The Schizosaccharomyces pombe Corepressor Tup11 Interacts with the Iron-responsive Transcription Factor Fep1
2004; Elsevier BV; Volume: 279; Issue: 10 Linguagem: Inglês
10.1074/jbc.m312787200
ISSN1083-351X
AutoresSadri Znaidi, Benoit Pelletier, Yukio Mukai, Simon Labbé,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoThe 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 β-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. 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 β-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. Iron is required for a number of biological functions in most life forms, from microbes to mammals (1Kaplan J. Cell. 2002; 111: 603-606Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 2Eide D.J. Annu. Rev. Nutr. 1998; 18: 441-469Crossref PubMed Scopus (240) Google Scholar). Because its chemistry allows a reversible one-electron oxidation-reduction reaction, iron plays a crucial role in electron-transfer reactions (3Wessling-Resnick M. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 285-314Crossref PubMed Scopus (60) Google Scholar). 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 (4Touati D. Arch. Biochem. Biophys. 2000; 373: 1-6Crossref PubMed Scopus (664) Google Scholar). 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 (5Kaplan J. Semin. Hematol. 2002; 39: 219-226Crossref PubMed Scopus (21) Google Scholar). Although iron is an abundant transition metal on earth, under aerobic conditions, it is usually present as insoluble ferric hydroxides (6Boukhalfa H. Crumbliss A.L. Biometals. 2002; 15: 325-339Crossref PubMed Scopus (388) Google Scholar). 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 (7Rutherford J.C. Jaron S. Winge D.R. J. Biol. Chem. 2003; 278: 27636-27643Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 8Philpott C.C. Protchenko O. Kim Y.W. Boretsky Y. Shakoury-Elizeh M. Biochem. Soc. Trans. 2002; 30: 698-702Crossref PubMed Scopus (59) Google Scholar, 9Haas H. Appl. Microbiol. Biotechnol. 2003; 62: 316-330Crossref PubMed Scopus (234) Google Scholar, 10Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachmiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (461) Google Scholar). In the model organism Schizosaccharomyces pombe, two pathways for iron uptake have been identified (11Pelletier B. Beaudoin J. Philpott C.C. Labbé S. Nucleic Acids Res. 2003; 31: 4332-4344Crossref PubMed Scopus (70) Google Scholar, 12Askwith C. Kaplan J. J. Biol. Chem. 1997; 272: 401-405Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 13Roman D.G. Dancis A. Anderson G.J. Klausner R.D. Mol. Cell. Biol. 1993; 13: 4342-4350Crossref PubMed Scopus (110) Google Scholar). 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 (11Pelletier B. Beaudoin J. Philpott C.C. Labbé S. Nucleic Acids Res. 2003; 31: 4332-4344Crossref PubMed Scopus (70) Google Scholar). 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 (11Pelletier B. Beaudoin J. Philpott C.C. Labbé S. Nucleic Acids Res. 2003; 31: 4332-4344Crossref PubMed Scopus (70) Google Scholar). The second pathway for iron assimilation in S. pombe involves three components, Frp1, Fio1, and Fip1 (12Askwith C. Kaplan J. J. Biol. Chem. 1997; 272: 401-405Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 13Roman D.G. Dancis A. Anderson G.J. Klausner R.D. Mol. Cell. Biol. 1993; 13: 4342-4350Crossref PubMed Scopus (110) Google Scholar). 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 (13Roman D.G. Dancis A. Anderson G.J. Klausner R.D. Mol. Cell. Biol. 1993; 13: 4342-4350Crossref PubMed Scopus (110) Google Scholar, 14Dancis A. J. Pediatr. 1998; 132 (part 2): S24-S29Abstract Full Text Full Text PDF PubMed Google Scholar). Once reduced, iron is transported by two high affinity iron uptake proteins encoded by the fio1+ and fip1+ genes (12Askwith C. Kaplan J. J. Biol. Chem. 1997; 272: 401-405Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The Fio1 protein is a multicopper ferroxidase that converts Fe2+ to Fe3+, which is then transported by Fip1, a transmembrane iron permease (12Askwith C. Kaplan J. J. Biol. Chem. 1997; 272: 401-405Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). 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 (12Askwith C. Kaplan J. J. Biol. Chem. 1997; 272: 401-405Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 13Roman D.G. Dancis A. Anderson G.J. Klausner R.D. Mol. Cell. Biol. 1993; 13: 4342-4350Crossref PubMed Scopus (110) Google Scholar, 15Labbé S. Peña M.M.O. Fernandes A.R. Thiele D.J. J. Biol. Chem. 1999; 274: 36252-36260Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). As expected, str1+, str2+, and str3+ gene expression is up-regulated under conditions of iron starvation and down-regulated under conditions of iron repletion (11Pelletier B. Beaudoin J. Philpott C.C. Labbé S. Nucleic Acids Res. 2003; 31: 4332-4344Crossref PubMed Scopus (70) Google Scholar). It was recently shown that the transcription factor Fep1 1The 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.1The 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. mediates the iron-dependent repression of str1+, str2+, str3+, frp1+, fio1+, and fip1+ transcription (11Pelletier B. Beaudoin J. Philpott C.C. Labbé S. Nucleic Acids Res. 2003; 31: 4332-4344Crossref PubMed Scopus (70) Google Scholar, 16Pelletier B. Beaudoin J. Mukai Y. Labbé S. J. Biol. Chem. 2002; 277: 22950-22958Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). 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 (17Oberegger H. Zadra I. Schoeser M. Abt B. Parson W. Haas H. Biochem. Soc. Trans. 2002; 30: 781-783Crossref PubMed Google Scholar, 18Haas H. Zadra I. Stoffler G. Angermayr K. J. Biol. Chem. 1999; 274: 4613-4619Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 19Harrison K.A. Marzluf G.A. Biochemistry. 2002; 41: 15288-15295Crossref PubMed Scopus (47) Google Scholar, 20Zhou L.W. Haas H. Marzluf G.A. Mol. Gen. Genet. 1998; 259: 532-540Crossref PubMed Scopus (70) Google Scholar, 21An Z. Mei B. Yuan W.M. Leong S.A. EMBO J. 1997; 16: 1742-1750Crossref PubMed Scopus (69) Google Scholar, 22Voisard C. Wang J. McEvoy J.L. Xu P. Leong S.A. Mol. Cell. Biol. 1993; 13: 7091-7100Crossref PubMed Scopus (102) Google Scholar). 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′ (16Pelletier B. Beaudoin J. Mukai Y. Labbé S. J. Biol. Chem. 2002; 277: 22950-22958Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Thus, Fep1 is related to a family of GATA-type transcription factors (23Scazzocchio C. Curr. Opin. Microbiol. 2000; 3: 126-131Crossref PubMed Scopus (166) Google Scholar). 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 (16Pelletier B. Beaudoin J. Mukai Y. Labbé S. J. Biol. Chem. 2002; 277: 22950-22958Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Elimination of either Tup11 or Tup12 alone was not sufficient to annihilate the iron-mediated repression of fio1+ (16Pelletier B. Beaudoin J. Mukai Y. Labbé S. J. Biol. Chem. 2002; 277: 22950-22958Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). 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 (24Greenall A. Hadcroft A.P. Malakasi P. Jones N. Morgan B.A. Hoffman C.S. Whitehall S.K. Mol. Biol. Cell. 2002; 13: 2977-2989Crossref PubMed Scopus (37) Google Scholar). The S. pombe Tup11 and Tup12 proteins exhibit 39.9 and 43.5% identity to Saccharomyces cerevisiae Tup1, respectively (25Janoo R.T.K. Neely L.A. Braun B.R. Whitehall S.K. Hoffman C.S. Genetics. 2001; 157: 1205-1215Crossref PubMed Google Scholar). Based on the extended homology of Tup11 and Tup12 to Tup1, putative functional domains have been designated for the Tup1 homologues in S. pombe (26Mukai Y. Matsuo E. Roth S.Y. Harashima S. Mol. Cell. Biol. 1999; 19: 8461-8468Crossref PubMed Scopus (39) Google Scholar). 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 (26Mukai Y. Matsuo E. Roth S.Y. Harashima S. Mol. Cell. Biol. 1999; 19: 8461-8468Crossref PubMed Scopus (39) Google Scholar). Within their C termini, Tup11 and Tup12 contain seven copies of a repeated amino acid motif, named WD40 repeat (27Smith T.F. Gaitatzes C. Saxena K. Neer E.J. Trends Biochem. Sci. 1999; 24: 181-185Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar), found in Tup1 and other Tup1-like corepressors as well as in other proteins that are unlinked to transcription like the β-subunit of the trimeric G-proteins (28Smith R.L. Johnson A.D. Trends Biochem. Sci. 2000; 25: 325-330Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Based on the x-ray crystal structures of three proteins with seven WD40 repeats, including the Tup1 corepressor, each repeat folds into four antiparallel β strands, forming a blade structure (29Pickles L.M. Roe S.M. Hemingway E.J. Stifani S. Pearl L.H. Structure. 2002; 10: 751-761Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 30Steele M.R. McCahill A. Thompson D.S. MacKenzie C. Isaacs N.W. Houslay M.D. Bolger G.B. Cell. Signal. 2001; 13: 507-513Crossref PubMed Scopus (63) Google Scholar, 31Sprague E.R. Redd M.J. Johnson A.D. Wolberger C. EMBO J. 2000; 19: 3016-3027Crossref PubMed Scopus (99) Google Scholar). Each WD40 repeat unit is interconnected to the other by a loop to form a seven-bladed β-propeller, structure that is highly symmetrical and assumes an overall donut shape (29Pickles L.M. Roe S.M. Hemingway E.J. Stifani S. Pearl L.H. Structure. 2002; 10: 751-761Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 31Sprague E.R. Redd M.J. Johnson A.D. Wolberger C. EMBO J. 2000; 19: 3016-3027Crossref PubMed Scopus (99) Google Scholar). Although biochemical data has shown that the sides of the β-propeller are capable of establishing protein-protein contacts, the flat upper surface has been shown to bind to some proteins (30Steele M.R. McCahill A. Thompson D.S. MacKenzie C. Isaacs N.W. Houslay M.D. Bolger G.B. Cell. Signal. 2001; 13: 507-513Crossref PubMed Scopus (63) Google Scholar, 32Komachi K. Johnson A.D. Mol. Cell. Biol. 1997; 17: 6023-6028Crossref PubMed Scopus (85) Google Scholar). Previous studies have shown that S. cerevisiae Tup1 and Ssn6 proteins physically interact in vivo to form a corepressor complex (33Williams F.E. Varanasi U. Trumbly R.J. Mol. Cell. Biol. 1991; 11: 3307-3316Crossref PubMed Scopus (174) Google Scholar). Furthermore, it has been demonstrated that Tup1 forms a tetramer with one Ssn6 molecule (34Varanasi U.S. Klis M. Mikesell P.B. Trumbly R.J. Mol. Cell. Biol. 1996; 16: 6707-6714Crossref PubMed Scopus (101) Google Scholar). 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 (28Smith R.L. Johnson A.D. Trends Biochem. Sci. 2000; 25: 325-330Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). A number of potential mechanisms by which the Ssn6-Tup1 complex can repress gene expression have been described (28Smith R.L. Johnson A.D. Trends Biochem. Sci. 2000; 25: 325-330Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). 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 (35Papamichos-Chronakis M. Conlan R.S. Gounalaki N. Copf T. Tzamarias D. J. Biol. Chem. 2000; 275: 8397-8403Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Second, the Ssn6-Tup1 may promote the formation of a repressive chromatin structure (36Davie J.K. Trumbly R.J. Dent S.Y.R. Mol. Cell. Biol. 2002; 22: 693-703Crossref PubMed Scopus (63) Google Scholar, 37Deckert J. Struhl K. Mol. Cell. Biol. 2001; 21: 2726-2735Crossref PubMed Scopus (177) Google Scholar). 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 (38Mukai Y. Davie J.K. Dent S.Y.R. J. Biol. Chem. 2003; 278: 18895-18901Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Based on our previous findings that the S. pombe Tup11 protein has the potential to regulate the expression of the fio1+ iron transport gene (16Pelletier B. Beaudoin J. Mukai Y. Labbé S. J. Biol. Chem. 2002; 277: 22950-22958Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), 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 β-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. Yeast Strains and Media—The S. cerevisiae strain L40 (Matahis3Δ200 trp1-901 leu2-3, 112 ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lacZ) (39Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1654) Google Scholar) 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 (40Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2526) Google Scholar). For routine growth, yeast extract (1%), bactopeptone (2%), dextrose (2%) (YPD) medium was used (41Sikorski R.S. Boeke J.D. Methods Enzymol. 1991; 194: 302-318Crossref PubMed Scopus (489) Google Scholar). The wild-type S. pombe strain used in this study was JY741 (h- leu1-32 ura4-Δ18 ade6-M210) (16Pelletier B. Beaudoin J. Mukai Y. Labbé S. J. Biol. Chem. 2002; 277: 22950-22958Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The fission yeast strain was maintained on yeast extract plus supplement (YES) medium (42Bezanilla M. Forsburg S.L. Pollard T.D. Mol. Biol. Cell. 1997; 8: 2693-2705Crossref PubMed Scopus (144) Google Scholar, 43Alfa C. Fantes P. Hyams J. McLeod M. Warbrick E. Experiments with Fission Yeasts: Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993Google Scholar). Under selective conditions, S. pombe cells were grown on Edinburgh minimal medium lacking specific nutrients required for plasmid selection (43Alfa C. Fantes P. Hyams J. McLeod M. Warbrick E. Experiments with Fission Yeasts: Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993Google Scholar). 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 (OD600 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 (16Pelletier B. Beaudoin J. Mukai Y. Labbé S. J. Biol. Chem. 2002; 277: 22950-22958Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 44Beaudoin J. Mercier A. Langlois R. Labbé S. J. Biol. Chem. 2003; 278: 14565-14577Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). 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 (26Mukai Y. Matsuo E. Roth S.Y. Harashima S. Mol. Cell. Biol. 1999; 19: 8461-8468Crossref PubMed Scopus (39) Google Scholar, 38Mukai Y. Davie J.K. Dent S.Y.R. J. Biol. Chem. 2003; 278: 18895-18901Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). 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 (45Vojtek A.B. Cooper J.A. Hollenberg S.M. Bartel P. Fields S. The Yeast Two-hybrid System: A Practical Approach. Oxford University Press, New York1997: 29-42Google Scholar) 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 (46Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6771) Google Scholar). 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 (45Vojtek A.B. Cooper J.A. Hollenberg S.M. Bartel P. Fields S. The Yeast Two-hybrid System: A Practical Approach. Oxford University Press, New York1997: 29-42Google Scholar, 47Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (580) Google Scholar), 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 (48Fashena S.J. Serebriiskii I.G. Golemis E.A. Gene (Amst.). 2000; 328: 14-26Google Scholar). For quantitative measurements, β-galactosidase activity was determined using early logarithmic phase cultures (OD600 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 β-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-β-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 A420 nm was measured within the linear response range and expressed in standard units (49Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 352-355Google Scholar). Values shown are the average of triplicate assays of three independent transformants. In addition to liquid β-galactosidase assays, a riboprobe derived from the plasmid pKSlacZ (50Labbé S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) 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 (51Köhrer K. Domdey H. Methods Enzymol. 1991; 194: 398-405Crossref PubMed Scopus (501) Google Scholar). RNase protection analyses were carried out as described previously (52Beaudoin J. Labbé S. J. Biol. Chem. 2001; 276: 15472-15480Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) using the plasmid pKSACT1 (50Labbé S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) 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 (53Tasto J.J. Carnahan R.H. McDonald W.H. Gould K.L. Yeast. 2001; 18: 657-662Crossref PubMed Scopus (127) Google Scholar). 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 (54Gilbreth M. Yang P.R. Bartholomeusz G. Pimental R.A. Kansra S. Gadiraju R. Marcus S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14781-14786Crossref PubMed Scopus (61) Google Scholar). 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
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