2 and 6, respectively, were exposed on the upper surface of the -propeller. We tested the roles of these residues in Tup11-Fep1 interaction by replacing Tyr³⁶² with cysteine or Leu⁵⁴² with serine and found that these mutations abolished the ability of Tup11 to interact with Fep1. Although we have not ascertained the contributions of each of the seven WD40 repeats that comprise the putative -propeller, taken together, these data suggest that proteins with WD40 repeats may use different blades of the propeller to provide binding sites for different partners. Indeed, the WD40-repeat protein RACK1 interacts with the PDE4D5 protein through its WD40 repeats 5-7, while the S. cerevisiae Tup1 can associate with 2 using only WD40 repeat 1 (30, 78).

It is currently unknown how sequence-specific DNA-interacting proteins are capable of recruiting the Tup corepressor complex. Recent studies have suggested a mechanism whereby relief from repression can be achieved (79, 80). In S. cerevisiae, under non-osmotic stress conditions, the Tup1 corepressor complex and its interacting partner protein Sko1 are required to negatively regulate expression of a subset of osmotic stress-responsive genes (81). It was shown that the Hog1 kinase can overcome the repressive action of the Tup1 corepressor complex by phosphorylating the DNA-binding transcription factor Sko1 (80, 81). Under osmotic stress, the phosphorylation of Sko1 by Hog1 converts the Sko1-Tup1 corepressor complex into an activator that recruits the SAGA histone acetylase and SWI/SNF nucleosome remodeling complexes to derepress osmotic-inducible promoters (80). Interestingly, S. cerevisiae Hog1 and S. pombe Spc1 (also named StyI) share a high percentage of sequence identity (82%).² Both are homologs of the mammalian stress-activated protein kinases (82). The Hog1 kinase is activated in response to osmotic stress, whereas activation of Spc1 is mediated by a wide range of stimuli including oxidative stress, nutrient limitation, and heavy metal toxicity (83). We tested the possibility that spc1⁺ plays a role in the regulation of the genes that are known to be involved in iron uptake in S. pombe. Using cells bearing a disruption of the sty1⁺ gene, we have been unable to demonstrate a loss of expression or lack of iron regulation of mRNA levels in each of these genes.³ Nevertheless, this observation leaves open the possibility that an iron starvation-activated cellular kinase may be involved in modulating iron-responsive gene expression of the fission yeast iron transport genes through the Fep1-Tup11 corepressor complex.

	FOOTNOTES

 
Tup11, allows the propeller of Tup11 (B and C, shown in green) to overlay remarkably well with that of Tup1. Predicted regions of Tup11 that differ from Tup1 are shown in red (B and C). Using the DeepView program (59), ribbon representations (B and C) of the three-dimensional structure of Tup11 were obtained. B, view from the top surface of the -propeller. The blades are numbered according to the Tup1 propeller structure. The positions of the mutations that affect the Tup11-Fep1 interaction are indicated with arrows. C, view from the side highlighting the fact that Tyr³⁶² and Leu⁵⁴² residues are exposed and located on the same surface of the -propeller. D, space-filled representation of the C-terminal WD40 domain of Tup11 emphasizing the accessibility of the Tyr³⁶² and Leu⁵⁴² residues (shown in green) for interaction with Fep1.

^* This study was supported by the NSERC of Canada Grant 238238-01 (to S. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported in part by the Université de Sherbrooke Faculty of Medicine.

^¶ Recipient of studentships from the NSERC of Canada and Fonds de la Recherche en Santé du Québec.

^** A New Investigator Scholar from the Canadian Institutes of Health Research. To whom correspondence should be addressed: Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, 3001 12e Ave Nord, Sherbrooke (Québec) J1H 5N4 Canada. Tel.: 819-820-6868 (ext. 15460); Fax: 819-564-5340; E-mail: Simon.Labbe{at}USherbrooke.ca.

¹ The abbreviations used are: Fep1, Fe protein 1; AD, activation domain; BPS, bathophenanthrolinedisulfonic acid; GST, glutathione S-transferase; MBP, maltose-binding protein; TAP, tandem affinity purification; PCNA, proliferating cell nuclear antigen.

² S. Znaidi, B. Pelletier, and S. Labbé, unpublished data.

³ B. Pelletier, and S. Labbé, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Maria M. O. Peña for comments on the article. We are grateful to Kevin A. Morano and Anne B. Vojtek for their generous gifts of S. cerevisiae strain L40 and plasmids. We would like to thank Kathleen L. Gould for the pREP1-NTAP plasmid used in this study. We thank Bruno Lamontagne and Sherif Abou Elela for helpful discussions regarding GST pull-down and coimmunoprecipitation experiments. Infrastructure equipment essential for conducting this investigation was obtained through the Canada Foundation for Innovation Grant NOF-3754 (to S. L.).

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