Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human
2015; Elsevier BV; Volume: 15; Issue: 1 Linguagem: Inglês
10.1074/mcp.m115.052449
ISSN1535-9484
AutoresJoshua J. Hamey, Daniel L. Winter, Daniel Yagoub, Christopher M. Overall, Gene Hart‐Smith, Marc R. Wilkins,
Tópico(s)RNA and protein synthesis mechanisms
ResumoEukaryotic elongation factor 1A (eEF1A) is an essential, highly methylated protein that facilitates translational elongation by delivering aminoacyl-tRNAs to ribosomes. Here, we report a new eukaryotic protein N-terminal methyltransferase, Saccharomyces cerevisiae YLR285W, which methylates eEF1A at a previously undescribed high-stoichiometry N-terminal site and the adjacent lysine. Deletion of YLR285W resulted in the loss of N-terminal and lysine methylation in vivo, whereas overexpression of YLR285W resulted in an increase of methylation at these sites. This was confirmed by in vitro methylation of eEF1A by recombinant YLR285W. Accordingly, we name YLR285W as elongation factor methyltransferase 7 (Efm7). This enzyme is a new type of eukaryotic N-terminal methyltransferase as, unlike the three other known eukaryotic N-terminal methyltransferases, its substrate does not have an N-terminal [A/P/S]-P-K motif. We show that the N-terminal methylation of eEF1A is also present in human; this conservation over a large evolutionary distance suggests it to be of functional importance. This study also reports that the trimethylation of Lys79 in eEF1A is conserved from yeast to human. The methyltransferase responsible for Lys79 methylation of human eEF1A is shown to be N6AMT2, previously documented as a putative N(6)-adenine-specific DNA methyltransferase. It is the direct ortholog of the recently described yeast Efm5, and we show that Efm5 and N6AMT2 can methylate eEF1A from either species in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. Including the present work, yeast eEF1A is now documented to be methylated by five different methyltransferases, making it one of the few eukaryotic proteins to be extensively methylated by independent enzymes. This implies more extensive regulation of eEF1A by this posttranslational modification than previously appreciated. Eukaryotic elongation factor 1A (eEF1A) is an essential, highly methylated protein that facilitates translational elongation by delivering aminoacyl-tRNAs to ribosomes. Here, we report a new eukaryotic protein N-terminal methyltransferase, Saccharomyces cerevisiae YLR285W, which methylates eEF1A at a previously undescribed high-stoichiometry N-terminal site and the adjacent lysine. Deletion of YLR285W resulted in the loss of N-terminal and lysine methylation in vivo, whereas overexpression of YLR285W resulted in an increase of methylation at these sites. This was confirmed by in vitro methylation of eEF1A by recombinant YLR285W. Accordingly, we name YLR285W as elongation factor methyltransferase 7 (Efm7). This enzyme is a new type of eukaryotic N-terminal methyltransferase as, unlike the three other known eukaryotic N-terminal methyltransferases, its substrate does not have an N-terminal [A/P/S]-P-K motif. We show that the N-terminal methylation of eEF1A is also present in human; this conservation over a large evolutionary distance suggests it to be of functional importance. This study also reports that the trimethylation of Lys79 in eEF1A is conserved from yeast to human. The methyltransferase responsible for Lys79 methylation of human eEF1A is shown to be N6AMT2, previously documented as a putative N(6)-adenine-specific DNA methyltransferase. It is the direct ortholog of the recently described yeast Efm5, and we show that Efm5 and N6AMT2 can methylate eEF1A from either species in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. Including the present work, yeast eEF1A is now documented to be methylated by five different methyltransferases, making it one of the few eukaryotic proteins to be extensively methylated by independent enzymes. This implies more extensive regulation of eEF1A by this posttranslational modification than previously appreciated. Protein methylation is emerging as one of the most prominent posttranslational modifications in the eukaryotic cell (1.Khoury G.A. Baliban R.C. Floudas C.A. Proteome-wide post-translational modification statistics: Frequency analysis and curation of the Swiss-prot database.Sci. Rep. 2011; 1 (Article number: 90)Crossref PubMed Scopus (593) Google Scholar). Often showing high evolutionary conservation, it is increasingly recognized for its role in modulating protein–protein interactions (2.Erce M.A. Pang C.N. Hart-Smith G. Wilkins M.R. The methylproteome and the intracellular methylation network.Proteomics. 2012; 12: 564-586Crossref PubMed Scopus (70) Google Scholar). Indeed, it has been documented in protein interaction codes (3.Winter D.L. Erce M.A. Wilkins M.R. A web of possibilities: Network-based discovery of protein interaction codes.J. Proteome Res. 2014; 13: 5333-5338Crossref PubMed Scopus (15) Google Scholar), such as those of the histones and p53 (4.Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7633) Google Scholar, 5.Gu B. Zhu W.G. Surf the post-translational modification network of p53 regulation.Int. J. Biol. Sci. 2012; 8: 672-684Crossref PubMed Scopus (162) Google Scholar), where it shows interplay with modifications such as acetylation and phosphorylation. Despite this, there remains a paucity of understanding of the enzymes that catalyze protein methylation. Many of the known methyltransferases target histones. However, many other methyltransferases have been discovered recently that act on nonhistone proteins (6.Clarke 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). While protein methylation predominantly occurs on lysine and arginine residues, it is also known to occur on glutamine, asparagine, glutamate, histidine, cysteine, and the N- and C termini of proteins. Although the presence of N-terminal methylation on numerous proteins has been known for decades (7.Stock A. Clarke S. Clarke C. Stock J. N-terminal methylation of proteins: Structure, function and specificity.FEBS Lett. 1987; 220: 8-14Crossref PubMed Scopus (78) Google Scholar), the first enzymes responsible for this methylation have only recently been discovered (8.Tooley C.E. Petkowski J.J. Muratore-Schroeder T.L. Balsbaugh J.L. Shabanowitz J. Sabat M. Minor W. Hunt D.F. Macara I.G. NRMT is an alpha-N-methyltransferase that methylates RCC1 and retinoblastoma protein.Nature. 2010; 466: 1125-1128Crossref PubMed Scopus (83) Google Scholar, 9.Webb K.J. Lipson R.S. Al-Hadid Q. Whitelegge J.P. Clarke S.G. Identification of protein N-terminal methyltransferases in yeast and humans.Biochemistry. 2010; 49: 5225-5235Crossref PubMed Scopus (69) Google Scholar). The Saccharomyces cerevisiae protein Tae1 and its human ortholog N-terminal methyltransferase 1 (NTMT1) catalyze N-terminal methylation of proteins with an N-terminal [A/P/S]-P-K motif (after methionine removal). Yet there is evidence that these enzymes may recognize a more general N-terminal motif (10.Petkowski J.J. Schaner Tooley C.E. Anderson L.C. Shumilin I.A. Balsbaugh J.L. Shabanowitz J. Hunt D.F. Minor W. Macara I.G. Substrate specificity of mammalian N-terminal alpha-amino methyltransferase NRMT.Biochemistry. 2012; 51: 5942-5950Crossref PubMed Scopus (37) Google Scholar). Human NTMT2 is a monomethyltransferase that methylates the same substrates as NTMT1 and may prime substrate proteins with monomethylation to assist subsequent trimethylation by NTMT1 (11.Petkowski J.J. Bonsignore L.A. Tooley J.G. Wilkey D.W. Merchant M.L. Macara I.G. Schaner Tooley C.E. NRMT2 is an N-terminal monomethylase that primes for its homologue NRMT1.Biochem. J. 2013; 456: 453-462Crossref PubMed Scopus (28) Google Scholar). The biological function of N-terminal methylation on some proteins has been recently revealed. For example, N-terminal methylation of regulator of chromatin condensation protein 1 (RCC1) is known to affect its binding to chromatin and thereby the correct chromosomal segregation during mitosis (12.Chen T. Muratore T.L. Schaner-Tooley C.E. Shabanowitz J. Hunt D.F. Macara I.G. N-terminal alpha-methylation of RCC1 is necessary for stable chromatin association and normal mitosis.Nat. Cell Biol. 2007; 9: 596-603Crossref PubMed Scopus (107) Google Scholar, 13.Hao Y. Macara I.G. Regulation of chromatin binding by a conformational switch in the tail of the Ran exchange factor RCC1.J. Cell Biol. 2008; 182: 827-836Crossref PubMed Scopus (44) Google Scholar), and N-terminal methylation of DNA damage-binding protein 2 (DDB2) is important for its role in UV-damaged DNA repair (14.Cai Q. Fu L. Wang Z. Gan N. Dai X. Wang Y. alpha-N-methylation of damaged DNA-binding protein 2 (DDB2) and its function in nucleotide excision repair.J. Biol. Chem. 2014; 289: 16046-16056Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Interestingly, there is evidence of interplay between N-terminal methylation and other posttranslational modifications (15.Bailey A.O. Panchenko T. Sathyan K.M. Petkowski J.J. Pai P.J. Bai D.L. Russell D.H. Macara I.G. Shabanowitz J. Hunt D.F. Black B.E. Foltz D.R. Posttranslational modification of CENP-A influences the conformation of centromeric chromatin.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 11827-11832Crossref PubMed Scopus (93) Google Scholar), suggesting that, like lysine and arginine methylation, it may be incorporated into protein interaction codes (3.Winter D.L. Erce M.A. Wilkins M.R. A web of possibilities: Network-based discovery of protein interaction codes.J. Proteome Res. 2014; 13: 5333-5338Crossref PubMed Scopus (15) Google Scholar). N-terminal methylation therefore appears to be a modification of functional importance in the cell. Eukaryotic elongation factor 1A (eEF1A), and its bacterial ortholog EF-Tu, is an essential translation elongation factor that is found in all living organisms. Its canonical function is in facilitating delivery of aminoacyl-tRNAs to the ribosome; however, it is also known to have a role in many other cellular functions, such as actin bundling, nuclear export, and proteasomal degradation (16.Mateyak 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). A number of methyltransferases have been discovered in both S. cerevisiae and human that target translation elongation factors. In yeast, four of these elongation factor methyltransferases (EFMs) act on eEF1A, namely Efm1, Efm4, Efm5, and Efm6, generating monomethylated Lys30, dimethylated Lys316, trimethylated Lys79, and monomethylated Lys390, respectively (17.Lipson 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, 18.Dzialo M.C. Travaglini K.J. Shen S. Loo J.A. Clarke S.G. A new type of protein lysine methyltransferase trimethylates Lys-79 of elongation factor 1A.Biochem. Biophys. Res. Commun. 2014; 455: 382-389Crossref PubMed Scopus (14) Google Scholar, 19.Jakobsson M.E. Davydova E. Malecki J. Moen A. Falnes P.Ø. Saccharomyces cerevisiae eukaryotic elongation factor 1A (eEF1A) is methylated at Lys-390 by a METTL21-like methyltransferase.PLoS ONE. 2015; 10: e0131426Crossref PubMed Scopus (29) Google Scholar). Human METTL10 is the ortholog of Efm4 in that it trimethylates eEF1A at Lys318, which is equivalent to Lys316 in yeast (20.Shimazu T. Barjau J. Sohtome Y. Sodeoka M. Shinkai Y. Selenium-based S-adenosylmethionine analog reveals the mammalian seven-beta-strand methyltransferase METTL10 to be an EF1A1 lysine methyltransferase.PloS One. 2014; 9: e105394Crossref PubMed Scopus (69) Google Scholar). Interestingly, eukaryotic elongation factor 2 (eEF2) is also methylated by a number of lysine methyltransferases. In yeast, Efm2 and Efm3 act on eEF2, generating dimethylated Lys613 and trimethylated Lys509, respectively (21.Couttas 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, 22.Zhang 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, 23.Davydova E. Ho A.Y. Malecki J. Moen A. Enserink J.M. Jakobsson M.E. Loenarz C. Falnes P.Ø. Identification and characterization of a novel evolutionarily conserved lysine-specific methyltransferase targeting eukaryotic translation elongation factor 2 (eEF2).J. Biol. Chem. 2014; 289: 30499-30510Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 24.Dzialo M.C. Travaglini K.J. Shen S. Roy K. Chanfreau G.F. Loo J.A. Clarke S.G. Translational roles of elongation factor 2 protein lysine methylation.J. Biol. Chem. 2014; 289: 30511-30524Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Human eEF2-KMT is the ortholog of Efm3 in that it trimethylates eEF2 at Lys525, which is equivalent to Lys509 in yeast eEF2 (23.Davydova E. Ho A.Y. Malecki J. Moen A. Enserink J.M. Jakobsson M.E. Loenarz C. Falnes P.Ø. Identification and characterization of a novel evolutionarily conserved lysine-specific methyltransferase targeting eukaryotic translation elongation factor 2 (eEF2).J. Biol. Chem. 2014; 289: 30499-30510Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Here, we report the N-terminal methylation of eEF1A in S. cerevisiae and the identification of the methyltransferase that catalyzes this event. Using parallel reaction monitoring and MS/MS/MS (MS3), we unambiguously localize the modification to the N-terminal glycine and show it is conserved in the human cell. We also show that YLR285W, which we rename elongation factor methyltransferase 7 (Efm7), is responsible for this modification in yeast, as well as dimethylation at the adjacent lysine. We also characterize the methyltransferases responsible for methylation of lysine 79 in eEF1A. Human N6AMT2 is shown to be the ortholog of yeast Efm5 through its capacity to methylate yeast and human eEF1A at Lys79 in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. The wild-type yeast strain used in this study was BY4741 (Open Biosystems, Huntsville, AL). Single gene deletion strains (ΔYLR285W, ΔYNL024C, ΔYGR001C, and ΔYNL092W) were obtained from (Euroscarf, Frankfurt, Germany). Strains were cultured and lysates obtained according to previous methods (22.Zhang 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). The BG1805-YLR285W plasmid was modified for overexpression of YLR285W by site-direct, ligase-independent mutagenesis (25.Chiu J. Tillett D. Dawes I.W. March P.E. Site-directed, ligase-independent mutagenesis (SLIM) for highly efficient mutagenesis of plasmids greater than 8kb.J. Microbiol. Methods. 2008; 73: 195-198Crossref PubMed Scopus (62) Google Scholar) to remove the HA-tag and the ZZ domain, leaving YLR285W with the C-terminal 6xHisTag and a short linker. Site-direct, ligase-independent mutagenesis was used again on this plasmid to remove the YLR285W gene, leaving only the 6xHisTag and the linker, to generate the empty vector control plasmid. Overexpression in BY4741 was done as per Mok et al. (26.Mok J. Im H. Snyder M. Global identification of protein kinase substrates by protein microarray analysis.Nat. Protoc. 2009; 4: 1820-1827Crossref PubMed Scopus (40) Google Scholar), except that the induction was done overnight. Lysates were separated by SDS-PAGE on 4–12% NuPAGE® Novex® gels (Life Technologies, Waltham, MA), fixed with 10% acetic acid/25% isopropanol (v/v) and stained with QC Colloidal Coomassie Stain (Bio-Rad, Hercules, CA). Gel bands were excised, destained with 50% acetonitrile (ACN)/50% 20 mm ammonium bicarbonate (v/v) and dehydrated with 100% ACN. Gel bands were then rehydrated with 5 μl sequencing-grade trypsin (Promega, Fitchburg, WI 10 ng/μl), 10 μl endoproteinase AspN (Promega, 10 ng/μl) or LysargiNase (27.Huesgen P.F. Lange P.F. Rogers L.D. Solis N. Eckhard U. Kleifeld O. Goulas T. Gomis-Rüth F.X. Overall C.M. LysargiNase mirrors trypsin for protein C-terminal and methylation-site identification.Nat. Methods. 2015; 12: 55-58Crossref PubMed Scopus (110) Google Scholar) (40 ng/μl), and made up to 50 μl with 20 mm ammonium bicarbonate (for AspN and trypsin) or 20 mm ammonium bicarbonate/12.5 mm CaCl2 (for LysargiNase, to give a final concentration of 10 mm CaCl2). Digests were left at 37 °C overnight and peptides extracted from the gel bands with 50% ACN/50% 0.1% formic acid (v/v) for 30 min and then 100% ACN for 10 min. Peptides were dried in a SpeedVacTM (Thermo Fisher Scientific, Waltham, MA) for 2 h before being resuspended in 0.1% formic acid (v/v). Peptide samples were separated by nano-LC and eluting peptides ionized by nano-ESI following previously described methods (28.Hart-Smith G. Raftery M.J. Detection and characterization of low abundance glycopeptides via higher-energy C-trap dissociation and Orbitrap mass analysis.J. Am. Soc. Mass. Spectrom. 2012; 23: 124-140Crossref PubMed Scopus (66) Google Scholar) before analysis on either an LTQ Orbitrap Velos Pro or a Q Exactive Plus (Thermo Fisher Scientific) for all mass spectrometric analyses. For analyses on the LTQ Orbitrap Velos Pro, survey scans m/z 350–1,750 were acquired in the Orbitrap (resolution = 30,000 at m/z 400) with an initial accumulation target value of 1 × 106 ions in the linear ion trap. The instrument was set to operate in data-dependent acquisition mode in combination with an inclusion list containing the m/z values of the relevant peptides for the methylation site(s) of interest. The five most abundant ions (>5,000 counts) within 10 ppm of any m/z value on the inclusion list were sequentially isolated and fragmented, followed by up to the five most abundant ions not within 10 ppm of any m/z value on the inclusion list. Precursor ions were fragmented via collision-induced dissociation with an activation time of 30 ms, normalized collision energy of 30% and at a target value of 10,000 ions, for analysis in the linear ion trap. Dynamic exclusion was enabled with an exclusion duration of 30 s. For analyses on the Q Exactive Plus, survey scans m/z 350–1,750 (MS automated gain control target = 3 × 106) were acquired in the Orbitrap (resolution = 70,000 at m/z 200). The instrument was set to operate in data-dependent acquisition mode in combination with an inclusion list containing the m/z values of the relevant peptides for the methylation site(s) of interest. The 10 most abundant ions within 10 ppm of any m/z value on the inclusion list were sequentially isolated and fragmented via Higher-energy collisional dissociation (HCD) using the following parameters: normalized collision energy = 35%, maximum injection time = 125 ms, and automated gain control target = 1 × 105. Fragment ions were then analyzed in the Orbitrap (resolution = 17,500). "If idle" was set as "pick others" for most analyses, allowing selection and fragmentation of ions not within 10 ppm of any m/z value on the inclusion list, only after all inclusion list-selected ions have been selected if less than 10 consecutive MS2 scans have been performed. Dynamic exclusion was enabled for most analyses with an exclusion duration of 30 s. All data were converted to Mascot Generic Format (.mgf) using either MassMatrix MS Data File Conversion (v. 3.9) or msConvert from the ProteoWizard Library and Tools collection (29.Kessner D. Chambers M. Burke R. Agus D. Mallick P. ProteoWizard: Open source software for rapid proteomics tools development.Bioinformatics. 2008; 24: 2534-2536Crossref PubMed Scopus (1218) Google Scholar). Data were then searched against the SwissProt database (2014_08, 546,238 sequences through to 2015_08, 549,008 sequences) using Mascot (v. 2.4, Matrix Sciences) hosted by the Walter and Eliza Hall Institute for Medical Research (Melbourne, Australia) with the following settings: enzyme: trypsin (trypsin-digested samples), AspN (AspN-digested samples), or none (LysargiNase-digested samples); two allowed missed cleavages; precursor tolerance: 4 ppm; fragment ion tolerance: 10 mmu (Q Exactive Plus) or 0.4 Da (LTQ Orbitrap Velos Pro); peptide charge: 2+, 3+, 4+ (except when analyzing the singly charged KFETS peptide); Instrument: Q-Exactive_Gen (Q Exactive Plus) or ESI-TRAP (LTQ Orbitrap Velos Pro); Variable modifications: oxidation (M), methyl (K), dimethyl (K), trimethyl (K), and methyl (DE). For samples pertaining to N-terminal methylation the following changes were made: Allowed missed cleavages was set to 0 and the following variable modifications were added: methyl (N-term), dimethyl (N-term), and propyl (N-term) (in lieu of any trimethyl N-term variable modification). Peptides were generally identified with Mascot scores >35. and then spectra and elution times were manually inspected and compared between different methylation states to confirm identification; the exception being the very short methylated KFETS peptides, that produced low Mascot scores due to their length (scores of 28–31), which were verified by manual inspection for additional, confirmatory peaks falling within 10 ppm of theoretical fragment ion m/z values. All Q Exactive Plus-derived MS2 spectra were manually annotated to only include fragment ions with 10 ppm error or less. Extracted ion chromatograms for peptides were obtained using Thermo Xcalibur Qual Browser 2.2 SP1.48 by setting mass windows of ±10 ppm of the relevant m/z value, and applying a scan filter to only analyze MS1 scans. Relevant data have been deposited to the ProteomeXchange Consortium (30.Vizcaíno J.A. Deutsch E.W. Wang R. Csordas A. Reisinger F. Ríos D. Dianes J.A. Sun Z. Farrah T. Bandeira N. Binz P.A. Xenarios I. Eisenacher M. Mayer G. Gatto L. Campos A. Chalkley R.J. Kraus H.J. Albar J.P. Martinez-Bartolomé S. Apweiler R. Omenn G.S. Martens L. Jones A.R. Hermjakob H. ProteomeXchange provides globally coordinated proteomics data submission and dissemination.Nat. Biotechnol. 2014; 32: 223-226Crossref PubMed Scopus (2071) Google Scholar) via the PRIDE partner repository with the dataset identifier PXD002941. For analysis of the abundance of y14 ions, samples were analyzed by parallel-reaction monitoring on a Q Exactive Plus using the Targeted MS2 method. An inclusion list containing the m/z values of all possible methylation states of the doubly charged N-terminal AspN peptide GKEKSHINVVVIGHV (unmethylated to pentamethylated) (List A) was used to generate full MS2 scans of precursors at these m/z values every ∼0.6 s across the entire LC run. The default MS2 scan settings were used except for the following changes: automated gain control target = 5 × 104, maximum injection time = 50 ms, isolation window = 1.6 m/z, normalized collision energy = 30%. Transitions from the methylated precursors to y14+1 ions were analyzed using Qual Browser. Mass windows of ±10 ppm for each y14 ion were analyzed with the relevant precursor ion set in the scan filter setting. Human eEF1A samples were analyzed in the same way except that the inclusion list contained only the m/z value of the doubly charged trimethylated N-terminal AspN peptide GKEKTHINIVVIGHV, and mass windows for y14 transitions were set at ±20 ppm. For MS3 analysis of y14 ions, samples were analyzed on an LTQ Orbitrap Velos Pro using the Data Dependent Product MS3 method with the following settings: collision-induced dissociation activation (normalized collision energy = 30%, activation time = 10 ms, isolation width = 2.5 m/z, default charge state = 2+ (MS2) or 1+ (MS3)), minimum signal threshold = 5,000 (MS2) or 200 (MS3) ion counts. The MS2 event was set to use List A as its parent mass list. For the MS3 event, the product mass list was set to contain the m/z values of all possible methylation states of the y14+1 ion (unmethylated to pentamethylated). Data were analyzed by manual assignment of fragment peaks falling within 0.4 Da of theoretical fragment m/z values for MS2 precursors falling within 0.4 Da of theoretical precursor m/z values. Wild-type yeast cells were grown in synthetic complete media (2 g/l histidine and methionine drop-out mix (US Biological, Salem, MA), 1.7 g/l yeast nitrogen base without amino acids or ammonium sulfate (BD Biosciences, Franklin Lakes, NJ), 5 g/l ammonium sulfate, 20 g/l glucose, 82 mg/l histidine), with 82 mg/l unlabeled (light), or 13CD3-labeled (heavy) methionine (Sigma-Aldrich, St. Louis, MO). Cell lysates were prepared as above, protein concentration quantified, and lysates mixed 3:1 (light:heavy). Mixed lysates were separated by SDS-PAGE, and the band corresponding to eEF1A excised, digested by AspN, and analyzed by LC-MS/MS as above. All relevant ORFs were cloned into pET15b for bacterial expression by Gibson assembly with the Gibson Assembly® Cloning Kit (New England BioLabs, Ipswich, MA). Primers were designed to insert a 6x HisTag at the N terminus for yeast EFM5 and human N6AMT2 and at the C terminus for all other ORFs. ORFs were amplified from wild-type yeast gDNA (YLR285W and SceEF1A 1The abbreviations used are:SceEF1ASaccharomyces cerevisiae eukaryotic elongation factor 1AHseEF1AHomo sapiens eukaryotic elongation factor 1AKMTlysine methyltransferaseSILACstable isotope labeling by amino acids in cell culture. (TEF1)), wild-type yeast cDNA (EFM5), or plasmids RC205604 (N6AMT2) and RC209697 (HseEF1A1) (Origene, Rockville, MD). SceEF1A truncations were cloned directly from gBlocksTM constructs (Integrated DNA Technologies, Coralville, IA) designed with C-terminal 6xHisTags and flanking regions for Gibson Assembly into pET15b. All plasmids were transformed into Escherichia coli Rosetta DE3 and recombinant proteins expressed and purified according to previous methods (22.Zhang 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). Saccharomyces cerevisiae eukaryotic elongation factor 1A Homo sapiens eukaryotic elongation factor 1A lysine methyltransferase stable isotope labeling by amino acids in cell culture. Purified SceEF1A (full length or truncated forms) or HseEF1A1 from E. coli (∼10 μm) were incubated with purified methyltransferase (Efm5, N6AMT2, or YLR285W/Efm7, all at ∼5 μm) in the presence of 500 μm S-adenosyl l-methionine (AdoMet) in in vitro methylation buffer (50 mm HEPES-KOH, 20 mm NaCl, 1 mm EDTA, pH 7.4) at 30 °C for 5 h (Efm5 and N6AMT2) or overnight (YLR285W/Efm7). GDP and a nonhydrolysable analog of GTP (guanosine 5′-[γ-thio] triphosphate) were added to a final concentration of 1 mm for the relevant assays. No enzyme was added for the negative controls. 6x SDS sample buffer (350 mm Tris-HCl, pH 6.8, 30% glycerol (v/v), 10% SDS (v/v), 0.6 m DTT, 0.012% bromphenol blue (w/v)) was added to stop reactions, which were then resolved by SDS-PAGE and prepared for mass spectrometry as above. For over 20 years, yeast eEF1A has been known to have four different sites of methylation (31.Cavallius J. Zoll W. Chakraburtty K. Merrick W.C. Characterization of yeast EF-1 alpha: Non-conservation of post-translational modifications.Biochim. Biophys. Acta. 1993; 1163: 75-80Crossref PubMed Scopus (59) Google Scholar). It was therefore surprising to see two tryptic peptides of 682.39 and 691.73 m/z, from the N terminus of eEF1A but lacking the initiator methionine, carrying three or five methyl groups (Supplemental Fig. S1, Supplemental Table S1). To localize the methylation, we further analyzed eEF1A using AspN. This generated the N-terminal peptide GKEKSHINVVVIGHV, whereby the N-terminal glycine is numbered Gly2 and the adjacent lysine Lys3. Fragmentation revealed up to five methyl groups in this peptide (Fig. 1A; see Supplemental Table S1 for details of additional methylation states), whereby three of the five methyl groups were localized to Gly2 and the two others to Lys3. Heavy methyl SILAC confirmed that this methylation is enzyme-mediated (Supplemental Fig. S2). To further investigate the combinations of methylation present, we analyzed the y14 ion corresponding to the fragmentation of peptide GKEKSHINVVVIGHV after its N-terminal glycine. Parallel reaction monitoring and MS/MS/MS (MS3) revealed that the di- and trimethylated forms of the peptide only produced unmethylated y14 ions of 1,558.90 m/z (Figd. 1B and 1C, Supplemental Table S2). Furthermore, the tetra- and pentamethylated forms of the AspN peptide only produced mono- and dimethylated y14 ions, of 1,572.92 and 1,586.94 m/z respectively. Tri-, tetra-, and pentamethylated y14 ions were not observed in any case. Overall, this indicates that the N terminus of eEF1A is trimethylated at Gly2, following which—in some cases—it is further mono- and dimethylated on Lys3 to generate the tetra- and pentamethylated forms. Having established that the N-terminal Gly2 and Lys3 of eEF1A are methylated, we sought to identify the methyltransferase(s) responsible. Trypsinized eEF1A from knockouts of the putative protein methyltransferases YLR285W, YGR001C, YNL024C, and YNL092W (32.Szczepińska T. Kutner J. Kopczynski M. Pawlowski K. Dziembowski A. Kudlicki A. Ginalski K. Rowicka M. Probabilistic approach to predicting substrate specificity of methyltransferases.PLoS Comput. Biol. 2014; 10: e1003514Crossref PubMed Scopus (16) Google Scholar) only revealed a loss of N-terminal Gly2 and Lys3 methylation in ΔYLR285W (Supplemental Fig. S3). AspN digests of eEF1A from wild-type and ΔYLR285W strains confirmed this, clearly showing a complete los
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