Title: A Novel SET Domain Methyltransferase Modifies Ribosomal Protein Rpl23ab in Yeast
Abstract: In vivo studies have shown that the ribosomal large subunit protein L23a (Rpl23ab) in Saccharomyces cerevisiae is methylated at lysine residues. However, the gene encoding the methyltransferase responsible for the modification has not been identified. We show here that the yeast YPL208w gene product, a member of the SET domain family of methyltransferases, catalyzes the reaction, and we have now designated it Rkm1 (ribosomal lysine (K) methyltransferase 1). Yeast strains with deletion mutations in candidate SET domain-containing genes were in vivo labeled with S-adenosyl-l-[methyl-3H]methionine. [3H]Methyl radioactivity was determined after lysates were fractionated by SDS gel electrophoresis. When compared with the parent strain or other candidate deletion strains, a loss of a radiolabeled 15-kDa species was observed in the rkm1 (Δypl208w) knock-out strain. Treatment of wild-type cell extracts with RNase or proteinase K demonstrated that the methyl-accepting substrate is a protein. Cellular lysates from parent and knockout strains were fractionated using high salt sucrose gradients. Analysis of the gradient fractions by SDS gel electrophoresis demonstrated that the 15-kDa methyl-accepting substrate elutes with the large ribosomal subunit. In vitro methylation experiments using purified ribosomes confirmed that the methyl-accepting substrate is a ribosomal protein. Amino acid analysis of the in vivo labeled 15 kDa polypeptide showed that it contains ϵ-[3H]dimethyllysine residues. Mass spectrometry of tryptic peptides of the 15 kDa polypeptide identified it as Rpl23ab. Analysis of the intact masses of the large ribosomal subunit proteins by electrospray mass spectrometry confirmed that the substrate is Rpl23ab and that it is specifically dimethylated at two distinct sites by Rkm1. These results show that SET domain methyltransferases can be involved in translational roles as well as in the previously described transcriptional roles. In vivo studies have shown that the ribosomal large subunit protein L23a (Rpl23ab) in Saccharomyces cerevisiae is methylated at lysine residues. However, the gene encoding the methyltransferase responsible for the modification has not been identified. We show here that the yeast YPL208w gene product, a member of the SET domain family of methyltransferases, catalyzes the reaction, and we have now designated it Rkm1 (ribosomal lysine (K) methyltransferase 1). Yeast strains with deletion mutations in candidate SET domain-containing genes were in vivo labeled with S-adenosyl-l-[methyl-3H]methionine. [3H]Methyl radioactivity was determined after lysates were fractionated by SDS gel electrophoresis. When compared with the parent strain or other candidate deletion strains, a loss of a radiolabeled 15-kDa species was observed in the rkm1 (Δypl208w) knock-out strain. Treatment of wild-type cell extracts with RNase or proteinase K demonstrated that the methyl-accepting substrate is a protein. Cellular lysates from parent and knockout strains were fractionated using high salt sucrose gradients. Analysis of the gradient fractions by SDS gel electrophoresis demonstrated that the 15-kDa methyl-accepting substrate elutes with the large ribosomal subunit. In vitro methylation experiments using purified ribosomes confirmed that the methyl-accepting substrate is a ribosomal protein. Amino acid analysis of the in vivo labeled 15 kDa polypeptide showed that it contains ϵ-[3H]dimethyllysine residues. Mass spectrometry of tryptic peptides of the 15 kDa polypeptide identified it as Rpl23ab. Analysis of the intact masses of the large ribosomal subunit proteins by electrospray mass spectrometry confirmed that the substrate is Rpl23ab and that it is specifically dimethylated at two distinct sites by Rkm1. These results show that SET domain methyltransferases can be involved in translational roles as well as in the previously described transcriptional roles. Methyltransferases containing the SET domain have been shown to post-translationally modify cytochrome c, Rubisco, and histones H3 and H4 at the ϵ-amino groups of lysine side chains (1Aravind L. Iyer L.M. Cell Cycle. 2004; 2: 369-376Google Scholar). The SET domain was first identified in the Drosophila heterochromatin-associated proteins Su(var), Enhancer of zeste, and Trithorax, species later determined to be histone lysine methyltransferases (2Tschiersch B. Hofmann A. Krauss V. Dorn R. Korge G. Reuter G. EMBO J. 1994; 13: 3822-3831Crossref PubMed Scopus (470) Google Scholar, 3Jones R.S. Gelbart W.M. Mol. Cell. Biol. 1993; 13: 6357-6366Crossref PubMed Scopus (200) Google Scholar, 4Stassen M.J. Bailey D. Nelson S. Chinwalla V. Harte P.J. Mech. Dev. 1995; 52: 209-223Crossref PubMed Scopus (115) Google Scholar, 5Jenuwein T. Laible G Dorn R Reuter G. Cell. Mol. Life Sci. 1998; 54: 80-93Crossref PubMed Scopus (300) Google Scholar). The SET domain is an S-adenosylmethionine (AdoMet) 3The abbreviations used are: AdoMet, S-adenosyl-l-methionine; Rkm1, ribosomal lysine(K) methyltransferase 1. 3The abbreviations used are: AdoMet, S-adenosyl-l-methionine; Rkm1, ribosomal lysine(K) methyltransferase 1. binding domain that does not resemble the canonical seven β-strand AdoMet-binding fold seen in the majority of methyltransferases whose structures are presently known (6Schubert H.L. Blumenthal R.M. Cheng X. Trends Biochem. Sci. 2003; 28: 329-335Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar). In the yeast Saccharomyces cerevisiae, three SET domain methyltransferases have been identified, two of which are involved in regulating transcription by methylating histone proteins in chromatin and one is the cytochrome c lysine methyltransferase Ctm1p (7Boa S. Coert C. Patterton H.-G. Yeast. 2003; 20: 827-835Crossref PubMed Scopus (52) Google Scholar, 8Roguev A Schaft D Shevchenko A. Pim Pijnappel W.W.M. Wilm M. Aasland R. Stewart A.F. EMBO J. 2001; 20: 7137-7148Crossref PubMed Scopus (454) Google Scholar, 9Shrahl B.D. Grant P.A. Briggs S.D. Sun Z.-W. Bone J.R. Baldwell J.A. Mollah S. Cook R.G. Shabanowitz J. Hunt D.F. Allis C.D. Mol. Cell. Biol. 2002; 22: 1298-1306Crossref PubMed Scopus (428) Google Scholar, 10Polevoda B. Martzen M.R. Das B. Phizicky E.M. Sherman F. J. Biol. Chem. 2000; 275: 20508-20513Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The two SET domain methyltransferases involved in transcription are designated Set1 and Set2. Set1 is a histone H3 lysine-4 methyltransferase involved in transcriptional activation, while Set2 is a histone H3 lysine-36 methyltransferase involved in transcriptional repression (7Boa S. Coert C. Patterton H.-G. Yeast. 2003; 20: 827-835Crossref PubMed Scopus (52) Google Scholar, 8Roguev A Schaft D Shevchenko A. Pim Pijnappel W.W.M. Wilm M. Aasland R. Stewart A.F. EMBO J. 2001; 20: 7137-7148Crossref PubMed Scopus (454) Google Scholar, 9Shrahl B.D. Grant P.A. Briggs S.D. Sun Z.-W. Bone J.R. Baldwell J.A. Mollah S. Cook R.G. Shabanowitz J. Hunt D.F. Allis C.D. Mol. Cell. Biol. 2002; 22: 1298-1306Crossref PubMed Scopus (428) Google Scholar). In higher organisms homologous SET domain protein histone methyltransferases have also been identified and their roles have also been linked to both the activation and repression of transcription (11Lachner M. Jenuwein T. Curr. Opin. Cell Biol. 2002; 14: 286-298Crossref PubMed Scopus (693) Google Scholar). Ctm1p was found to specifically trimethylate lysine-72 of iso-1-cytochrome c. The functional role of this modification is not understood (10Polevoda B. Martzen M.R. Das B. Phizicky E.M. Sherman F. J. Biol. Chem. 2000; 275: 20508-20513Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), although it has been suggested that the trimethyl group on lysine-72 may aid in abrogating the pro-apoptotic activity of cytochrome c (12Kluck R.M. Ellerby L.M. Ellerby H.M. Naiem S. Yaffe M.P. Margoliash E. Bredesen D. Mauk A.G. Sherman F. Newmeyer D.D. J. Biol. Chem. 2000; 275: 16127-16133Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In contrast to the methylation of histones, the methylation of cytochrome c has only been found to occur in plants and fungi and not in higher animals (10Polevoda B. Martzen M.R. Das B. Phizicky E.M. Sherman F. J. Biol. Chem. 2000; 275: 20508-20513Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 13DeLange R.J. Glazer A.N. Smith E.L. J. Biol. Chem. 1970; 245: 3325-3327Abstract Full Text PDF PubMed Google Scholar). Other cellular processes may also be regulated by methylation, including translation. In a mass spectral analysis of the large ribosomal proteins of S. cerevisiae, it was found that six of the proteins are post-translationally modified by the addition of methyl groups including L1ab, L3, L12ab, L23ab, L42ab, and L43ab (14Lee S.W. Berger S.J. Marinovic S. Pasa-Tolic L. Anderson G.A. Shen Y. Zhao R. Smith R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5942-5947Crossref PubMed Scopus (165) Google Scholar). The yeast ribosomal protein L23a or YL32 is encoded as identical amino acid sequences by the RPL23a and RPL23b genes and is designated here Rpl23ab. Rpl23ab was shown to be one of the three most highly methylated proteins in the large ribosomal subunit (15Lhoest J. Lobet Y. Costers E. Colson C. Eur. J. Biochem. 1984; 141: 585-590Crossref PubMed Scopus (30) Google Scholar, 16Lobet Y. Lhoest J. Colson C. Biochim. Biophys. Acta. 1989; 997: 224-231Crossref PubMed Scopus (12) Google Scholar), modified in vivo and in vitro by dimethylation at the side chain of one or more lysine residues (15Lhoest J. Lobet Y. Costers E. Colson C. Eur. J. Biochem. 1984; 141: 585-590Crossref PubMed Scopus (30) Google Scholar). The small subunit of the ribosome is also modified by methylation (17Kruiswijk T. Kunst A. Planta R.J. Mager W.H. Biochemistry. 1978; 175: 221-225Crossref Scopus (25) Google Scholar). However, the physiological roles of ribosomal protein methylation are poorly understood (18Toledo H. Amaro A.M. Sanhueza S. Jerez C.A. Arch. Biol. Med. Exp. 1988; 21: 219-229PubMed Google Scholar). Currently, only two genes have been identified that encode ribosomal protein methyltransferases. In S. cerevisiae, the RMT2 gene encodes an enzyme that monomethylates the δ nitrogen of arginine 67 in the L12 protein of the large subunit (19Chern M.-K. Chang K.-N. Liu L.-F. Tam T.-C.S. Liu Y.-C. J. Biol. Chem. 2002; 277: 15345-15353Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In the fission yeast Schizosaccharomyces pombe, the protein-arginine methyltransferase 3 homolog (Prmt3) asymmetrically dimethylates an arginine residue in the small ribosomal subunit protein S2 (20Bachand F. Silver P.A. EMBO J. 2004; 23: 2641-2650Crossref PubMed Scopus (131) Google Scholar). Deleting the PRMT3 gene resulted in an accumulation of free 60 S subunits (20Bachand F. Silver P.A. EMBO J. 2004; 23: 2641-2650Crossref PubMed Scopus (131) Google Scholar). The mammalian PRMT3 gene product can also specifically methylate the S2 protein (21Swiercz R. Person M.D. Bedford M.T. Biochem. J. 2005; 386: 85-91Crossref PubMed Scopus (129) Google Scholar). Biochemical efforts have been made to identify the methyltransferase responsible for modifying Rpl23ab. However, purification to homogeneity was not achieved due largely to the instability of the activity (16Lobet Y. Lhoest J. Colson C. Biochim. Biophys. Acta. 1989; 997: 224-231Crossref PubMed Scopus (12) Google Scholar). Nevertheless, these efforts did lead to the estimation of the native molecular mass of the partially purified enzyme at 82 kDa by size exclusion chromatography and an isoelectric point of 4.45 by isoelectric focusing (16Lobet Y. Lhoest J. Colson C. Biochim. Biophys. Acta. 1989; 997: 224-231Crossref PubMed Scopus (12) Google Scholar). The genes and proteins for the methyltransferases that modify the remaining ribosomal proteins have not yet been identified. Here we have determined that the gene encoding the Rpl23ab lysine-N-methyltransferase is YPL208w, which we now refer to as RKM1 (ribosomal protein lysine (K) methyltransferase 1). In Vivo Labeling of Potential SET Methyltransferase Mutants with [3H]AdoMet—Yeast strains were obtained from Invitrogen (Carlsbad, CA) in which the gene encoding a potential SET methyltransferase was deleted. Genotypes of strains used in this study are described in TABLE ONE. These strains were grown at 30 °C in YPD media (1% bacto-yeast extract, 2% bacto-peptone, 2% dextrose) to an optical density of 0.7–0.9 at 600 nm. Once the cells reached the desired optical density, 7 A600 nm units of each culture were harvested by centrifugation at 5,000 × g for 5 min at 4 °C and washed twice with 1 ml of sterile water. The pellet was resuspended in 924 μl of fresh YPD and 76 μl of [3H]AdoMet; Amersham Biosciences, 1 miCi/ml, 70–81 Ci/mmol, in dilute HCl/ethanol (9:1, v/v), pH 2 to 2.5). Cells were labeled for 30 min at 30 °C with shaking, pelleted at 5,000 × g for 5 min at 4 °C, washed twice with water, and lysed in 100 μl of 1% SDS and 0.7 mm phenylmethylsulfonyl fluoride. Lysis was performed by vortexing the cells for 1 min in the presence of 0.2 g of baked zirconium beads (Biospec Products; Bartlesville, OK), followed by cooling on ice for 1 min, for a total of 7 cycles. The lysate obtained for each mutant was then centrifuged for 15 min at 12,000 × g followed by a 10-min centrifugation at 17,000 × g, both at 4 °C. 10 μlof the resulting lysate was mixed with an equal volume of 2× SDS gel sample buffer (180 mm Tris/HCl, pH 6.8, 4% SDS, 10% β-mercaptoethanol, 20% glycerol, and 0.002% bromphenol blue) and heated at 100 °C for 3 min. Samples were then electrophoresed at 30 mA for 5 h using a Laemmli buffer system (24Laemmli A.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206620) Google Scholar) on a gel prepared with 12.6% acrylamide and 0.43% N,N-methylene-bisacrylamide (unless otherwise stated) (1.5-mm thick, 10.5-cm long resolving gel, 2-cm long stacking gel). Gels were stained with Coomassie Brilliant Blue R-250 for 1 h and destained in 10% methanol and 5% acetic acid overnight. For fluorography, the gels were treated with EN3HANCE (PerkinElmer Life Sciences) for 1 h, followed by a 20-min wash in water. Gels were dried at 70 °C for 2 h in vacuo and allowed to cool for 1 h in vacuo. The gels were exposed to Kodak X-Omat AR scientific imaging film at –80 °C.TABLE ONEStrains used Strain Genotype Source BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 aStrains were purchased from the Saccharomyces Genome Deletion Project (www.sequence.stanford.edu/group/yeast_deletion_project/deletions3.html). Δypl208w (BY4741) BY4741, Δypl208w::Kanr aStrains were purchased from the Saccharomyces Genome Deletion Project (www.sequence.stanford.edu/group/yeast_deletion_project/deletions3.html). BY4742 MATα his3D1 leu2Δ0 lys2Δ0 ura3Δ0 aStrains were purchased from the Saccharomyces Genome Deletion Project (www.sequence.stanford.edu/group/yeast_deletion_project/deletions3.html). Δybr030w BY4742, Δybr030w::Kanr aStrains were purchased from the Saccharomyces Genome Deletion Project (www.sequence.stanford.edu/group/yeast_deletion_project/deletions3.html). Δydr257c BY4742, Δydr257c::Kanr aStrains were purchased from the Saccharomyces Genome Deletion Project (www.sequence.stanford.edu/group/yeast_deletion_project/deletions3.html). Δyhl039w BY4742, Δyhl039w::Kanr aStrains were purchased from the Saccharomyces Genome Deletion Project (www.sequence.stanford.edu/group/yeast_deletion_project/deletions3.html). Δyhr207c BY4742, Δyhr207c::Kanr aStrains were purchased from the Saccharomyces Genome Deletion Project (www.sequence.stanford.edu/group/yeast_deletion_project/deletions3.html). Δyjl105w BY4742, Δyjl105w::Kanr aStrains were purchased from the Saccharomyces Genome Deletion Project (www.sequence.stanford.edu/group/yeast_deletion_project/deletions3.html). Δypl165c BY4742, Δypl165c::Kanr aStrains were purchased from the Saccharomyces Genome Deletion Project (www.sequence.stanford.edu/group/yeast_deletion_project/deletions3.html). Δypl208w (BY4742) BY4742, Δypl208w::Kanr aStrains were purchased from the Saccharomyces Genome Deletion Project (www.sequence.stanford.edu/group/yeast_deletion_project/deletions3.html). YIT617(CB012) MATa ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 pep4Δ::HIS3 prc1Δ::hisG bThe YIT617 and YIT613 strains were kindly provided by Dr. Toshifumi Inada (40). YIT613 YIT617 rpl25::LEU2 [pRPL25-FH-URA3CEN] bThe YIT617 and YIT613 strains were kindly provided by Dr. Toshifumi Inada (40).a Strains were purchased from the Saccharomyces Genome Deletion Project (www.sequence.stanford.edu/group/yeast_deletion_project/deletions3.html).b The YIT617 and YIT613 strains were kindly provided by Dr. Toshifumi Inada (40Inada T. Winstall E. Tarun Jr, S.Z. Yates J.R. Schieltz D. Sachs A.B. RNA. 2002; 8: 948-958Crossref PubMed Scopus (97) Google Scholar). Open table in a new tab Cellular Fractionation by High Salt Sucrose Gradients—The procedure of Lhoest et al (15Lhoest J. Lobet Y. Costers E. Colson C. Eur. J. Biochem. 1984; 141: 585-590Crossref PubMed Scopus (30) Google Scholar) and Hardy et al. (25Hardy S.J. Kurland C.G. Voynow P. Mora G. Biochemistry. 1969; 8: 2897-2905Crossref PubMed Scopus (807) Google Scholar) was used with a few modifications. Briefly, 500-ml cultures were grown in YPD to an optical density of 0.5–0.8 at 600 nm, harvested and washed as described above and in vivo labeled for 30 min at 30 °C in the presence of 152 μl of [3H]AdoMet and 20 ml of fresh YPD medium. The amount of radioactivity used in this label is 20-fold less than the amount used in the in vivo label procedure. After labeling the cells were again harvested by centrifugation at 5,000 × g for 5 min, washed twice with water, lysed in 1.5 ml of buffer A (20 mm Tris/HCl, 15 mm magnesium acetate, 60 mm KCl, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, pH 7.4) and 1.5 g of baked zirconium beads using the method described above. After lysis, the beads were washed with an additional 1.5 ml of buffer to maximize the yield of lysate. The combined lysate was centrifuged twice at 12,000 × g for 5 min at 4 °C, followed by one centrifugation at 20,000 × g for 15 min at 4 °C. In each case the pellet was discarded. The resulting supernatant was then centrifuged at 100,000 × g for 2 h at 4°C using a Beckman type Ti 65 rotor. The ribosomal pellet obtained was resuspended in 500 μl of buffer B (50 mm Tris-HCl 5 mm magnesium acetate, 500 mm KCl, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, pH 7.4) and layered onto a 7–25% sucrose gradient made in the presence of buffer B. The gradients were centrifuged at 60,000 × g for 16 h at 4 °C, using a Beckman type SW41 rotor. Fractions were then collected from the bottom and the A260 nm and A280 nm were measured. Fractions containing either the 60 S or 40 S ribosomal subunits, or cytosolic proteins, were individually pooled and ethanol precipitated by adding 0.7 volumes of cold ethanol in the presence of 15 mm magnesium acetate. The proteins were allowed to precipitate for 24 h at –20 °C. Proteins were pelleted by centrifugation at 12,000 × g for 20 min at 4 °C. The pelleted proteins were resuspended in 200 μl of water, and the RNA was extracted by the addition of 400 μl of glacial acetic acid and 20 μlof1 m magnesium chloride, in rapid succession. The mixture was stirred for 45 min in an ice bath, after which the precipitated RNA was removed by spinning at 20,000 × g for 10 min at 4 °C. The supernatant was dialyzed against 2% acetic acid, and lyophilized. The presence of the ribosomal subunits was confirmed by phenol extracting the RNA and determining the content of the 25 S- and 18 S-rRNA species after agarose gel electrophoresis using methods described previously (26Collart M. Oliviero S. Current Protocols in Microbiology. John Wiley & Sons, Inc., Hoboken, NJ1993: unit 13.12Google Scholar, 27Mueller E.G. Buck C.J. Palenchar P.M. Barnhart L.E. Paulson J.L. Nucleic Acids Res. 1998; 26: 2606-2610Crossref PubMed Scopus (85) Google Scholar). Electrospray-Ionization Mass Spectrometry—Acetic acid-extracted lyophilized ribosomal proteins were dissolved in 90% formic acid and immediately injected for reverse-phase liquid chromatography with electrospray-ionization mass spectrometry and fraction collection (LC-MS+). The procedure has been described in detail (28Whitelegge J.P. Methods Mol. Biol. 2004; 251: 323-339PubMed Google Scholar); briefly, the stationary phase used is polymeric (PLRP/S, Polymer Laboratories) and buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile) are used for equilibration (95% A; 5% B) and an extended gradient. The mass spectrometer (API III+, PE Sciex) was tuned and calibrated as described (29Whitelegge J.P. Gundersen C. Faull K.F. Protein Sci. 1998; 7: 1423-1430Crossref PubMed Scopus (166) Google Scholar) yielding mass accuracy of 0.01% (±1.5 Da at 15 kDa). Approximately 50% of the column eluent was directed to a fraction collector using a T flow splitter. Fractions were stored at –20 °C for further processing. Purification of Tap-Tagged Rkm1—Tap-tagged Rkm1 (Open Biosystems; Huntsville, AL) was purified by slightly modifying the method used by Puig et al. (30Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. Methods. 2001; 24: 218-229Crossref PubMed Scopus (1418) Google Scholar). Briefly, 2 liters of tap-tagged Rkm1-containing cells were grown to 2 OD at an absorbance of 600 nm. The cells were harvested by centrifugation at 5,000 × g for 5 min and washed twice with 50 ml of water. The cell pellet was stored at –20 °C overnight. The next day the cells were thawed and lysed in 10 ml of Tap Buffer A (10 mm K-HEPES, 10 mm KCl, 1.5 mm magnesium chloride, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, pH 7.9) and an equal volume of baked zirconium beads by seven vortexing/icing cycles. After lysis the KCl concentration was adjusted to 0.2 m by adding 1:9 volume of 2 m KCl. The lysate was centrifuged at 25,000 × g for 30 min and the supernatant was recovered. This material was dialyzed against Tap buffer D (20 mm K-HEPES, 50 mm KCl, 0.2 mm EDTA, 0.5 mm dithiothreitol, 20% glycerol, and 0.5 mm phenylmethylsulfonyl fluoride, pH 7.9) for 3 h at 4 °C. Purification with IgG affinity beads (Amersham Biosciences) and cleavage from beads with TEV protease (Invitrogen) was performed as described in Puig et al. (30Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. Methods. 2001; 24: 218-229Crossref PubMed Scopus (1418) Google Scholar). The TEV protease was then separated from the purified protein using calmodulin-Sepharose 4B chromatography (Amersham Biosciences) as described previously (30Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. Methods. 2001; 24: 218-229Crossref PubMed Scopus (1418) Google Scholar). As described in TABLE TWO, the genome of the yeast S. cerevisiae appears to encode at least eleven SET domain-containing proteins. Two of these genes (SET1 and SET2) have been shown to encode histone protein lysine methyltransferases, one gene (SET3) encodes a histone deacetylase, and another gene (CTM1) encodes the cytochrome c lysine methyltransferase (7Boa S. Coert C. Patterton H.-G. Yeast. 2003; 20: 827-835Crossref PubMed Scopus (52) Google Scholar, 8Roguev A Schaft D Shevchenko A. Pim Pijnappel W.W.M. Wilm M. Aasland R. Stewart A.F. EMBO J. 2001; 20: 7137-7148Crossref PubMed Scopus (454) Google Scholar, 9Shrahl B.D. Grant P.A. Briggs S.D. Sun Z.-W. Bone J.R. Baldwell J.A. Mollah S. Cook R.G. Shabanowitz J. Hunt D.F. Allis C.D. Mol. Cell. Biol. 2002; 22: 1298-1306Crossref PubMed Scopus (428) Google Scholar, 10Polevoda B. Martzen M.R. Das B. Phizicky E.M. Sherman F. J. Biol. Chem. 2000; 275: 20508-20513Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 31Pijnappel W.W. Schaft D. Roguev A. Shevchenko A. Tekotte H. Wilm M. Rigaut G. Seraphin B. Aasland R. Stewart A.F. Genes Dev. 2001; 15: 2991-3004Crossref PubMed Scopus (210) Google Scholar). The remaining seven SET domain-encoding genes were identified by the Pfam protein family data base (www.sanger.ac.uk/gi-bin/Pfam; version of 31 October 2002) (TABLE TWO). The function of these seven gene products is unknown. In this work, we investigated the possible role of these species as protein methyltransferases.TABLE TWOSET domain proteins in Saccharomyces cerevisiae Gene name ORF name Function Localization Length of protein/pI SET-domain residues Other domains Ref. SET1 YHR119w Histone H3-K4 MTaMT designates methyltransferase. Nuclear 1,080/9.68 932-1061 7Boa S. Coert C. Patterton H.-G. Yeast. 2003; 20: 827-835Crossref PubMed Scopus (52) Google Scholar, 8Roguev A Schaft D Shevchenko A. Pim Pijnappel W.W.M. Wilm M. Aasland R. Stewart A.F. EMBO J. 2001; 20: 7137-7148Crossref PubMed Scopus (454) Google Scholar SET2 YJL168c Histone H3-K36 MT Nuclear 733/8.56 114-243 9Shrahl B.D. Grant P.A. Briggs S.D. Sun Z.-W. Bone J.R. Baldwell J.A. Mollah S. Cook R.G. Shabanowitz J. Hunt D.F. Allis C.D. Mol. Cell. Biol. 2002; 22: 1298-1306Crossref PubMed Scopus (428) Google Scholar SET3 YKR029c NAD-dependent histone deacetylase Nuclear 751/9.12 315-463 PHD: 119-166 31Pijnappel W.W. Schaft D. Roguev A. Shevchenko A. Tekotte H. Wilm M. Rigaut G. Seraphin B. Aasland R. Stewart A.F. Genes Dev. 2001; 15: 2991-3004Crossref PubMed Scopus (210) Google Scholar SET4 YJL105w Unknown Unknown 560/8.76 340-482 PHD: 162-210 SET5 YHR207c Unknown Cytoplasmic/Nuclear 526/6.43 106-140 & 364-409 TD: 236-253 SET6 YPL165c Unknown Unknown 373/7.59 297-345 TD: 232-260 SET7 (RMS1) YDR257c Unknown Nuclear 494/4.80 19-271 TD: 166-189 & 344-372 YBR030w YBR030w Unknown Nuclear 552/4.37 10-341 TD: 74-92 YHL039w YHL039w Unknown Cytoplasmic 585/6.64 8-287 TD: 345-362 & 457-473 YPL208w YPL208w Unknown Cytoplasmic/Nuclear 583/4.86 TD: 196-224 CTM1 YHR109w Cytochrome c lysine MT Cytoplasmic 585/4.72 10Polevoda B. Martzen M.R. Das B. Phizicky E.M. Sherman F. J. Biol. Chem. 2000; 275: 20508-20513Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholara MT designates methyltransferase. Open table in a new tab We first compared the methylation of size-fractionated polypeptides derived from parent cells and cells with deletions in the seven SET domain-containing genes. In S. cerevisiae, AdoMet is readily taken up from the external media through a plasma membrane transporter (32Murphy J.T. Spence K.D. J. Bacteriol. 1972; 109: 499-504Crossref PubMed Google Scholar, 33Rouillon A. Surdin-Kerjan Y. Thomas D. J. Biol. Chem. 1999; 274: 28096-28105Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and it is possible to directly label species methylated by incubating with intact cells [3H]AdoMet. Here, [3H]methyl groups are transferred by the endogenous yeast methyltransferases to their methyl-accepting substrates. When cell extracts were fractionated by SDS gel electrophoresis and analyzed for [3H]methyl groups by autoradiography, a number of radiolabeled species were found (Fig. 1). We detected little or no difference between the parent BY4742 strain and six of the deletion mutants. However, when analyzing the Δypl208w deletion strain, we noted the loss of a 15-kDa radiolabeled species (Fig. 1). It does not appear that the loss of methylation at 15 kDa is caused by the loss of the YPL208w gene product as a methyl-accepting protein because its encoded polypeptide has a mass of 67.2 kDa. We further confirmed these results by analyzing the independently-derived Δypl208w deletion strain obtained in the BY4741 background. Here, we also observed a similar loss of methylation at 15 kDa (Fig. 2). The Δypl208w deletion in both strains was confirmed by PCR analysis (data not shown). These results suggest that the YPL208w gene encodes a methyltransferase active on a 15-kDa species.FIGURE 2Proteinase K and RNase treatment of [3H]AdoMet-labeled cell extracts. In vivo labeled lysate (from 0.35 A600 nm units of cells) was incubated with either 2.4 mg/ml proteinase K for 18 h at 30 °C (lanes 5–8), 0.2 mg/ml RNase A for 30 min at 30 °C (lanes 9–12), or not treated, and incubated for 30 min at 30 °C (lanes 13–16). As a control, fresh lysate was loaded without incubation and without treatment (lanes 1–4). Lysates obtained from the wild-type BY4741 and BY4742 strains were analyzed in addition to their respective Δypl208w deletion strains. The reaction mixtures were adjusted to final concentrations of 9.6 mm Tris/HCl, 12 mm NaCl, and 0.24 mm EDTA, pH 7.5. The reactions were quenched by the addition of an equal volume of SDS gel sample buffer, electrophoresed, and analyzed by fluorography as described in the legend to Fig. 1 above with a film exposure of 4 days. The arrow indicates the migration position of the Ypl208w-methyl-accepting substrate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because tRNA molecules are also modified by AdoMet-dependent methyltransferases and because they can migrate on an SDS gel in the position of 15–25 kDa polypeptides (34Hrycyna C.A. Yang M.C. Clarke S. Biochemistry. 1994; 33: 9806-9812Crossref PubMed Scopus (22) Google Scholar), we wanted to determine if the YPL208w-dependent 15-kDa labeled species represented a polypeptide or an RNA-methylated species. Lysates from in vivo labeled cells were treated with either RNase, which would be expected to degrade all of the RNA in the sample, or proteinase K, which would be expected to degrade all polypeptides. After SDS gel electrophoresis, we found that the samples treated with protei