The Fission Yeast Protein Ker1p Is an Ortholog of RNA Polymerase I Subunit A14 in Saccharomyces cerevisiae and Is Required for Stable Association of Rrn3p and RPA21 in RNA Polymerase I
2005; Elsevier BV; Volume: 280; Issue: 12 Linguagem: Inglês
10.1074/jbc.m411150200
ISSN1083-351X
AutoresYukiko Imazawa, Koji Hisatake, Hiroshi Mitsuzawa, Masahito Matsumoto, Tohru Tsukui, Kaori Nakagawa, Tomoyoshi Nakadai, Miho Shimada, Akira Ishihama, Yasuhisa Nogi,
Tópico(s)RNA and protein synthesis mechanisms
ResumoA heterodimer formed by the A14 and A43 subunits of RNA polymerase (pol) I in Saccharomyces cerevisiae is proposed to correspond to the Rpb4/Rpb7 and C17/C25 heterodimers in pol II and pol III, respectively, and to play a role(s) in the recruitment of pol I to the promoter. However, the question of whether the A14/A43 heterodimer is conserved in eukaryotes other than S. cerevisiae remains unanswered, although both Rpb4/Rpb7 and C17/C25 are conserved from yeast to human. To address this question, we have isolated a Schizosaccharomyces pombe gene named ker1+ using a yeast two-hybrid system, including rpa21+, which encodes an ortholog of A43, as bait. Although no homolog of A14 has previously been found in the S. pombe genome, functional characterization of Ker1p and alignment of Ker1p and A14 showed that Ker1p is an ortholog of A14. Disruption of ker1+ resulted in temperature-sensitive growth, and the temperature-sensitive deficit of ker1Δ was suppressed by overexpression of either rpa21+ or rrn3+, which encodes the rDNA transcription factor Rrn3p, suggesting that Ker1p is involved in stabilizing the association of RPA21 and Rrn3p in pol I. We also found that Ker1p dissociated from pol I in post-log-phase cells, suggesting that Ker1p is involved in growth-dependent regulation of rDNA transcription. A heterodimer formed by the A14 and A43 subunits of RNA polymerase (pol) I in Saccharomyces cerevisiae is proposed to correspond to the Rpb4/Rpb7 and C17/C25 heterodimers in pol II and pol III, respectively, and to play a role(s) in the recruitment of pol I to the promoter. However, the question of whether the A14/A43 heterodimer is conserved in eukaryotes other than S. cerevisiae remains unanswered, although both Rpb4/Rpb7 and C17/C25 are conserved from yeast to human. To address this question, we have isolated a Schizosaccharomyces pombe gene named ker1+ using a yeast two-hybrid system, including rpa21+, which encodes an ortholog of A43, as bait. Although no homolog of A14 has previously been found in the S. pombe genome, functional characterization of Ker1p and alignment of Ker1p and A14 showed that Ker1p is an ortholog of A14. Disruption of ker1+ resulted in temperature-sensitive growth, and the temperature-sensitive deficit of ker1Δ was suppressed by overexpression of either rpa21+ or rrn3+, which encodes the rDNA transcription factor Rrn3p, suggesting that Ker1p is involved in stabilizing the association of RPA21 and Rrn3p in pol I. We also found that Ker1p dissociated from pol I in post-log-phase cells, suggesting that Ker1p is involved in growth-dependent regulation of rDNA transcription. There are three distinct types of eukaryotic nuclear RNA polymerases: RNA polymerase (pol) 1The abbreviations used are: pol, RNA polymerase; 3-AT, 3-amino-1,2,4-triazole; HA, hemagglutinin; Gal4DB, Gal4 DNA-binding domain; CMV, cytomegalovirus; GFP, green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole. I, pol II, and pol III. Among eukaryotic organisms, the structure and function of RNA polymerases in Saccharomyces cerevisiae have been studied fairly extensively (1Thuriaux P. Sentenac A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces. 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 1-48Google Scholar, 2Armache K.J. Kettenberger H. Cramer P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6964-6968Crossref PubMed Scopus (195) Google Scholar, 3Bushnell D.A. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6969-6973Crossref PubMed Scopus (222) Google Scholar, 4Bischler N. Brino L. Carles C. Riva M. Tschochner H. Mallouh V. Schultz P. EMBO J. 2002; 21: 4136-4144Crossref PubMed Scopus (46) Google Scholar). S. cerevisiae pol I consists of 14 subunits. The core structure contains 10 subunits (A190, A135, AC40, AC19, Rpb5, Rpb6, Rpb8, Rpb10, Rpb12, and A12.2) and is believed to be sufficient for nonspecific transcription, but not for accurate initiation of transcription (5Edwards A.M. Kane C.M. Young R.A. Kornberg R.D. J. Biol. Chem. 1991; 266: 71-75Abstract Full Text PDF PubMed Google Scholar). In fact, pol I requires four specific subunits (A49, A43, A34.5, and A14) for specific transcription of rDNA. A43 is also essential for cell growth (6Thuriaux P. Mariotte S. Buhler J.M. Sentenac A. Vu L. Lee B.S. Nomura M. J. Biol. Chem. 1995; 270: 24252-24257Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), whereas A49 (7Liljelund P. Mariotte S. Buhler J.M. Sentenac A. Proc. Natl. Acad. Sci. U. S. 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J.): 1137-1143Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) heterodimers in pol II, pol III, and archaeal RNA polymerases, respectively. It should be noted that Rpb4, C17, and RpoF have mutual sequence similarity and are grouped into a gene family, but no obvious homolog of A14 has been found in available data bases. A14 and Rpb4 are required for the stable assembly of A43 and Rpb7, respectively, in their respective RNA polymerases, suggesting a functional similarity of A14 to Rpb4 (5Edwards A.M. Kane C.M. Young R.A. Kornberg R.D. J. Biol. Chem. 1991; 266: 71-75Abstract Full Text PDF PubMed Google Scholar, 11Peyroche G. Levillain E. Siaut M. Callebaut I. Schultz P. Sentenac A. Riva M. Carles C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14670-14675Crossref PubMed Scopus (45) Google Scholar, 15Sheffer A. Varon M. Choder M. Mol. Cell. Biol. 1999; 19: 2672-2680Crossref PubMed Scopus (57) Google Scholar, 16Lanzendorfer M. Smid A. Klinger C. Schultz P. Sentenac A. Carles C. Riva M. Genes Dev. 1997; 11: 1037-1047Crossref PubMed Scopus (23) Google Scholar). The position of A14/A43 in the three-dimensional structure of pol I has been deduced to be similar to that of Rpb4/Rpb7, forming an upstream interface with the C-terminal domain of Rpb1 to interact with transcription factor IIB for pol II recruitment to the pol II promoter (4Bischler N. Brino L. Carles C. Riva M. Tschochner H. Mallouh V. Schultz P. EMBO J. 2002; 21: 4136-4144Crossref PubMed Scopus (46) Google Scholar) and, furthermore, playing a role in the processing of the nascent RNA transcript (17Orlicky S. Tran P.T. Sayre M.H. Edwards A.M. J. Biol. Chem. 2001; 276: 10097-10102Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Consistent with the proposed position in pol I, A14/A43 also interacts with an rDNA-specific transcription factor (Rrn3p) for pol I recruitment to the rDNA promoter (10Peyroche G. Milkereit P. Bischer N. Tschochner H. Schultz P. Sentenac A. Carles C. Riva M. EMBO J. 2000; 19: 5473-5482Crossref PubMed Scopus (140) Google Scholar) and is able to bind to single-stranded RNA (18Meka H. Daoust G. Arnvig K.B. Werner F. Brick P. Onesti S. Nucleic Acids Res. 2003; 31: 4391-4400Crossref PubMed Scopus (25) Google Scholar). Interestingly, C17/C25 in pol III is also reported to interact with transcription factor IIIB, which recruits pol III to the pol III promoter (19Ferri M.L. Peyroche G. Siaut M. Lefebvre Q. Carles C. Conesa C. Sentenac A. Mol. Cell. Biol. 2000; 20: 488-495Crossref PubMed Scopus (66) Google Scholar). The mechanism of the down-regulation of rDNA transcription (20Moss T. Stefanovsky V.Y. Cell. 2002; 109: 545-548Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 21Ju Q. Warner J.R. Yeast. 1994; 10: 151-157Crossref PubMed Scopus (84) Google Scholar, 22Zaragoza D. Ghavidel A. Heitman J. Schultz M. Mol. Cell. Biol. 1998; 18: 4463-4470Crossref PubMed Scopus (184) Google Scholar, 23Bodem J. Dobreva G. 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EMBO J. 2001; 20 (J. C. B. M.): 1373-1382Crossref PubMed Scopus (148) Google Scholar). A43 in pol I is responsible for associating with Rrn3p (10Peyroche G. Milkereit P. Bischer N. Tschochner H. Schultz P. Sentenac A. Carles C. Riva M. EMBO J. 2000; 19: 5473-5482Crossref PubMed Scopus (140) Google Scholar), and the association of A43 with Rrn3p is inhibited in post-log-phase cells (including nutrient-starved or growth-arrested cells) (24Cavanaugh A.H. Hirschler-Laszkiewicz I. Hu Q. Dundr M. Smink T. Misteli T. Rothblum L.I. J. Biol. Chem. 2002; 277: 27423-27432Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 26Milkereit P. Tschochner H. EMBO J. 1998; 17: 3692-3703Crossref PubMed Scopus (119) Google Scholar), resulting in a drastic decrease in pol I recruitment to the promoter (29Claypool J.A. French S.L. Johzuka K. Eliason K. Vu L. Dodd J.A. Beyer A.L. Nomura M. Mol. Biol. Cell. 2004; 15: 946-956Crossref PubMed Scopus (140) Google Scholar). Thus, the molecular function of A43 and Rrn3p deserves further study to resolve long-standing questions regarding growth-dependent transcription of rDNA (30Grummt I. Genes Dev. 2003; 17: 1691-1702Crossref PubMed Scopus (433) Google Scholar). It is firmly established that all 12 and all 17 subunits of S. cerevisiae pol II and pol III, respectively, are conserved in human pol II and pol III (31Woychik N.A. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 311-317Crossref PubMed Scopus (22) Google Scholar, 32Hu P. Wu S. Sun Y. Yuan C. Kobayashi R. Myers M.P. Hernandez N. Mol. Biol. Cell. 2003; 22: 8044-8055Crossref Scopus (69) Google Scholar). However, it is not clear whether all 14 subunits identified in S. cerevisiae pol I are conserved in other eukaryotes (33Seither P. Iben S. Thiry M. Grummt I. Biol. Chem. 2001; 382: 1163-1170Crossref PubMed Scopus (12) Google Scholar, 34Yamamoto K. Yamamoto M. Hanada K. Nogi Y. Matsuyama T. Muramatsu M. Mol. Cell. Biol. 2004; 24: 6525-6535Crossref PubMed Scopus (30) Google Scholar). To gain further insight into the structure and function of pol I, we have been studying pol I of Schizosaccharomyces pombe, which is only distantly related to S. cerevisiae, but is amenable to genetic analysis (35Alfa C. Fantes P. Hyams J. McLeod M. Warbrick E. Experiments with Fission Yeast. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar). To date, it is known that S. pombe pol I consists of at least 12 subunits. The two largest, RPA190 and RPA140, are homologous to A190 and A135, respectively (36Yamagishi M. Nomura M. Gene (Amst.). 1988; 74: 503-515Crossref PubMed Scopus (28) Google Scholar, 37Hirano T. Konoha G. Toda T. Yanagida M. J. Cell Biol. 1989; 108: 243-253Crossref PubMed Scopus (72) Google Scholar). The two smaller subunits, RPA42 and RPA17, correspond to AC40 and AC19, respectively (38Imazawa Y. Imai K. Fukushima A. Hisatake K. Muramatsu M. Nogi Y. Mol. Gen. Genet. 1999; 262: 749-757PubMed Google Scholar, 39Imai K. Imazawa Y. Yao Y. Yamamoto K. Hisatake K. Muramatsu M. Nogi Y. Mol. Gen. Genet. 1999; 261: 364-373Crossref PubMed Scopus (9) Google Scholar). Five common subunits (Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12) are shared by pol II and pol III (12Sakurai H. Mitsuzawa H. Kimura M. Ishihama A. Mol. Cell. Biol. 1999; 19: 7511-7518Crossref PubMed Scopus (49) Google Scholar), and SpRPA12 is a functional homolog of A12.2 (40Imazawa Y. Imai K. Yao Y. Yamamoto K. Hisatake K. Muramatsu M. Nogi Y. Mol. Gen. Genet. 2001; 264: 852-859Crossref PubMed Scopus (8) Google Scholar). Thus, the 10-subunit core structure of pol I has been well conserved between the two yeasts through evolution. Moreover, the two specific subunits in S. pombe, RPA21 and RPA51, have been identified to be related to A43 and as a functional homolog of A49, respectively, suggesting that the pol I architecture in S. pombe is likely to be analogous to that in S. cerevisiae (41Imazawa Y. Hisatake K. Nakagawa K. Muramatsu M. Nogi Y. Genes Genet. Syst. 2002; 77: 147-157Crossref PubMed Scopus (6) Google Scholar, 42Nakagawa K. Hisatake K. Imazawa Y. Ishiguro A. Matsumoto M. Pape L. Ishihama A. Nogi Y. Genes Genet. Syst. 2003; 78: 199-209Crossref PubMed Scopus (12) Google Scholar). In this study, we demonstrate that a newly isolated protein, Ker1p, is an ortholog of A14 and that the Ker1p/RPA21 heterodimer in S. pombe is the functional counterpart of A14/A43 in S. cerevisiae. We also show novel aspects of Ker1p that have not been previously observed in A14 and suggest that Ker1p is involved in growth-dependent transcription of rDNA. Media, Strains, and Genetic Techniques—The yeast plasmids and strains used are listed in Table I. Minimal medium with or without thiamine and supplemented with appropriate amino acids and yeast extract/dextrose medium were prepared to grow S. pombe cells as described previously (35Alfa C. Fantes P. Hyams J. McLeod M. Warbrick E. Experiments with Fission Yeast. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar). Yeast extract/peptone/dextrose medium and synthetic dextrose medium were prepared as described previously (43Sherman F. Fink G.R. Hicks J.B. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986: 163-167Google Scholar). Synthetic dextrose medium lacking tryptophan and leucine and synthetic dextrose medium containing 25 mm 3-amino-1,2,4-triazole (3-AT) were used. Minimal medium containing 0.1–0.4 μg/ml aureobasidin A was also used. Disruption of chromosomal ker1+ was carried out as follows. Diploid cells (a cross between JY742 and JY745 cells) were transformed with the 4.5-kb XhoI-SacI linear fragment containing ker1Δ::ura4+ from pYI186. To replace Ker1p with Ker1p-HA3, a 5.0-kb XhoI-SacI fragment from pYI176 (see below) was transformed into strain IZ2, resulting in YI28.Table IYeast strains and plasmidsStrains and plasmidsDescriptionStrainsS. pombeJY742h+ ade6-M216 ura4-D18 leu1JY745hj- ade6-M210 ura4-D18 leu1IZ2Derivative of JY742 expressing RPA140 tagged with His6-FLAG (42Nakagawa K. Hisatake K. Imazawa Y. Ishiguro A. Matsumoto M. Pape L. Ishihama A. Nogi Y. Genes Genet. Syst. 2003; 78: 199-209Crossref PubMed Scopus (12) Google Scholar)YI28Derivative of IZ2 expressing ker1+-HA3YI29Diploid (crossing JY742 with JY745) carrying ker1Δ::ura4+/ker1+YI30h- ker1Δ::ura4+ ade6 ura4-D18 leu1S. cerevisiaeY190MATa ade2-101 ura3-52 his3-Δ200 lys2-801 trp1-901 leu2-3, 112 gal4Δ gal80Δ LYS2::GAL1-HIS3 URA3::GAL1-lacZPlasmidspKI45Derivative of KS(+) cloned with full-length cDNA of rpa21+ between XhoI and EcoRI sitespYI77Derivative of pAS2-1 expressing Gal4DB-RPA21, TRP1, 2μmpKS406Derivative of pSGA (47Sawin K.E. Nurse P. Proc. Natl. Acad. Sci. U. S. A. 1996; 94: 15146-15151Crossref Scopus (77) Google Scholar) expressing GFP fusion proteins under control of nmt1 promoter, ars1, LEU2pYI193Derivative of pKS406 expressing GFP-Ker1ppYI200Derivative of pKS406 expressing GFP-fibrillarinpYN1235Derivative of pGEM3ZpBR322ura4+ pBR322 with BamHI site introduced into HindIII site (55Waddel S. Jenkins J.R. Nucleic Acids Res. 1995; 23: 1836-1837Crossref PubMed Scopus (35) Google Scholar)pYN1237Derivative of KS(+); HA3-TAG sequence (triple-HA epitope tagged with TAG) inserted between SmaI and SpeI sites; 2.5-kb BamHI-BamHI ura4+ fragment excised from pYN1235 inserted at BglII site created downstream of HA3-TAG sequencepAUR224Expression vector under control of CMV promoter, aur1r, ars1pYI195Derivative of pAUR224 expressing full-length Ker1p under control of CMV promoterpYI176Derivative of pYN1237 carrying 1.0-kb fragment of 5′-flanking and coding sequence of Ker1p-HA3, ura4+, and 1.0-kb fragment of 3′-flanking sequence of ker1+pGK100Derivative of pDB248 carrying nuc1+/rpa190+ (37Hirano T. Konoha G. Toda T. Yanagida M. J. Cell Biol. 1989; 108: 243-253Crossref PubMed Scopus (72) Google Scholar)pYI185Derivative of KS(+) carrying 0.88-kb fragment of 5′-flanking region of ker1+ between XhoI and BamHI sites and 1.0-kb fragment of 3′-flanking region of ker1+ between NotI and SacI sitespYI186Derivative of pYI185 with ura4+ inserted at BamHI sitepREP41Expression vector under control of modified nmt1 promoter, LEU2, ars1 (56Basi G. Schmid E. Maundrell K. Gene (Amst.). 1993; 123: 131-136Crossref PubMed Scopus (568) Google Scholar)pREP81Expression vector under control of modified nmt1 promoter, LEU2, ars1 (56Basi G. Schmid E. Maundrell K. Gene (Amst.). 1993; 123: 131-136Crossref PubMed Scopus (568) Google Scholar)pYI40Derivative of pREP81 with NdeI site converted to BglII site (41Imazawa Y. Hisatake K. Nakagawa K. Muramatsu M. Nogi Y. Genes Genet. Syst. 2002; 77: 147-157Crossref PubMed Scopus (6) Google Scholar)pYI82Derivative of pYI40 expressing rpa21+ cDNA under control of weak nmt1 promoter (41Imazawa Y. Hisatake K. Nakagawa K. Muramatsu M. Nogi Y. Genes Genet. Syst. 2002; 77: 147-157Crossref PubMed Scopus (6) Google Scholar)pKI27Derivative of pREP81 expressing full-length rrn3+ under control of nmt1 promoterpYI210Derivative of pYI40 expressing full-length ker1+ under control of nmt1 promoter Open table in a new tab Plasmids—For two-hybrid screening, Gal4DB-rpa21+ (pYI77) was constructed as follows. 0.52-kb rpa21+ cDNA was amplified by PCR using pKI45 containing a full-length 0.52-kb cDNA of rpa21+ (41Imazawa Y. Hisatake K. Nakagawa K. Muramatsu M. Nogi Y. Genes Genet. Syst. 2002; 77: 147-157Crossref PubMed Scopus (6) Google Scholar) as a template and cloned between the SmaI and XhoI sites of pAS2-1 (44Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5239) Google Scholar) to fuse Gal4DB to RPA21 in-frame. To construct deletion derivatives of rpa21+, pKI45 was also used as template DNA for PCR amplification. pYI106 expresses RPA21 with the N-terminal 56 amino acids truncated fused to Gal4DB, whereas pYI105 expresses RPA21 with the C-terminal 60 amino acids truncated fused to Gal4DB. To replace chromosomal ker1+ with ker1+-HA3, a PCR-amplified 1317-bp XhoI-SmaI fragment of the 5′-untranslated region and open reading frame of ker1+ and a 1017-bp NotI-SacI fragment of the 3′-flanking region of ker1+ were cloned successively between the XhoI and SmaI sites and the NotI and SacI sites of pYN1237, generating pYI176. To express Ker1p under the control of the cytomegalovirus (CMV) promoter in S. pombe, full-length ker1+ (441 bp) was amplified from JY742 DNA by PCR and cloned between the XhoI and BamHI sites of pAUR222 (TaKaRa), resulting in pYI195. To study the cellular localization of Ker1p, full-length ker1+ was amplified from JY742 DNA, and the 441-bp fragment was cloned between the NotI and BamHI sites of pKS406 to express a GFP-Ker1p fusion protein, resulting in pYI193. A GFP-fibrillarin fusion construct was made using a 912-bp fragment of the fib gene encoding fibrillarin, which was amplified by PCR from JY742 DNA and cloned between the NotI and BamHI sites of pKS406, resulting in pYI200. To construct a disrupted ker1Δ::ura4+ allele, we amplified the 880-bp 5′-untranslated sequence of ker1+ flanked by the XhoI and BamHI sites and the 1.0-kb 3′-untranslated sequence of ker1+ flanked by the NotI and SacI sites from the JY742 genome. Each PCR product was cloned successively between the XhoI and BamHI sites and between the NotI and SacI sites of pBluescript II KS(+), resulting in pYI185. Then, the 2.5-kb BamHI-BamHI DNA fragment of ura4+ obtained from pYN1235 was cloned into the BamHI site of the resulting plasmid, generating pYI186. To construct pKI27, full-length rrn3+ was amplified by PCR from JY742 DNA and cloned between the SalI and SmaI sites of pREP81. To express ker1+ under the control of the nmt1 promoter, full-length ker1+ was amplified by PCR from JY742 DNA and cloned between the SalI and BamHI sites of pYI40, generating pYI210. Pfu DNA polymerase was used for PCR, and DNA sequencing analysis was used to confirm the PCR product. Two-hybrid Screening—pYI77 expressing a Gal4DB-RPA21 bait was transformed into the reporter strain Y190. Y190 carrying pYI77 was transformed with an S. pombe cDNA library fused to Gal4 activation domain in pGAD-GH (Clontech). The 3-AT-resistant and His+ transformants were screened on synthetic dextrose medium plates without Trp and Leu and containing 25 mm 3-AT. lacZ activation was examined by a filter lifting assay (38Imazawa Y. Imai K. Fukushima A. Hisatake K. Muramatsu M. Nogi Y. Mol. Gen. Genet. 1999; 262: 749-757PubMed Google Scholar). Fluorescence Microscopy of GFP Fusion Proteins—To visualize the nuclear chromatin region, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) at 1 mg/ml. Fluorescent images were obtained with a Fujix HC-2500 CCD camera using a Zeiss Axioskop fluorescence microscope. Immunoprecipitation—S. pombe cells were grown in yeast extract/dextrose medium and harvested in mid-log phase. Preparation of cell extracts and immunoprecipitation with anti-HA epitope monoclonal antibody 12CA5 (Roche Applied Science) and anti-RPA190 antibody were carried out as described by Mitsuzawa et al. (45Mitsuzawa H. Seino H. Yamao F. Ishihama A. J. Biol. Chem. 2001; 276: 17117-17124Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Immunoblotting was performed essentially as described previously (39Imai K. Imazawa Y. Yao Y. Yamamoto K. Hisatake K. Muramatsu M. Nogi Y. Mol. Gen. Genet. 1999; 261: 364-373Crossref PubMed Scopus (9) Google Scholar) using polyclonal antibodies against RPA190, RPA140, RPA21, and Rpb1 (pol II) (41Imazawa Y. Hisatake K. Nakagawa K. Muramatsu M. Nogi Y. Genes Genet. Syst. 2002; 77: 147-157Crossref PubMed Scopus (6) Google Scholar, 46Ishiguro A. Nogi Y. Hisatake K. Muramatsu M. Ishihama A. Mol. Cell. Biol. 2000; 20: 1263-1270Crossref PubMed Scopus (31) Google Scholar). Biochemical Fractionation of pol I—pol I was partially purified as described previously (46Ishiguro A. Nogi Y. Hisatake K. Muramatsu M. Ishihama A. Mol. Cell. Biol. 2000; 20: 1263-1270Crossref PubMed Scopus (31) Google Scholar). Whole cell extract from strain YI28 was loaded onto a nickel-nitrilotriacetic acid-agarose column. The proteins eluted with 200 mm imidazole were loaded onto a DEAE-Sephadex A25 column and eluted with a 50–620 mm ammonium sulfate gradient. Fractions were examined by SDS-PAGE, followed by Western blotting using antibodies against RPA190, RPA21, and HA. (Ker1p was tagged with HA3 in strain YI28.) Phosphatase Treatment—Whole cell extract was prepared form strain YI28, and 1.6 mg of protein was immunoprecipitated with anti-RPA190 antibody (10 μl of antiserum) as described above. The precipitates were washed three times with 20 mm HEPES-KOH (pH 7.6), 150 mm potassium acetate, 20% glycerol, 0.1% Nonidet P-40, and 1 mm dithiothreitol and once with HM buffer (50 mm HEPES-KOH (pH 7.6) and 1 mm MgCl2). The pellet was resuspended in 1 ml of HM buffer, divided into four aliquots, centrifuged again, resuspended with 100 μlof HM buffer, and incubated for 10 min at 30 °C. Calf intestine alkaline phosphatase (30 units, 1.5 μl; Roche Applied Science) was added to one tube and incubated for 20 min at 30 °C. The reaction was stopped by addition of SDS sample buffer and heating at 95 °C for 5 min. In controls, sodium pyrophosphate (final concentration of 5.4 mm) was added with or without alkaline phosphatase, and the sample was then treated as described above. No treatment was performed for the fourth sample. All samples were subjected to 8% SDS-PAGE, followed by immunoblot analysis with anti-HA antibody. Identification of a Novel Protein (Ker1p) That Interacts with the RPA21 Subunit—To identify protein(s) that interact with RPA21, we generated a Gal4DB-RPA21 fusion construct in pAS2-1 (pYI77) and introduced it into the S. cerevisiae two-hybrid reporter strain Y190. Subsequently, we introduced an S. pombe cDNA library fused to the Gal4 activation domain into the Y190 strain carrying pYI77. We selected ∼107 Leu+ transformants and screened colonies showing 3-AT resistance and a lacZ-positive phenotype. In total, 27 transformants showing 3-AT resistance and the lacZ-positive phenotype were obtained, and the responsible plasmids carrying cDNA fused to the Gal4 activation domain were retrieved (data not shown). Nucleotide sequencing of the retrieved plasmids indicated that all of the cDNAs encoding the protein shown to interact with RPA21 were derived from the same gene; one group lacked the C-terminal 30 amino acids, and another retained the full-length gene, indicating that the C-terminal 30 amino acids are not required for interaction with RPA21 in the yeast two-hybrid method. The gene isolated by the two-hybrid system encodes a protein of 147 amino acids with a calculated molecular mass of 16,976 Da and a calculated pI of 6.25. The predicted protein is very hydrophilic and contains many charged amino acids: 21 lysine residues, 9 arginine residues, 24 glutamic acid residues, and 7 aspartic acid residues (see Fig. 7). Therefore, we have named this protein Ker1p (for lysine (K) and glutamic acid (E)-rich protein 1) and the gene encoding it ker1+. No proteins homologous to Ker1p were observed in an initial data base search. Apparent Molecular Mass of Ker1p-HA3—To determine the apparent molecular mass of Ker1p, a YI28 strain expressing Ker1p-HA3 was constructed. Whole cell extracts prepared from YI28 and the parental strain IZ2, in which Ker1p had not been tagged, were subjected to SDS-PAGE, followed by immunoblotting with anti-HA monoclonal antibody 12CA5. Fig. 1A shows that Ker1p-HA3 was detected as a doublet of bands at 30 and 32 kDa, including a triple-HA sequence (4.3 kDa). Since the calculated molecular mass of Ker1p is ∼17 kDa, it appears that Ker1p-HA3 migrates abnormally on SDS-polyacrylamide gel, for unknown reasons. Ker1p Is Phosphorylated—The predicted amino acid sequence of Ker1p suggested that it contains many consensus phosphorylation sites for protein kinase A (Ser14), protein kinase C (Ser14, Ser22, and Ser94), casein kinase I (Ser45), casein kinase II (Ser41 and Thr89), and glycogen synthase kinase I (Ser41, Ser45, Ser94, and Thr89) (see Fig. 7). We considered the possibility that Ker1p is phosphorylated and that both phosphorylated and non-phosphorylated forms were detected as doublet bands by immunoblotting in Fig. 1A. Therefore, Ker1p-HA3 was first immunoprecipitated with anti-HA antibody, and the immunoprecipitates were then treated with alkaline phosphatase in the absence or presence of a phosphatase inhibitor. As shown in Fig. 1B, phosphatase treatment resulted in the appearance of only the faster moving 30-kDa band (lane 2). No treatment (lane 1), treatment with phosphatase and an inhibitor (lane 3), or treatment with the inhibitor only (lane 4) generated two bands of 30 and 32 kDa, similar to those observed in Fig. 1A (lane 2). Therefore, we conclude that the 30-kDa band represents non-phosphorylated Ker1p-HA3 and that the 32-kDa band represents phosphorylated Ker1p-HA3. Ker1p Is Localized Predominantly in the Nucleolus—Because Ker1p interacts with RPA21 of pol I, which localizes specifically in the nucleolus, we examined whether Ker1p also localizes in the nucleolus using a GFP-Ker1p fusion protein. Fig. 2 (A–C) shows that GFP-Ker1p formed a dense, crescent-shaped structure that occupied one side of the nucleus and that the crescent-shaped region was not stained well by DAPI. The observed crescent-shaped structure with much reduced DNA staining is the most obvious charact
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