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• W2020267592 abstract "The general transcription factor IID consists of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). Here we report the isolation of two related TAF genes from the fission yeast Schizosaccharomyces pombe as multicopy suppressors of a temperature-sensitive mutation in the ubiquitin-conjugating enzyme gene ubcP4 +. The ubcP4 ts mutation causes cell cycle arrest in mitosis, probably due to defects in ubiquitination mediated by the anaphase-promoting complex/cyclosome. One multicopy suppressor is the previously reported gene taf72 +, whereas the other is a previously unidentified gene namedtaf73 +. We show that thetaf73 + gene, liketaf72 +, is essential for cell viability. Thetaf72 + and taf73 + genes encode proteins homologous to WD repeat-containing TAFs such as human TAF100, Drosophila TAF80/85, and Saccharomyces cerevisiae TAF90. We demonstrate that TAF72 and TAF73 proteins are present in the same complex with TBP and other TAFs and that TAF72, but not TAF73, is associated with the putative histone acetylase Gcn5. We also show that overexpression of TAF72 or TAF73 suppresses the cell cycle arrest in mitosis caused by a mutation in the anaphase-promoting complex/cyclosome subunit gene cut9 +. These results suggest that TAF72 and TAF73 may regulate the expression of genes involved in ubiquitin-dependent proteolysis during mitosis. Our study thus provides evidence for a possible role of WD repeat-containing TAFs in the expression of genes involved in progression through the M phase of the cell cycle.AB039954 The general transcription factor IID consists of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). Here we report the isolation of two related TAF genes from the fission yeast Schizosaccharomyces pombe as multicopy suppressors of a temperature-sensitive mutation in the ubiquitin-conjugating enzyme gene ubcP4 +. The ubcP4 ts mutation causes cell cycle arrest in mitosis, probably due to defects in ubiquitination mediated by the anaphase-promoting complex/cyclosome. One multicopy suppressor is the previously reported gene taf72 +, whereas the other is a previously unidentified gene namedtaf73 +. We show that thetaf73 + gene, liketaf72 +, is essential for cell viability. Thetaf72 + and taf73 + genes encode proteins homologous to WD repeat-containing TAFs such as human TAF100, Drosophila TAF80/85, and Saccharomyces cerevisiae TAF90. We demonstrate that TAF72 and TAF73 proteins are present in the same complex with TBP and other TAFs and that TAF72, but not TAF73, is associated with the putative histone acetylase Gcn5. We also show that overexpression of TAF72 or TAF73 suppresses the cell cycle arrest in mitosis caused by a mutation in the anaphase-promoting complex/cyclosome subunit gene cut9 +. These results suggest that TAF72 and TAF73 may regulate the expression of genes involved in ubiquitin-dependent proteolysis during mitosis. Our study thus provides evidence for a possible role of WD repeat-containing TAFs in the expression of genes involved in progression through the M phase of the cell cycle. AB039954 transcription factor TATA-binding protein TBP-associated factor (prefixes y, h, and d indicate yeast (S. cerevisiae), human, andDrosophila, respectively) Spt-Ada-Gcn5 acetyltransferase TBP-free TAFII-containing p300/CBP-associated factor PCAF-associated factor anaphase-promoting complex or cyclosome polymerase chain reaction Edinburgh minimal medium kilobase pair(s) hemagglutinin yeast extract poly(A)+ RNA transport The general transcription factor (TF)1 IID plays a critical role in transcription initiation of protein-coding genes by RNA polymerase II. TFIID is a multiprotein complex comprising the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs), which have been well conserved from yeast to humans (1Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (625) Google Scholar, 2Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). TBP specifically recognizes TATA elements, whereas certain TAFs directly interact with initiator or downstream promoter elements. In addition to a role in core promoter recognition, TAFs have been proposed to function as targets of activators. Subsets of TAFs have also been found in histone acetylase complexes distinct from TFIID (3Struhl K. Moqtaderi Z. Cell. 1998; 94: 1-4Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 4Brown C.E. Lechner T. Howe L. Workman J.L. Trends Biochem. Sci. 2000; 25: 15-19Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). To assess the requirement of individual TAFs for transcription in vivo, yeast (Saccharomyces cerevisiae) mutants have been used (5Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (213) Google Scholar, 6Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 7Apone L.M. Virbasius C.A. Reese J.C. Green M.R. Genes Dev. 1996; 10: 2368-2380Crossref PubMed Scopus (130) Google Scholar, 8Apone L.M. Virbasius C.A. Holstege F.C.P. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 9Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 10Moqtaderi Z. Bai Y. Poon D. Weil P.A. Struhl K. Nature. 1996; 383: 188-191Crossref PubMed Scopus (251) Google Scholar, 11Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 12Sanders S.L. Klebanow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 13Komarnitsky P.B. Michel B. Buratowski S. Genes Dev. 1999; 13: 2484-2489Crossref PubMed Scopus (53) Google Scholar). Interestingly, inactivation of some TAFs results in cell cycle phenotypes. yTAF130/145 inactivation leads to G1arrest, whereas inactivation of yTAF90 or yTAF150/TSM1 results in G2/M arrest (5Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (213) Google Scholar, 7Apone L.M. Virbasius C.A. Reese J.C. Green M.R. Genes Dev. 1996; 10: 2368-2380Crossref PubMed Scopus (130) Google Scholar). Genome-wide expression analyses have identified sets of genes whose expression depends on yTAF17, yTAF23/25, yTAF60, yTAF61/68, yTAF90, or yTAF130/145 (14Holstege F.C.P. Jennings E.G. Wyrick J.J. Lee T.I. Hengartner C.J. Green M.R. Golub T.R. Lander E.S. Young R.A. Cell. 1998; 95: 717-728Abstract Full Text Full Text PDF PubMed Scopus (1596) Google Scholar, 15Lee T.I. Causton H.C. Holstege F.C.P. Shen W.-C. Hannett N. Jennings E.G. Winston F. Green M.R. Young R.A. Nature. 2000; 405: 701-704Crossref PubMed Scopus (300) Google Scholar). For example, upon inactivation of yTAF90, ∼8% of all yeast genes show a significant decrease in expression. yTAF90 (16Reese J.C. Apone L. Walker S.S. Griffin L.A. Green M.R. Nature. 1994; 371: 523-527Crossref PubMed Scopus (148) Google Scholar, 17Poon D. Bai Y. Campbell A.M. Bjorklund S. Kim Y.-J. Zhou S. Kornberg R.D. Weil P.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8224-8228Crossref PubMed Scopus (118) Google Scholar) and its human and Drosophila homologs, hTAF100 (18Dubrovskaya V. Lavigne A.-C. Davidson I. Acker J. Staub A. Tora L. EMBO J. 1996; 15: 3702-3712Crossref PubMed Scopus (78) Google Scholar, 19Tanese N. Saluja D. Vassallo M.F. Chen J.-L. Admon A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13611-13616Crossref PubMed Scopus (118) Google Scholar, 20Tao Y. Guermah M. Martinez E. Oelgeschläger T. Hasegawa S. Takada R. Yamamoto T. Horikoshi M. Roeder R.G. J. Biol. Chem. 1997; 272: 6714-6721Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and dTAF80/85 (21Dynlacht B.D. Weinzierl R.O.J. Admon A. Tjian R. Nature. 1993; 363: 176-179Crossref PubMed Scopus (101) Google Scholar, 22Kokubo T. Gong D.-W. Yamashita S. Takada R. Roeder R.G. Horikoshi M. Nakatani Y. Mol. Cell. Biol. 1993; 13: 7859-7863Crossref PubMed Scopus (38) Google Scholar), respectively, all contain WD repeats. The WD repeat is a conserved sequence motif usually ending with Trp-Asp (WD), and WD repeat-containing proteins are implicated in a wide variety of cellular functions (23Smith T.F. Gaitatzes C. Saxena K. Neer E.J. Trends Biochem. Sci. 1999; 24: 181-185Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar). yTAF90 and hTAF100 are also components of histone acetylase complexes distinct from TFIID such as the Spt-Ada-Gcn5 acetyltransferase (SAGA) and TBP-free TAFII-containing (TFTC) complexes (24Grant P.A. Schieltz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates III, J.R. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 25Brand M. Yamamoto K. Staub A. Tora L. J. Biol. Chem. 1999; 274: 18285-18289Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). In addition, an hTAF100-related protein, PAF65β, is present in the p300/CBP-associated factor (PCAF) and TFTC complexes (25Brand M. Yamamoto K. Staub A. Tora L. J. Biol. Chem. 1999; 274: 18285-18289Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 26Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). Ubiquitin-dependent proteolysis has been shown to play a key role in progression through the cell cycle (27King R.W. Deshaies R.J. Peters J.-M. Kirschner M.W. Science. 1996; 274: 1652-1659Crossref PubMed Scopus (1117) Google Scholar). A ubiquitin-protein ligase complex known as the anaphase-promoting complex or cyclosome (APC/C) promotes the metaphase-to-anaphase transition and the exit from mitosis by mediating ubiquitination of anaphase inhibitors and mitotic cyclins, leading to their destruction by the 26 S proteasome (28Zachariae W. Nasmyth K. Genes Dev. 1999; 13: 2039-2058Crossref PubMed Scopus (575) Google Scholar). In the fission yeastSchizosaccharomyces pombe, the ubiquitin-conjugating enzyme UbcP4 seems to be involved in APC/C-mediated proteolysis (29Osaka F. Seino H. Seno T. Yamao F. Mol. Cell. Biol. 1997; 17: 3388-3397Crossref PubMed Scopus (44) Google Scholar). First, depletion of UbcP4, like mutations in APC/C subunit genes such ascut9 +, blocks the initiation of anaphase. Second, overexpression of UbcP4 suppresses a cut9 mutation. Finally, among the family of ubiquitin-conjugating enzymes, UbcP4 is most closely related to clam E2-C, Xenopus UBCx, and human UbcH10, all of which are involved in ubiquitination of mitotic cyclins. We report here the isolation of two related TAF genes,taf72 + and taf73 +, fromS. pombe as multicopy suppressors of a temperature-sensitiveubcP4 mutation. TAF72 and TAF73 proteins have homology to WD repeat-containing TAFs such as hTAF100, dTAF80/85, and yTAF90. We show that both TAF72 and TAF73 are associated with TBP and other TAFs, whereas only TAF72 is associated with Gcn5, a putative histone acetylase. We also show that taf72 + andtaf73 + suppress a mutation in thecut9 + gene. These results suggest that TAF72 and TAF73 may regulate the expression of genes involved in progression through the M phase of the cell cycle. The following S. pombe strains were used in this study: JY741 (h − ade6-M216 leu1 ura4-D18), JY746 (h + ade6-M210 leu1 ura4-D18), ubcP4ts (h − ade6-M210 ura4 leu1 ubcP4-140::ura4 +) (this study), cut9ts (h − leu1 cut9-665; a gift from Mitsuhiro Yanagida) (30Samejima I. Yanagida M. J. Cell Biol. 1994; 127: 1655-1670Crossref PubMed Scopus (90) Google Scholar), and mts2ts(h + leu1-32 mts2-1; a gift from Colin Gordon) (31Gordon C. McGurk G. Dillon P. Rosen C. Hastie N.D. Nature. 1993; 366: 355-357Crossref PubMed Scopus (210) Google Scholar). S. pombe media were prepared as described (32Moreno S. Klar A. Nurse P. Methods Enzymol. 1991; 194: 795-823Crossref PubMed Scopus (3143) Google Scholar,33Alfa C. Fantes P. Hyams J. McLeod M. Warbrick E. Experiments with Fission Yeast. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar). Standard methods were used for molecular genetic analysis ofS. pombe (32Moreno S. Klar A. Nurse P. Methods Enzymol. 1991; 194: 795-823Crossref PubMed Scopus (3143) Google Scholar, 33Alfa C. Fantes P. Hyams J. McLeod M. Warbrick E. Experiments with Fission Yeast. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar). AnXhoI-XbaI fragment containing theubcP4 + gene was cloned into theSalI/XbaI site of pUC19ΔSS, a pUC19 derivative lacking the SspI site. A ura4 +fragment was inserted into the SspI site located 440 base pairs downstream of the ubcP4 + gene. AubcP4 +::ura4 +fragment was amplified by PCR in the presence of 0.5 mmMnCl2 and used to transform an S. pombe ura4strain. Ura+ transformants selected at 25 °C were replica-plated onto EMM + AdeLeu containing phloxine B (Sigma), and the plates were incubated at 36 °C. Inability of one clone to grow at 36 °C was complemented by the pREP81-ubcP4 plasmid, indicating that the clone contains a recessive, temperature-sensitive mutation in the ubcP4 + gene, which was designatedubcP4-140. Replacement of ubcP4 + byubcP4-140::ura4 + was confirmed by Southern analysis. Sequencing of the ubcP4-140allele identified two amino acid substitutions: isoleucine by threonine at position 80 and threonine by alanine at position 129. A ubcP4-140 strain was transformed with an S. pombe genomic library constructed with the multicopy plasmid pSP1 (34Cottarel G. Beach D. Deuschle U. Curr. Genet. 1993; 23: 547-548Crossref PubMed Scopus (77) Google Scholar). Leu+ transformants selected on EMM + Ade at 25 °C were replica-plated onto EMM + Ade containing phloxine B, and the plates were incubated at 35 °C. Plasmids were recovered from white colonies grown at 35 °C and used to retransform the ubcP4-140 strain. Subcloning of inserts from two plasmids (pSP1-7 and pSP1-19) resulted in 2.6-kbSacI-HindIII and 2.2-kbSalI-HindIII fragments capable of suppression (see Fig. 3 A). Sequencing followed by data base searches using the BLAST program revealed that the former contained thetaf72 + gene (35Yamamoto T. Poon D. Weil P.A. Horikoshi M. Genes Cells. 1997; 2: 245-254Crossref PubMed Scopus (15) Google Scholar) and the latter contained a related but previously unidentified gene. This gene was namedtaf73 + and analyzed further.taf73 + cDNA was amplified by reverse transcription-PCR, cloned into pGEM-T (Promega), and sequenced. A 3′-portion of taf73 + cDNA was also amplified by 3′-rapid amplification of cDNA ends and sequenced. A 3.0-kb genomic DNA fragment containing thetaf73 + gene was amplified by PCR and cloned into pGEM-T to generate pGEM-T(taf73). The entire vector sequence flanked by 5′- and 3′-noncoding sequences of taf73 + was amplified from pGEM-T(taf73) by PCR, digested with XhoI andSmaI, and ligated with a 1.8-kbXhoI-SmaI ura4 + fragment to generate pGEM-T(Δtaf73::ura4). A 2.7-kb blunt-end Δtaf73::ura4 + fragment was amplified from pGEM-T(Δtaf73::ura4) by PCR with Vent DNA polymerase (New England Biolabs) and used for the one-step gene disruption (36Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2026) Google Scholar). After transformation, Ura+ colonies were screened for sensitivity to 5-fluoroorotic acid (Toronto Research Chemicals). Correct disruption was confirmed by PCR. Complementation by a taf73 + plasmid (Table I) also confirmed correct disruption.Table IOverexpression of TAF72 does not suppress Δtaf73PlasmidNo. of tetrads for the indicated no. of viable sporesNo. of spores01234Ura+ 1-aUra+ spores are presumed to be Δtaf73∷ura4 +segregants. All viable Ura+ spores were also Leu+.Leu+ 1-bLeu+ spores are segregants carrying the plasmid.Vector6940006taf73 +15950615taf72 + 1-cFor unknown reasons, thetaf72 + plasmid increased the spore viability oftaf73 + segregants.071200011Ataf73 +/Δtaf73∷ura4 +diploid was transformed with multicopy plasmids carrying thetaf72 + or taf73 + gene. Transformants with pSP1 (Vector), pSP1–7 (taf72 +), or pSP1–19 (taf73 +) were sporulated and subjected to tetrad analysis.1-a Ura+ spores are presumed to be Δtaf73∷ura4 +segregants. All viable Ura+ spores were also Leu+.1-b Leu+ spores are segregants carrying the plasmid.1-c For unknown reasons, thetaf72 + plasmid increased the spore viability oftaf73 + segregants. Open table in a new tab Ataf73 +/Δtaf73∷ura4 +diploid was transformed with multicopy plasmids carrying thetaf72 + or taf73 + gene. Transformants with pSP1 (Vector), pSP1–7 (taf72 +), or pSP1–19 (taf73 +) were sporulated and subjected to tetrad analysis. To construct S. pombe strains expressing FLAG or HA epitope-tagged TAF protein, DNA fragments that encode epitope-tagged TAF were amplified by PCR using primers with overlapping extension (37Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene ( Amst. ). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar) and used to replace the chromosome segment by transplacement (38Winston F. Chumley F. Fink G.R. Methods Enzymol. 1983; 101: 211-228Crossref PubMed Scopus (159) Google Scholar). Strains expressing FLAG-tagged TAF72 or TAF73, in which a FLAG epitope (DYKDDDDK) was inserted at the N terminus of the TAF72 or TAF73 protein, were constructed as follows. DNA fragments containing both a 5′-noncoding region and a 5′-portion of the coding sequence with the FLAG sequence immediately after the initiation codon were amplified by PCR and cloned into pBluescript SK (Stratagene) carrying aura4 + fragment. The resulting plasmids were linearized at a unique restriction site within thetaf genes (AatII fortaf72 + and SpeI fortaf73 +) (see Fig. 3 A) and used to transform strain JY741. Integration into the taf locus on the chromosome results in a full-length taf gene with the FLAG sequence and a 3′-truncated taf gene that are separated by the ura4 + plasmid sequence. Correct integration was confirmed by PCR. Recombination that occurs upstream of the FLAG sequence leaves only a FLAG-TAF gene on the chromosome. 5-Fluoroorotic acid-resistant segregants were screened by PCR for the presence of the FLAG sequence to obtaintaf FLAG strains. Similarly, an S. pombe strain expressing HA-tagged TAF73, in which three copies of an HA epitope (YPYDVPDYA) were inserted at the N terminus of the TAF73 protein, was constructed using strain JY746. Thistaf73 HA strain was crossed with the wild-type,taf72 FLAG, and taf73 FLAGstrains described above to construct diploid strains expressing HA-TAF73; HA-TAF73 and FLAG-TAF72; and HA-TAF73 and FLAG-TAF73, respectively. S. pombe strains expressing HA-tagged Gcn5, in which three copies of an HA epitope were inserted at the N terminus of the Gcn5 protein, were constructed using the wild-type,taf72 FLAG, and taf73 FLAGstrains. A DNA fragment containing a 5′-noncoding region and a 5′-portion of the coding sequence with the triple HA sequence immediately after the initiation codon was cloned into theura4 + plasmid. Linearization withNruI followed by integration into the S. pombechromosome resulted in strains expressing HA-Gcn5; HA-Gcn5 and FLAG-TAF72; and HA-Gcn5 and FLAG-TAF73, respectively. S. pombe strains were grown to an A600 of 0.5 (1 × 107 cells/ml) in 200 ml of YE + AdeUra medium at 30 °C. Cells were harvested, washed with H2O, transferred to a 2.0-ml tube, and frozen at −80 °C. The cells (∼350 mg) were resuspended in 700 μl of buffer A (20 mmHEPES-KOH (pH 7.6), 150 mm potassium acetate, 20% glycerol, 0.1% Nonidet P-40, and 1 mm dithiothreitol) with 1 mm phenylmethylsulfonyl fluoride (or 4-(2-aminoethyl)benzenesulfonyl fluoride) and protease inhibitor mixture (Roche Molecular Biochemicals) and then disrupted with 1 ml of glass beads (∼0.5 mm in diameter) six times for 1 min using a BeadBeater (BioSpec Products). Cell lysates were recovered through a small hole punched at the bottom of the tube and clarified twice by centrifugation at 18,000 × g for 10 min. The protein concentration of the extracts was typically ∼20 mg/ml. For immunoprecipitation with anti-FLAG antibody, 300–500 μl of whole-cell extract was mixed with 0.2 volume of a slurry of anti-FLAG M2-agarose and incubated for 2–4 h at 4 °C on a rotating wheel. For immunoprecipitation with anti-TBP antibody, 200 μl of whole-cell extract was mixed with 10 μl of anti-S. pombe TBP serum or preimmune serum and incubated for 1 h on ice, and then 40 μl of a slurry of protein A-Sepharose (Amersham Pharmacia Biotech) was added and incubated for 1 h at 4 °C on a rotating wheel. The beads were washed three times with 1 ml of buffer A, resuspended in 1.5× SDS gel loading buffer, and frozen at −80 °C. Proteins that had derived from 750 or 1500 μg of total protein were separated by 7.5 or 10% SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and probed with antibodies. Immune complexes were detected by chemiluminescence. In some cases, a blot was stripped before reprobing. Rabbit anti-S. pombe TBP, anti-S. pombe TAF130, and anti-S. pombe PTR6 polyclonal antibodies were kindly provided by Tetsuro Kokubo (Nara Institute of Science and Technology). Mouse anti-FLAG M2 monoclonal antibody and anti-FLAG M2-agarose were purchased from Sigma, and mouse anti-HA monoclonal antibody (16B12) was purchased from BAbCO. Peroxidase-conjugated goat anti-rabbit IgG (Cappel) and peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) were used as secondary antibodies. The sequence of thetaf73 + gene has been submitted to the DDBJ/EMBL/GenBankTM Data Bank under accession numberAB039954. In the course of this study, we noticed that the sequence of a genomic DNA fragment containing the taf73 +gene was determined by the S. pombe Genome Sequencing Project. Our taf73 + sequence is identical to the sequence of the predicted gene SPBC15D4.14 on cosmid c15D4 (accession number AL031349). Depletion of the UbcP4 protein, anS. pombe ubiquitin-conjugating enzyme, blocks the initiation of anaphase in mitosis, suggesting a role of UbcP4 in cell cycle progression through mitosis (29Osaka F. Seino H. Seno T. Yamao F. Mol. Cell. Biol. 1997; 17: 3388-3397Crossref PubMed Scopus (44) Google Scholar). To confirm and extend this result, we isolated a temperature-sensitive mutation in theubcP4 + gene (designated ubcP4-140 orubcP4 ts) as described under “Experimental Procedures.” A ubcP4-140 strain showed a rapid cessation of cell growth when the culture was shifted from 25 to 36 °C (Fig.1 A). After the shift to 36 °C, the following types of cells accumulated: metaphase-arrested cells with condensed chromosomes, septated cells without chromosome segregation, and cells undergoing cytokinesis without chromosome segregation (a cut phenotype) (Fig. 1 B). At 6 h after the shift, ∼30% of the cells showed septation or cytokinesis without chromosome segregation. Thus, theubcP4 ts mutation seems to block the initiation of anaphase, thereby causing uncoordinated mitosis where septation or cytokinesis occurs without chromosome segregation. This phenotype closely resembles those caused by mutations in the cut genes that encode components of APC/C such as cut9-665 (30Samejima I. Yanagida M. J. Cell Biol. 1994; 127: 1655-1670Crossref PubMed Scopus (90) Google Scholar) andcut4-533 (39Yamashita Y.M. Nakaseko Y. Samejima I. Kumada K. Yamada H. Michaelson D. Yanagida M. Nature. 1996; 384: 276-279Crossref PubMed Scopus (138) Google Scholar). It is therefore most likely that UbcP4, in conjunction with APC/C, functions in ubiquitination of proteins required for progression through mitosis, including the anaphase inhibitor (securin) Cut2 and the mitotic cyclin Cdc13 (40Yanagida M. Trends Cell Biol. 1998; 8: 144-149Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The ubcP4-140 mutation was used for isolation of multicopy suppressors that enable growth at 35 °C (see “Experimental Procedures”) (Fig. 2). Screening of anS. pombe genomic library unexpectedly identified two related genes that encode proteins with homology to WD repeat-containing TAFs such as hTAF100, dTAF80/85, and yTAF90 (Fig.3). One gene (represented by five clones) was found to be taf72 +, a putative TAF gene isolated on the basis of sequence similarity (35Yamamoto T. Poon D. Weil P.A. Horikoshi M. Genes Cells. 1997; 2: 245-254Crossref PubMed Scopus (15) Google Scholar). Thetaf72 + gene encodes a protein of 643 amino acids with a predicted molecular mass of 72.4 kDa, but its association with TBP has not been demonstrated. The other gene (represented by two clones) was a previously unidentified gene, which has been namedtaf73 +. A comparison between the genomic DNA and cDNA sequences of the taf73 + gene revealed that there is a 56-base pair intron (nucleotides 1474–1529) with consensus sequences for splicing (41Zhang M.Q. Marr T.G. Nucleic Acids Res. 1994; 22: 1750-1759Crossref PubMed Scopus (64) Google Scholar). The taf73 + gene encodes a protein of 642 amino acids with a predicted molecular mass of 72.3 kDa. Like other WD repeat-containing TAFs, TAF73 contains six WD repeats (23Smith T.F. Gaitatzes C. Saxena K. Neer E.J. Trends Biochem. Sci. 1999; 24: 181-185Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar) in its C-terminal half (Fig. 3 B). The TAF73 protein is 45% identical to TAF72, 39% identical to yTAF90, 30% identical to hTAF100, and 29% identical to dTAF80/85. Similarity was observed throughout the proteins, although the C-terminal regions show higher degrees of conservation (Fig. 3 B). TAF72 is more similar to yTAF90, hTAF100, and dTAF80/85 than TAF73 is. We also compared TAF72 and TAF73 with PAF65β, an hTAF100-related protein present in the human histone acetylase complexes PCAF and TFTC (25Brand M. Yamamoto K. Staub A. Tora L. J. Biol. Chem. 1999; 274: 18285-18289Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 26Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). Both TAF72 and TAF73 are less related to PAF65β (27 and 22% identical, respectively) than to hTAF100 (34 and 30% identical). To test the possibility that the suppression of theubcP4 ts mutation by taf72 + ortaf73 + results from an increase inubcP4 expression, we carried out Northern analysis.ubcP4 mRNA levels were not affected by multicopy plasmids carrying the taf72 + ortaf73 + gene (Fig.4). We speculate that the suppression results from increased expression of some other genes involved in APC/C-mediated proteolysis (see “Discussion”). Suppression of theubcP4 ts mutation seems to be specific to thetaf72 + and taf73 + genes because no other TAF genes were isolated in our library screen. Thetaf72 + gene is essential for cell viability (35Yamamoto T. Poon D. Weil P.A. Horikoshi M. Genes Cells. 1997; 2: 245-254Crossref PubMed Scopus (15) Google Scholar). To determine whether taf73 + is also an essential gene, we constructed a diploid S. pombe strain in which one copy of taf73 + was disrupted. Thetaf73 +/Δtaf73::ura4 +cells were sporulated and subjected to tetrad analysis. Of 34 tetrads dissected, 0, 1, and 2 viable spores were observed for 2, 15, and 17 tetrads, respectively, and no tetrads with more than 2 viable spores were recovered (Fig. 5). Importantly, all the viable spores were Ura− and thus presumed to betaf73 +. Microscopic observation of the 34 Δtaf73::ura4 + spores revealed that most spores germinated and divided three times before they ceased growing (no spores divided more than four times). In addition, Δtaf73::ura4 +haploid cells carrying a taf73 plasmid, pREP81(taf73cDNA), did not lose the plasmid under nonselective conditions. These results indicate that thetaf73 + gene, like taf72 +, is essential for cell viability. The Δtaf73 strain carrying the plasmid pREP81(taf73cDNA) grew even under conditions that repress taf73 + expression (i.e.in the presence of thiamine), indicating that residual expression allows cells to grow. Consequently, whether depletion of TAF73 causes a cell cycle phenotype remains to be determined. We next examined whether overexpression of TAF72 suppresses Δtaf73. Thetaf73 +/Δtaf73::ura4 +diploid strain was transformed with multicopy plasmids carrying thetaf72 + or taf73 + gene, sporulated, and subjected to tetrad analysis. As shown in TableI, tetrads with more than two viable spores were recovered from the diploid carrying thetaf73 + plasmid, but not from the diploid carrying the taf72 + plasmid, indicating that overexpression of TAF72 did not suppress Δtaf73. Thus, TAF72 cannot substitute for TAF73. To detect TAF72 and TAF73 proteins, we constructed S. pombestrains expressing FLAG-tagged TAF72 or TAF73, in which the wild-type gene on the chromosome was replaced by a gene encoding the epitope-tagged protein (see “Experimental Procedures”). Whole-cell extracts were prepared from these strains, along with wild-type strain JY741, which did not express any FLAG-tagged protein. Immunoblotting with anti-FLAG antibody detected FLAG-TAF72 and FLAG-TAF73 proteins, which were absent from the extract of the wild-type strain (data not shown). FLAG-TAF72 and FLAG-TAF73 migrated on SDS-polyacrylamide gel with apparent molecular masses of ∼75 and ∼80 kDa, respectively. We next examined whether TAF72 and TAF73 are associated with TBP. The whole-cell extracts of the strains expressing FLAG-TAF72 or FLAG-TAF73 were used for immunoprecipitation with anti-FLAG antibody. Immunoblotting with anti-S. pombe TBP antibody revealed that TBP was co-immunoprecipitated with TAF72 and TAF73 (Fig.6 A, lanes 2 and3). TBP was no" @default.
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