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• W2085304602 abstract "Methylation of various components of the translational machinery has been shown to globally affect protein synthesis. Little is currently known about the role of lysine methylation on elongation factors. Here we show that in Saccharomyces cerevisiae, the product of the EFM3/YJR129C gene is responsible for the trimethylation of lysine 509 on elongation factor 2. Deletion of EFM3 or of the previously described EFM2 increases sensitivity to antibiotics that target translation and decreases translational fidelity. Furthermore, the amino acid sequences of Efm3 and Efm2, as well as their respective methylation sites on EF2, are conserved in other eukaryotes. These results suggest the importance of lysine methylation modification of EF2 in fine tuning the translational apparatus. Methylation of various components of the translational machinery has been shown to globally affect protein synthesis. Little is currently known about the role of lysine methylation on elongation factors. Here we show that in Saccharomyces cerevisiae, the product of the EFM3/YJR129C gene is responsible for the trimethylation of lysine 509 on elongation factor 2. Deletion of EFM3 or of the previously described EFM2 increases sensitivity to antibiotics that target translation and decreases translational fidelity. Furthermore, the amino acid sequences of Efm3 and Efm2, as well as their respective methylation sites on EF2, are conserved in other eukaryotes. These results suggest the importance of lysine methylation modification of EF2 in fine tuning the translational apparatus. Methylation of translational components has been shown to have a broad spectrum of functional consequences (1Graille M. Figaro S. Kervestin S. Buckingham R.H. Liger D. Heurgué-Hamard V. Methylation of class I translation termination factors: structural and functional aspects.Biochimie. 2012; 94: 1533-1543Crossref PubMed Scopus (13) Google Scholar2Jackman J.E. Alfonzo J.D. Transfer RNA modifications: nature's combinatorial chemistry playground.Wiley Interdiscip. Rev. RNA. 2013; 4: 35-48Crossref PubMed Scopus (194) Google Scholar, 3Liu J. Jia G. Methylation modifications in eukaryotic messenger RNA.J. Genet. Genomics. 2014; 41: 21-33Crossref PubMed Scopus (88) Google Scholar, 4Motorin Y. Helm M. RNA nucleotide methylation.Wiley Interdiscip. Rev. RNA. 2011; 2: 611-631Crossref PubMed Scopus (311) Google Scholar, 5Polevoda B. Sherman F. Methylation of proteins involved in translation.Mol. Microbiol. 2007; 65: 590-606Crossref PubMed Scopus (107) Google Scholar6Sharma S. Yang J. Watzinger P. Kötter P. Entian K.D. Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively.Nucleic Acids Res. 2013; 41: 9062-9076Crossref PubMed Scopus (118) Google Scholar). Methylation of rRNA plays a role in ribosome biogenesis (6Sharma S. Yang J. Watzinger P. Kötter P. Entian K.D. Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively.Nucleic Acids Res. 2013; 41: 9062-9076Crossref PubMed Scopus (118) Google Scholar), and modifications to tRNAs increase their stability or affect translational fidelity (2Jackman J.E. Alfonzo J.D. Transfer RNA modifications: nature's combinatorial chemistry playground.Wiley Interdiscip. Rev. RNA. 2013; 4: 35-48Crossref PubMed Scopus (194) Google Scholar). Protein modifications are also important and are found on various components, including ribosomal proteins, release factors, and elongation factors (1Graille M. Figaro S. Kervestin S. Buckingham R.H. Liger D. Heurgué-Hamard V. Methylation of class I translation termination factors: structural and functional aspects.Biochimie. 2012; 94: 1533-1543Crossref PubMed Scopus (13) Google Scholar, 5Polevoda B. Sherman F. Methylation of proteins involved in translation.Mol. Microbiol. 2007; 65: 590-606Crossref PubMed Scopus (107) Google Scholar, 7Clarke S.G. Protein methylation at the surface and buried deep: thinking outside the histone box.Trends Biochem. Sci. 2013; 38: 243-252Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). In a few cases, the functional consequences of these methylations have been established. For example, a 3-methyl histidine on ribosomal protein Rpl3 was recently shown to be involved in large ribosomal subunit biogenesis and translational fidelity (8Al-Hadid Q. Roy K. Munroe W. Dzialo M.C. Chanfreau G.F. Clarke S.G. Histidine methylation of yeast ribosomal protein Rpl3p is required for proper 60S subunit assembly.Mol. Cell Biol. 2014; 34: 2903-2916Crossref PubMed Scopus (28) Google Scholar). In prokaryotes and eukaryotes, release factor 1 is methylated on the conserved GGQ motif that enters the peptidyl transfer center. Loss of the methyltransferase in bacteria results in termination defects (1Graille M. Figaro S. Kervestin S. Buckingham R.H. Liger D. Heurgué-Hamard V. Methylation of class I translation termination factors: structural and functional aspects.Biochimie. 2012; 94: 1533-1543Crossref PubMed Scopus (13) Google Scholar); in yeast, the loss of the release factor 1 methylation site increases resistance to zymocin (9Studte P. Zink S. Jablonowski D. Bär C. von der Haar T. Tuite M.F. Schaffrath R. tRNA and protein methylase complexes mediate zymocin toxicity in yeast.Mol. Microbiol. 2008; 69: 1266-1277Crossref PubMed Scopus (49) Google Scholar). However, in most cases, the functional relevance of protein methylation in translation is not known. Methylation of elongation factors has been well established in Saccharomyces cerevisiae, and many of the modification sites are conserved in higher eukaryotes (10Cavallius J. Zoll W. Chakraburtty K. Merrick W.C. Characterization of yeast EF-1 α: non-conservation of post-translational modifications.Biochim. Biophys. Acta. 1993; 1163: 75-80Crossref PubMed Scopus (59) Google Scholar, 11Couttas T.A. Raftery M.J. Padula M.P. Herbert B.R. Wilkins M.R. Methylation of translation-associated proteins in Saccharomyces cerevisiae: identification of methylated lysines and their methyltransferases.Proteomics. 2012; 12: 960-972Crossref PubMed Scopus (46) Google Scholar). There are three protein elongation factors in budding yeast: the evolutionarily conserved EF1A and EF2 and the fungal-specific EF3. These three proteins guide tRNAs through the various active sites of the ribosome (12Belfield G.P. Tuite M.F. Translation elongation factor 3: a fungus-specific translation factor?.Mol. Microbiol. 1993; 9: 411-418Crossref PubMed Scopus (59) Google Scholar13Justice M.C. Hsu M.J. Tse B. Ku T. Balkovec J. Schmatz D. Nielsen J. Elongation factor 2 as a novel target for selective inhibition of fungal protein synthesis.J. Biol. Chem. 1998; 273: 3148-3151Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 14Kurata S. Shen B. Liu J.O. Takeuchi N. Kaji A. Kaji H. Possible steps of complete disassembly of post-termination complex by yeast eEF3 deduced from inhibition by translocation inhibitors.Nucleic Acids Res. 2013; 41: 264-276Crossref PubMed Scopus (17) Google Scholar15Mateyak M.K. Kinzy T.G. eEF1A: thinking outside the ribosome.J. Biol. Chem. 2010; 285: 21209-21213Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). EF1A ensures that correct codon matches occur between the aminoacyl-tRNA and the mRNA, whereas EF2 and EF3 help facilitate the timely translocation of peptidyl-tRNAs and removal of deacylated tRNAs. These three proteins together contain 10 methylated lysine residues (10Cavallius J. Zoll W. Chakraburtty K. Merrick W.C. Characterization of yeast EF-1 α: non-conservation of post-translational modifications.Biochim. Biophys. Acta. 1993; 1163: 75-80Crossref PubMed Scopus (59) Google Scholar, 11Couttas T.A. Raftery M.J. Padula M.P. Herbert B.R. Wilkins M.R. Methylation of translation-associated proteins in Saccharomyces cerevisiae: identification of methylated lysines and their methyltransferases.Proteomics. 2012; 12: 960-972Crossref PubMed Scopus (46) Google Scholar). The methyltransferases responsible for catalyzing the modification of only three of these residues have been identified (11Couttas T.A. Raftery M.J. Padula M.P. Herbert B.R. Wilkins M.R. Methylation of translation-associated proteins in Saccharomyces cerevisiae: identification of methylated lysines and their methyltransferases.Proteomics. 2012; 12: 960-972Crossref PubMed Scopus (46) Google Scholar, 16Lipson R.S. Webb K.J. Clarke S.G. Two novel methyltransferases acting upon eukaryotic elongation factor 1A in Saccharomyces cerevisiae.Arch. Biochem. Biophys. 2010; 500: 137-143Crossref PubMed Scopus (48) Google Scholar). Furthermore, the functional relevance of these modifications has been largely unexplored. In yeast, EF1A is the most heavily methylated of the elongation factors, containing two monomethyllysines (Lys-30 and Lys-390), one dimethyllysine (Lys-316), one trimethyllysine (Lys-79), and a C-terminal lysine α-carboxyl methyl ester (10Cavallius J. Zoll W. Chakraburtty K. Merrick W.C. Characterization of yeast EF-1 α: non-conservation of post-translational modifications.Biochim. Biophys. Acta. 1993; 1163: 75-80Crossref PubMed Scopus (59) Google Scholar, 17Zobel-Thropp P. Yang M.C. Machado L. Clarke S. A novel post-translational modification of yeast elongation factor 1A: methylesterification at the C terminus.J. Biol. Chem. 2000; 275: 37150-37158Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). EF2 contains trimethyl Lys-509 and dimethyl Lys-613, whereas EF3 has three trimethyllysines: Lys-187, Lys-196, and Lys-789 (11Couttas T.A. Raftery M.J. Padula M.P. Herbert B.R. Wilkins M.R. Methylation of translation-associated proteins in Saccharomyces cerevisiae: identification of methylated lysines and their methyltransferases.Proteomics. 2012; 12: 960-972Crossref PubMed Scopus (46) Google Scholar). With the exception of the C-terminal methyl ester, there is no evidence that these modifications are reversible. Although the functional role of these methylations during translation elongation is unclear, the locations of these modifications hint at their importance. Structural studies of EF2 and the 40 S ribosome subunit indicate that the Lys-509 site is in close contact with ribosomal protein Rps23b (18Spahn C.M. Gomez-Lorenzo M.G. Grassucci R.A. Jørgensen R. Andersen G.R. Beckmann R. Penczek P.A. Ballesta J.P. Frank J. Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation.EMBO J. 2004; 23: 1008-1019Crossref PubMed Scopus (325) Google Scholar). Lys-613 is in proximity to helix 33 of the 18 S rRNA (18Spahn C.M. Gomez-Lorenzo M.G. Grassucci R.A. Jørgensen R. Andersen G.R. Beckmann R. Penczek P.A. Ballesta J.P. Frank J. Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation.EMBO J. 2004; 23: 1008-1019Crossref PubMed Scopus (325) Google Scholar) and is on the same domain as the diphthamide modification at His-699 that aids in maintaining proper transcript frame (19Ortiz P.A. Ulloque R. Kihara G.K. Zheng H. Kinzy T.G. Translation elongation factor 2 anticodon mimicry domain mutants affect fidelity and diphtheria toxin resistance.J. Biol. Chem. 2006; 281: 32639-32648Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The potential for enhancing contact with ribosomal components suggests that these methylation sites could be crucial to maintain proper communication with the ribosome during translocation. Three elongation factor methyltransferases (EFMs) 3The abbreviations used are: EFMelongation factor methyltransferaseBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolAdoMetS-adenosyl-l-methionine[3H]AdoMetS-adenosyl-l-[methyl-3H]methionineMMKmonomethyllysineDMKdimethyllysineTMKtrimethyllysinePRM-MSparallel reaction-monitoring mass spectrometryPDBProtein Data Bank. have been identified in yeast. Efm1 monomethylates Lys-30 of EF1A (11Couttas T.A. Raftery M.J. Padula M.P. Herbert B.R. Wilkins M.R. Methylation of translation-associated proteins in Saccharomyces cerevisiae: identification of methylated lysines and their methyltransferases.Proteomics. 2012; 12: 960-972Crossref PubMed Scopus (46) Google Scholar, 16Lipson R.S. Webb K.J. Clarke S.G. Two novel methyltransferases acting upon eukaryotic elongation factor 1A in Saccharomyces cerevisiae.Arch. Biochem. Biophys. 2010; 500: 137-143Crossref PubMed Scopus (48) Google Scholar). See1, which we now refer to as Efm4, dimethylates Lys-316 of EF1A (11Couttas T.A. Raftery M.J. Padula M.P. Herbert B.R. Wilkins M.R. Methylation of translation-associated proteins in Saccharomyces cerevisiae: identification of methylated lysines and their methyltransferases.Proteomics. 2012; 12: 960-972Crossref PubMed Scopus (46) Google Scholar, 16Lipson R.S. Webb K.J. Clarke S.G. Two novel methyltransferases acting upon eukaryotic elongation factor 1A in Saccharomyces cerevisiae.Arch. Biochem. Biophys. 2010; 500: 137-143Crossref PubMed Scopus (48) Google Scholar). Efm2 has been shown to dimethylate Lys-613 on EF2, and indirect evidence suggests that it may trimethylate Lys-196 of EF3 (11Couttas T.A. Raftery M.J. Padula M.P. Herbert B.R. Wilkins M.R. Methylation of translation-associated proteins in Saccharomyces cerevisiae: identification of methylated lysines and their methyltransferases.Proteomics. 2012; 12: 960-972Crossref PubMed Scopus (46) Google Scholar). This leaves five methylation events with no known responsible enzyme. Because the majority of those sites are trimethylated, we sought to search for these enzymes through trimethyllysine immunoblot-based screens. elongation factor methyltransferase 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol S-adenosyl-l-methionine S-adenosyl-l-[methyl-3H]methionine monomethyllysine dimethyllysine trimethyllysine parallel reaction-monitoring mass spectrometry Protein Data Bank. In this study, we identified Yjr129c as the enzyme responsible for the trimethylation at lysine 509 on elongation factor 2. While this work was being prepared for publication, this finding was reported by another group, and the protein was designated Efm3 (20Zhang L. Hamey J.J. Hart-Smith G. Erce M.A. Wilkins M.R. Elongation factor methyltransferase 3: a novel eukaryotic lysine methyltransferase.Biochem. Biophys. Res. Commun. 2014; 451: 229-234Crossref PubMed Scopus (14) Google Scholar). In addition to mass spectrometric and immunoblot identification, we directly confirm the identity of this modification as a trimethyllysine by amino acid analysis. We then tested possible functions of elongation factor methylation, including Efm2-catalyzed modification of EF2. Deletion of EFM2 or EFM3 increased sensitivity to translation inhibitors, indicating changes in the ability for EF2 to interact and communicate with ribosomal components. Additionally, we found that translational fidelity is reduced in efm2Δ, indicating possible termination defects. S. cerevisiae strains used in this study are listed in Table 1. Growth media in this study include YPD (BD Difco 242810, 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) dextrose), SD −Ura (minimal synthetic defined medium lacking uracil; 0.07% (w/v) CSM-Ura powder (MP Biomedicals, 114511212), 0.17% (w/v) yeast nitrogen base without amino acids or ammonium sulfate, 0.5% (w/v) ammonium sulfate, 2% (w/v) dextrose), SC (synthetic complete; 0.07% (w/v) CSM (MP Biomedicals, 114500012), 0.17% (w/v) yeast nitrogen base without amino acids or ammonium sulfate, 0.5% (w/v) ammonium sulfate, with or without 2% (w/v) glucose).TABLE 1S. cerevisiae strains used in this studyStrainGenotypeBiological functionSourceBY4741MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0Wild typeOpen BiosystemsBY4742MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0Wild typeOpen Biosystemsefm3Δ aBY4741 backgroundPutative/elongation factor methyltransferaseOpen Biosystemsefm3Δ αBY4742 backgroundPutative/elongation factor methyltransferaseOpen Biosystemsefm2ΔBY4741 backgroundElongation factor methyltransferaseOpen Biosystemsefm4Δ(see1Δ)BY4741 backgroundElongation factor methyltransferaseOpen Biosystemsefm1ΔBY4741 backgroundElongation factor methyltransferaseOpen Biosystemsrkm1ΔBY4741 backgroundRibosomal protein lysine methyltransferaseOpen Biosystemsrkm2ΔBY4741 backgroundRibosomal protein lysine methyltransferaseOpen Biosystemsyjr093cΔBY4741 backgroundPutative methyltransferaseOpen Biosystemsykl162cΔBY4741 backgroundPutative methyltransferaseOpen Biosystemsynl024cΔBY4741 backgroundPutative methyltransferaseOpen Biosystemsymr209cΔBY4741 backgroundPutative methyltransferaseOpen Biosystemsyor021cΔBY4741 backgroundPutative methyltransferaseOpen Biosystemsylr063cΔBY4741 backgroundPutative methyltransferaseOpen Biosystemsymr310cΔBY4741 backgroundPutative methyltransferaseOpen Biosystemsygr283cΔBY4741 backgroundPutative methyltransferaseOpen Biosystems Open table in a new tab Overnight 5-ml cultures were used to inoculate cultures to an A600 of 0.1 or 0.15 and grown to values needed for the specific experiment. Cultures were grown in flasks on a rotary shaker (250 rpm) at 30 °C. Strains of interest were grown in YPD to an A600 of 0.7, and cells from 14 ml of the culture were harvested and washed twice with water. Lysis was performed using 0.2 g of glass beads (Biospec Products, 11079105) and 50 µl of lysis buffer (1% SDS, 0.7 mm PMSF). Samples were vortexed for 1 min and then incubated on ice for 1 min, repeated 10 times. Crude lysates were extracted, and beads were washed once with 50 µl of lysis buffer. Unbroken cells and membranes were pelleted by centrifugation at 12,000 × g for 15 min at 4 °C. Protein concentrations were determined using the Lowry method (21Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). 50 µg of protein from each sample was loaded onto a 4–12% BisTris gel (Invitrogen, NuPAGE Novex) and run at 200 V for 1 h with MOPS buffer. Rainbow full range molecular weight markers (GE Healthcare, RPN800E) were used as standards. Proteins were transferred to PVDF membrane (Hybond-P) at 30 V for 1 h. Membranes were blocked overnight at 4 °C in 5% dried nonfat milk in PBST (phosphate-buffered saline with 0.1% Tween 20 (v/v)). Membranes were washed in PBST and incubated with primary antibodies diluted into 1% dried nonfat milk in PBST for 1.5 h at room temperature and then with secondary antibodies diluted in the same solution for 1 h at room temperature. ECL was used to visualize bands (Amersham Biosciences ECL Prime Western blotting, GE Healthcare, RPN2232). After probing, membranes were stained with Ponceau (1% Ponceau S (w/v), 0.1% acetic acid (v/v)) to determine loading equality. Antibodies in this study include anti-trimethyllysine-HRP (1:5000; Immunechem, ICP0602), anti-di-/trimethyllysine (1:10,000; Upstate Biotechnology, Inc., 07-756), anti-pan-methyllysine (1:10,000; Abcam, ab7315), and anti-rabbit IgG-HRP (1:6666; Cell Signaling, 7074). The Immunechem and Upstate Biotechnology antibodies were kind gifts from Joanna Goldberg (Emory University). The Abcam “anti-pan methyllysine” antibody was prepared against calf histone H1 containing dimethyllysine residues. The Upstate Biotechnology antibody was raised against a synthetic peptide containing dimethyllysine at position 9 of human histone H3 and is listed by the manufacturer as an anti-di-/trimethyllysine antibody. In the figures, we have emphasized which modified form the antibody prefers in the yeast elongation factors by using boldface type and underlining the respective degree of lysine methylation. Thus, we describe the Abcam antibody as α-M/D/TMK and the Upstate Biotechnology antibody as α-D/TMK. Mouse tissue cytosolic extracts were a kind gift of Dr. Jonathan Lowenson from UCLA. Cultures of wild type and knock-out cells were grown in YPD to an A600 of 0.7, and cells from 14 ml of culture were harvested by centrifugation at 5000 × g for 5 min), resuspended in 1 ml of water, and transferred to a microcentrifuge tube. After centrifugation, cells were resuspended in 900 µl of YPD and 100 µl of S-adenosyl-l-[methyl-3H]methionine ([3H]AdoMet; 83.3 Ci/mmol; 0.55 mCi/ml in 10 mm H2SO4-ethanol (9:1); PerkinElmer Life Sciences). Cells were incubated for 30 min at 30 °C on a rotary shaker. Radiolabeled cells were washed twice with water and lysed using the glass bead method described above. Lysates from each strain were loaded onto a 4% stacking, 12% resolving SDS/Tris-glycine polyacrylamide gel (15 × 17 × 0.2 cm) and run at 35 mA through the stacking and 45 mA through the resolving gels. Gels were Coomassie-stained (50% methanol, 10% acetic acid, 40% water, 0.2% Brilliant Blue R-250 (w/v)) and destained overnight (10% methanol, 10% acetic acid, 80% water). The protein band running just above the 97 kDa marker was excised and placed into a 6 × 50-mm glass test tube. 100 µl of 6 n HCl was added to each slice, and tubes were placed in a reaction chamber (Eldex Laboratories, 1163) containing 500 µl of 6 n HCl. Chambers were heated for 20 h in vacuo at 109 °C in a Pico-Tag vapor phase apparatus (Waters). Residual HCl was removed by vacuum centrifugation. Dried gel slices were resuspended in 400 µl of cation exchange loading buffer (sodium citrate, 0.2 m Na+, pH 2.2). 2 µmol of each methyllysine standard was added to the sample (Sigma; Nϵ-methyl-l-lysine hydrochloride 04685, Nϵ,Nϵ-dimethyl-l-lysine monohydrochloride 19773, and Nϵ,Nϵ,Nϵ-trimethyllysine hydrochloride T1660) and loaded onto a cation exchange column (Beckman AA-15 sulfonated polystyrene resin, 0.9-cm inner diameter by 12-cm height) equilibrated with running buffer (sodium citrate, 0.3 m Na+) at 55 °C. For full separation of mono-, di-, and trimethyllysine, low pH buffer (pH 3.8) was used. To reduce run times when separation of mono- and dimethyllysine was not required, a pH 4.5 buffer was used. Buffer at pH 5.5 was used to analyze when the separation of mono-, di-, and trimethyllysine species was not needed. Amino acids were eluted in the equilibration buffer at 1 ml/min while collecting 1-min fractions at the expected elution position of the methyllysine standards. 50 µl of each fraction was added to a flat-bottom 96-well plate to detect standards by the ninhydrin method. Each well was mixed with 100 µl of ninhydrin reagent (2% ninhydrin (w/v), 0.3% hydrindantin (w/v), 75% dimethyl sulfoxide (v/v), 25% 4 m lithium acetate, pH 4.2 (v/v)), and the plate was heated at 100 °C for 15 min. Standards were detected by measuring absorbance at 570 nm using a SpectraMax M5 microplate reader. The remainder of each fraction was added to 5 ml of scintillation fluor (Safety Solve, Research Products International) in a 20-ml scintillation vial and counted for three 5-min cycles using a Beckman LS6500 instrument to detect 3H-methylated amino acids. Coomassie-stained gel slices from the 100-kDa region of fractionated polypeptides of yeast cell lysates were washed with 50 mm ammonium bicarbonate and destained by incubating in a solution of 50% 50 mm ammonium bicarbonate, 50% acetonitrile for 2–4 h until the gel slice became transparent. Slices were incubated in 100% acetonitrile and dried by vacuum centrifugation for 10 min. After incubating the dried slice in a minimal volume of 10 mm DTT in 50 mm ammonium bicarbonate for 1 h at 60 °C to reduce the disulfide bond, proteins were alkylated by treatment in 50 mm iodoacetamide in 50 mm ammonium bicarbonate for 45 min at 45 °C. Gel slices were washed by alternating 10-min incubations in 50 mm ammonium bicarbonate and 100% acetonitrile. Slices swelled on ice in a working stock solution of 20 ng/µl sequencing grade trypsin (Promega, V5111) for 45 min. Digests were performed for 16 h at 37 °C, and peptides were eluted using 50% acetonitrile, 1% trifluoroacetic acid in water. Peptides were dried by vacuum centrifugation and resuspended in 200 µl of 0.1% TFA in water. Tryptic peptides from the 100 kDa SDS-gel band of wild type, efm2Δ, and efm3Δ lysates were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an EASY-nLC 1000 system (Thermo Scientific, Waltham, MA) coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific) and an EASY-Spray nano-electrospray ionization source. Peptides were injected onto a 75 µm × 15-cm, 3µ, 100-Å PepMap C18 reversed-phase LC column and separated using a linear gradient from 5% solvent B (0.1% formic acid in acetonitrile), 95% solvent A (0.1% formic acid in water) to 50% solvent B in 45 min at a constant flow of 300 nl/min. Eluted peptides were analyzed with a top 10 data-dependent acquisition method and identified using Proteome Discoverer (version 1.4; Thermo Scientific) coupled with MASCOT (version 2.4.1; Matrix Science, London, UK). Orbitrap MS resolving power was set to 70,000 at m/z 200 for MS1 and 17,500 at m/z 200 for MS2. Tryptic peptides with up to one missed cleavage were searched against the SwissProt S. cerevisiae database (2013; 7798 sequences) with dynamic modifications for carbamidomethyl (C), oxidation (M), deamidation (N, Q) monomethyl (K), dimethyl (K), and trimethyl (K). Precursor and product ion mass tolerances were set to 10 ppm and 0.005 Da, respectively. Methylated EF2 peptides identified by MASCOT were manually examined and confirmed from the corresponding MS/MS spectra. Manually confirmed EF2 peptides with methylated lysine residues were further examined by targeted parallel reaction-monitoring mass spectrometry (PRM-MS) to explore the effects of EFM2 and EFM3 deletions on EF2 methylation. Samples (described above) were reanalyzed by a targeted MS/MS acquisition method using an inclusion list containing the doubly and triply charged mass-to-charge (m/z) values of the manually confirmed EF2 peptides from wild type lysate. Peaks corresponding to methylated EF2 peptides were visualized in Xcalibur Qual Browser software (Thermo Scientific) using precursor → fragment transitions extracted within 10 ppm mass accuracy. The following transitions (m/z) were used to identify methylated and unmethylated peptides of interest: 286.1920 → 372.2423 (LVEGLKTMK509R); 281.5201 → 251.1790 (LVEGLKDMK509R); 390.2060 → 402.2823 (DDFKDMK613AR); 383.1981 → 268.1712 (DDFKMMK613AR); 329.7103 → 446.2609 (LVEGLK); 376.1903 → 521.3194 (DDFKAR). Whole protein sequences for translocase (EF2 or EF-G) were aligned using Clustal Omega. A protein-protein BLAST search was performed using S. cerevisiae Efm3 (UniProt P47163) or Efm2 (UniProt P32324) as the query. Sequences were aligned using MUSCLE (multiple-sequence comparison by log-expectation) in MEGA 6. The evolutionary history was inferred using the neighbor-joining method to create phylogenetic trees. The evolutionary distances were computed using the p-distance method. All positions containing gaps and missing data were partially eliminated. In instances where poor homology was found, organisms were eliminated from the alignment or replaced by a different representative from the same kingdom. Changes in sensitivity to various translation inhibitors were determined using serial dilution spot test growth assays. Briefly, cells were grown at 30 °C in YPD medium to an A600 of ∼0.5. 1 ml of each culture was centrifuged down at 5000 × g for 5 min. Cells were washed with water, and the pellets were diluted to an A600 of 0.5. The cells were then diluted in a 5-fold series in water under sterile conditions. 3 µl of each dilution was spotted onto a 10-cm 2% agar plate containing YPD or YPD + antibiotic and incubated at 30 °C for 2–5 days. Antibiotics used were cycloheximide (Sigma, C7698), puromycin (VWR, 97064-280), paromomycin (Sigma, P9297), anisomycin (Sigma, A9789), tunicamycin (Sigma, T7765), and verrucarin A (Sigma, V4877). Levels of the drug transporter Pdr5 were measured by Northern blot as described previously (8Al-Hadid Q. Roy K. Munroe W. Dzialo M.C. Chanfreau G.F. Clarke S.G. Histidine methylation of yeast ribosomal protein Rpl3p is required for proper 60S subunit assembly.Mol. Cell Biol. 2014; 34: 2903-2916Crossref PubMed Scopus (28) Google Scholar). The dual luciferase systems were used as described previously (8Al-Hadid Q. Roy K. Munroe W. Dzialo M.C. Chanfreau G.F. Clarke S.G. Histidine methylation of yeast ribosomal protein Rpl3p is required for proper 60S subunit assembly.Mol. Cell Biol. 2014; 34: 2903-2916Crossref PubMed Scopus (28) Google Scholar, 22Salas-Marco J. Bedwell D.M. Discrimination between defects in elongation fidelity and termination efficiency provides mechanistic insights into translational readthrough.J. Mol. Biol. 2005; 348: 801-815Crossref PubMed Scopus (86) Google Scholar, 23Plant E.P. Nguyen P. Russ J.R. Pittman Y.R. Nguyen T. Quesinberry J.T. Kinzy T.G. Dinman J.D. Differentiating between near- and non-cognate codons in Saccharomyces cerevisiae.PLoS One. 2007; 2: e517Crossref PubMed Scopus (42) Google Scholar24Harger J.W. Meskauskas A. Dinman J.D. An “integrated model” of programmed ribosomal frameshifting.Trends Biochem. Sci. 2002; 27: 448-454Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Stop codon read-through and amino acid misincorporation reporters and control vectors were generously provided by Dr. David Bedwell and Ming Du (University of Alabama, Birmingham, AL). Frameshift reporter plasmids were generously provided by Dr. Jonathan Dinman (University of Maryland). All vectors (Table 2) were transformed into wild type, efm2Δ, and efm3Δ cells by the LiOAc-ssDNA-PEG method. The assay was performed as described with the Dual-Luciferase reporter assay system (Promega) using a SpectraMax M5 microplate reader.TABLE 2Vectors used for Dual-Luciferase assaysExperimental purposeControl vectorExperimental vectorSourceAmino acid misincorporationCTY775/luc CAAACTY775/luc CAAA FF K529 (AAA to AAT, K → N)Gift from Dr. David Bedwell (University of Alabama)Stop codon read-through, UAA" @default.
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• W2085304602 date "2014-10-01" @default.
• W2085304602 modified "2023-10-09" @default.
• W2085304602 title "Translational Roles of Elongation Factor 2 Protein Lysine Methylation" @default.
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