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• W2014976589 abstract "In mammalian mitochondria, protein methylation is a relatively uncommon post-transcriptional modification, and the extent of the mitochondrial protein methylome, the modifying methyltransferases, and their substrates have been little studied. As shown here, the β-subunit of the electron transfer flavoprotein (ETF) is one such methylated protein. The ETF is a heterodimer of α- and β-subunits. Lysine residues 199 and 202 of mature ETFβ are almost completely trimethylated in bovine heart mitochondria, whereas ETFα is not methylated. The enzyme responsible for the modifications was identified as methyltransferase-like protein 20 (METTL20). In human 143B cells, the methylation of ETFβ is less extensive and is diminished further by suppression of METTL20. Tagged METTL20 expressed in HEK293T cells specifically associates with the ETF and promotes the trimethylation of ETFβ lysine residues 199 and 202. ETF serves as a mobile electron carrier linking dehydrogenases involved in fatty acid oxidation and one-carbon metabolism to the membrane-associated ubiquinone pool. The methylated residues in ETFβ are immediately adjacent to a protein loop that recognizes and binds to the dehydrogenases. Suppression of trimethylation of ETFβ in mouse C2C12 cells oxidizing palmitate as an energy source reduced the consumption of oxygen by the cells. These experiments suggest that the oxidation of fatty acids in mitochondria and the passage of electrons via the ETF may be controlled by modulating the protein-protein interactions between the reduced dehydrogenases and the β-subunit of the ETF by trimethylation of lysine residues. METTL20 is the first lysine methyltransferase to be found to be associated with mitochondria. In mammalian mitochondria, protein methylation is a relatively uncommon post-transcriptional modification, and the extent of the mitochondrial protein methylome, the modifying methyltransferases, and their substrates have been little studied. As shown here, the β-subunit of the electron transfer flavoprotein (ETF) is one such methylated protein. The ETF is a heterodimer of α- and β-subunits. Lysine residues 199 and 202 of mature ETFβ are almost completely trimethylated in bovine heart mitochondria, whereas ETFα is not methylated. The enzyme responsible for the modifications was identified as methyltransferase-like protein 20 (METTL20). In human 143B cells, the methylation of ETFβ is less extensive and is diminished further by suppression of METTL20. Tagged METTL20 expressed in HEK293T cells specifically associates with the ETF and promotes the trimethylation of ETFβ lysine residues 199 and 202. ETF serves as a mobile electron carrier linking dehydrogenases involved in fatty acid oxidation and one-carbon metabolism to the membrane-associated ubiquinone pool. The methylated residues in ETFβ are immediately adjacent to a protein loop that recognizes and binds to the dehydrogenases. Suppression of trimethylation of ETFβ in mouse C2C12 cells oxidizing palmitate as an energy source reduced the consumption of oxygen by the cells. These experiments suggest that the oxidation of fatty acids in mitochondria and the passage of electrons via the ETF may be controlled by modulating the protein-protein interactions between the reduced dehydrogenases and the β-subunit of the ETF by trimethylation of lysine residues. METTL20 is the first lysine methyltransferase to be found to be associated with mitochondria. The post-translational methylation of proteins by methyltransferases with S-adenosylmethionine as the methyl donor occurs primarily on the side chains of lysine and arginine residues, but histidyl and glutamyl side chains and α-amino and α-carboxyl groups can be methylated also (1.Clarke S. Protein methylation.Curr. Opin. Cell Biol. 1993; 5: 977-983Crossref PubMed Scopus (199) Google Scholar, 2.Grillo M.A. Colombatto S. S-Adenosylmethionine and protein methylation.Amino Acids. 2005; 28: 357-362Crossref PubMed Scopus (47) Google Scholar). The ϵ-amino groups of lysine residues can carry one, two, or three methyl groups, and the guanidino moieties of arginines can be monomethylated, and dimethylated either symmetrically or asymmetrically. The remodeling of chromatin by methylation and demethylation of lysine and arginine residues in histones, together with acetylation and deacetylation of lysines, influences gene expression, DNA replication, and DNA repair and apoptosis among other processes (3.Bedford M.T. Clarke S.G. Protein arginine methylation in mammals: who, what, and why.Mol. Cell. 2009; 33: 1-13Abstract Full Text Full Text PDF PubMed Scopus (1258) Google Scholar, 4.Chang B. Chen Y. Zhao Y. Bruick R.K. JMJD6 is a histone arginine demethylase.Science. 2007; 318: 444-447Crossref PubMed Scopus (520) Google Scholar, 5.Martin C. Zhang Y. The diverse functions of histone lysine methylation.Nat. Rev. Mol. Cell Biol. 2005; 6: 838-849Crossref PubMed Scopus (1597) Google Scholar, 6.Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Shi Y. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (3164) Google Scholar, 7.Tsukada Y. Fang J. Erdjument-Bromage H. Warren M.E. Borchers C.H. Tempst P. Zhang Y. Histone demethylation by a family of JmjC domain-containing proteins.Nature. 2006; 439: 811-816Crossref PubMed Scopus (1610) Google Scholar), and methylation of lysine and arginine residues in non-histone proteins is being associated increasingly with the regulation of many other cellular activities (8.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 (130) Google Scholar, 9.Huang J. Berger S.L. The emerging field of dynamic lysine methylation of non-histone proteins.Curr. Opin. Genet. Dev. 2008; 18: 152-158Crossref PubMed Scopus (225) Google Scholar). Moreover, lysine residues can also be ubiquitinated and sumoylated, and the interplay between methylation, acetylation, and these other post-translational modifications, at the same and neighboring sites, adds complexity to the regulation of biological processes by post-translational modification (9.Huang J. Berger S.L. The emerging field of dynamic lysine methylation of non-histone proteins.Curr. Opin. Genet. Dev. 2008; 18: 152-158Crossref PubMed Scopus (225) Google Scholar, 10.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, 11.Wang H. Cao R. Xia L. Erdjument-Bromage H. Borchers C. Tempst P. Zhang Y. Purification and functional characterization of a histone H3-lysine 4-specific methyltransferase.Mol. Cell. 2001; 8: 1207-1217Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). The methylation reactions are catalyzed by methyltransferases, and 30% of known and putative methyltransferases are associated with diseases including cancer, inflammation, and metabolic disorders (12.Jakobsson M.E. Moen A. Bousset L. Egge-Jacobsen W. Kernstock S. Melki R. Falnes P.Ø. Identification and characterization of a novel human methyltransferase modulating Hsp70 function through lysine methylation.J. Biol. Chem. 2013; 288: 27752-27763Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 13.Petrossian T.C. Clarke S.G. Uncovering the human methyltransferasome.Mol. Cell. Proteomics. 2011; 10 (M110.000976)Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 14.Yang Y. Bedford M.T. Protein arginine methyltransferases and cancer.Nat. Rev. Cancer. 2013; 13: 37-50Crossref PubMed Scopus (722) Google Scholar). Hitherto, the methylation of lysine and arginine residues of proteins found in mitochondria has been little investigated. It is well known that lysine residues in apocytochrome c are methylated by Ctm1p in the cytoplasm of Saccharomyces cerevisiae (15.DeLange R.J. Glazer A.N. Smith E.L. Presence and location of an unusual amino acid, ϵ-N-trimethyllysine, in cytochrome c of wheat germ and Neurospora.J. Biol. Chem. 1969; 244: 1385-1388Abstract Full Text PDF PubMed Google Scholar, 16.Polevoda B. Martzen M.R. Das B. Phizicky E.M. Sherman F. Cytochrome c methyltransferase, Ctm1p, of yeast.J. Biol. Chem. 2000; 275: 20508-20513Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), but this modification has no clear role, and the mammalian orthologue is unmodified. Trimethyllysine residues have been characterized in three proteins isolated directly from mammalian mitochondria: they are citrate synthase (17.Bloxham D.P. Parmelee D.C. Kumar S. Walsh K.A. Titani K. Complete amino acid sequence of porcine heart citrate synthase.Biochemistry. 1982; 21: 2028-2036Crossref PubMed Scopus (58) Google Scholar), ADP/ATP translocase (18.Aquila H. Bogner W. Klingenberg M. ADP/ATP-translocator from beef heart mitochondria: amino acid sequence and surface labelling.Hoppe-Seyler's Z. Physiol. Chem. 1982; 363: 894Google Scholar), and the c-subunit in the rotor of ATP synthase (19.Chen R. Fearnley I.M. Palmer D.N. Walker J.E. Lysine 43 is trimethylated in subunit c from bovine mitochondrial ATP synthase and in storage bodies associated with Batten disease.J. Biol. Chem. 2004; 279: 21883-21887Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). A dimethylarginine residue has been characterized in the NDUFS2 subunit of complex I (20.Carroll J. Ding S. Fearnley I.M. Walker J.E. Post-translational modifications near the quinone binding site of mammalian complex I.J. Biol. Chem. 2013; 288: 24799-24808Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), and a complex pattern of methylation of three histidine residues has been found near the N terminus of the NDUFB3 subunit of complex I (21.Carroll J. Fearnley I.M. Skehel J.M. Runswick M.J. Shannon R.J. Hirst J. Walker J.E. The post-translational modifications of the nuclear encoded subunits of complex I from bovine heart mitochondria.Mol. Cell. Proteomics. 2005; 4: 693-699Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In addition to these five methylations characterized in proteins purified from mitochondria, other human proteins that locate to mitochondria have been reported in cell-wide methylome studies to contain methylated lysine and arginine residues (22.Cao X.-J. Arnaudo A.M. Garcia B.A. Large-scale global identification of protein lysine methylation in vivo.Epigenetics. 2013; 8: 477-485Crossref PubMed Scopus (107) Google Scholar, 23.Guo A. Gu H. Zhou J. Mulhern D. Wang Y. Lee K.A. Yang V. Aguiar M. Kornhauser J. Jia X. Ren J. Beausoleil S.A. Silva J.C. Vemulapalli V. Bedford M.T. Comb M.J. Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.Mol. Cell. Proteomics. 2014; 13: 372-387Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). With the exceptions of HEMK1 3The abbreviations used are: HEMK1heme biosynthesis gene KETDelectron transfer dissociationETFelectron transfer flavoproteinMETTL12methyltransferase-like protein 12METTL20methyltransferase-like protein 20NDUFAF7NADH dehydrogenase (ubiquinone) complex I assembly factor 7NDUFB3NADH dehydrogenase (ubiquinone) 1β subcomplex subunit 3NDUFS2NADH dehydrogenase (ubiquinone) iron-sulfur protein 2OCRoxygen consumption rateSILACstable isotope labeling by amino acids in cell cultureTMLtrimethyllysineDMLdimethyllysine. (heme biosynthesis gene K) and NDUFAF7, the mammalian methyltransferases responsible for the modifications of mitochondrial proteins, their subcellular locations, and the biological significance of the modifications are unknown. HEMK1 methylates a glutamine residue in the mitochondrial translation release factor MTRF1L (24.Ishizawa T. Nozaki Y. Ueda T. Takeuchi N. The human mitochondrial translation release factor HMRF1L is methylated in the GGQ motif by the methyltransferase HMPrmC.Biochem. Biophys. Res. Commun. 2008; 373: 99-103Crossref PubMed Scopus (20) Google Scholar), and NDUFAF7 symmetrically dimethylates Arg-85 in the mature NDUFS2 subunit of the multisubunit enzyme, complex I, an essential step in the assembly of the complex (25.Rhein V.F. Carroll J. Ding S. Fearnley I.M. Walker J.E. NDUFAF7 methylates arginine 85 in the NDUFS2 subunit of human complex I.J. Biol. Chem. 2013; 288: 33016-33026Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). NDUFAF7 is one of 35 known and possible mitochondrial methylases catalogued previously by bioinformatic analysis (25.Rhein V.F. Carroll J. Ding S. Fearnley I.M. Walker J.E. NDUFAF7 methylates arginine 85 in the NDUFS2 subunit of human complex I.J. Biol. Chem. 2013; 288: 33016-33026Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). heme biosynthesis gene K electron transfer dissociation electron transfer flavoprotein methyltransferase-like protein 12 methyltransferase-like protein 20 NADH dehydrogenase (ubiquinone) complex I assembly factor 7 NADH dehydrogenase (ubiquinone) 1β subcomplex subunit 3 NADH dehydrogenase (ubiquinone) iron-sulfur protein 2 oxygen consumption rate stable isotope labeling by amino acids in cell culture trimethyllysine dimethyllysine. As described below, we have found that another mitochondrial protein, the β-subunit of the electron transfer flavoprotein (ETF) is also methylated. The ETF is a heterodimeric complex of α- and β-subunits, known as ETFα and ETFβ, respectively (26.Gorelick R.J. Mizzer J.P. Thorpe C. Purification and properties of electron-transferring flavoprotein from pig kidney.Biochemistry. 1982; 21: 6936-6942Crossref PubMed Scopus (72) Google Scholar, 27.McKean M.C. Beckmann J.D. Frerman F.E. Subunit structure of electron transfer flavoprotein.J. Biol. Chem. 1983; 258: 1866-1870Abstract Full Text PDF PubMed Google Scholar, 28.Sato K. Nishina Y. Shiga K. Electron-transferring flavoprotein has an AMP-binding site in addition to the FAD-binding site.J. Biochem. 1993; 114: 215-222Crossref PubMed Scopus (41) Google Scholar, 29.Roberts D.L. Frerman F.E. Kim J.J. Three-dimensional structure of human electron transfer flavoprotein to 2.1-Å resolution.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 14355-14360Crossref PubMed Scopus (155) Google Scholar). It acts as a mobile electron carrier in the matrix of mitochondria, linking 11 different mitochondrial FAD-containing acyl-CoA dehydrogenases involved in fatty acid β-oxidation to the ubiquinone pool of the respiratory chain (30.Ghisla S. Thorpe C. Acyl-CoA dehydrogenases: a mechanistic overview.Eur. J. Biochem. 2004; 271: 494-508Crossref PubMed Scopus (240) Google Scholar, 31.He M. Pei Z. Mohsen A.W. Watkins P. Murdoch G. Van Veldhoven P.P. Ensenauer R. Vockley J. Identification and characterization of new long chain acyl-CoA dehydrogenases.Mol. Genet. Metab. 2011; 102: 418-429Crossref PubMed Scopus (64) Google Scholar). The ETF also accepts electrons from sarcosine dehydrogenase and dimethylglycine dehydrogenase, two enzymes of mitochondrial one-carbon metabolism (32.Steenkamp D.J. Husain M. The effect of tetrahydrofolate on the reduction of electron transfer flavoprotein by sarcosine and dimethylglycine dehydrogenases.Biochem. J. 1982; 203: 707-715Crossref PubMed Scopus (38) Google Scholar). A “recognition loop” in ETFβ recognizes and interacts with these 13 different dehydrogenases (33.Toogood H.S. van Thiel A. Basran J. Sutcliffe M.J. Scrutton N.S. Leys D. Extensive domain motion and electron transfer in the human electron transferring flavoprotein medium chain acyl-CoA dehydrogenase complex.J. Biol. Chem. 2004; 279: 32904-32912Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 34.Toogood H.S. Leys D. Scrutton N.S. Dynamics driving function: new insights from electron transferring flavoproteins and partner complexes.FEBS J. 2007; 274: 5481-5504Crossref PubMed Scopus (94) Google Scholar), and the sites of methylation are immediately adjacent to the recognition loop. Once bound to the recognition loop, the dehydrogenases reduce an FAD prosthetic group in the ETF. Then the reduced ETF carries the electrons to the ETF-quinone oxidoreductase bound in the inner mitochondrial membrane (35.Watmough N.J. Frerman F.E. The electron transfer flavoprotein: ubiquinone oxidoreductases.Biochim. Biophys. Acta. 2010; 1797: 1910-1916Crossref PubMed Scopus (171) Google Scholar). The precise mode of interaction of ETF with the quinone oxidoreductase is not known, but ETFβ is involved in the formation of this complex (36.Steenkamp D.J. Cross-linking of the electron-transfer flavoprotein to electron-transfer flavoprotein-ubiquinone oxidoreductase with heterobifunctional reagents.Biochem. J. 1988; 255: 869-876Crossref PubMed Scopus (17) Google Scholar). Finally, the reduced ETF-quinone oxidoreductase transfers the electrons to ubiquinone, and the reduced quinone enters the quinone pool, providing electrons for the cytochrome bc1 complex (complex III) to reduce cytochrome c. The location of the methylated lysine residues characterized in the current study, immediately adjacent to the recognition loop, suggests that their methylation may influence the interactions of the ETF with the dehydrogenases and possibly with ETF-quinone oxidoreductase, and that it could provide a mechanism of regulating the β-oxidation of fatty acids. We have also identified METTL20 (methyltransferase-like protein 20) as the mitochondrial methyltransferase that introduces methyl groups into two specific lysine residues in ETFβ. No protein lysine methyltransferase had been characterized previously in mitochondria. Human 143B osteosarcoma cells (ATCC number CRL8303) and mouse C2C12 myoblasts (Public Health England 91031101) were grown at 37 °C in Dulbecco's modified Eagle's medium (DMEM) containing 25 mm glucose and supplemented with fetal bovine serum (FBS, 10% v/v), penicillin (100 units/ml), and streptomycin (0.1 mg/ml) under an atmosphere of 5% CO2. The serum in the media for parental human embryonic kidney cells (HEK293T) and FLAG-strepII-tagged METTL20 inducible HEK293T cells was tetracycline-free, and the media included blasticidin (15 μg/ml) and zeocin (100 μg/ml), or blasticidin (15 μg/ml) and hygromycin (100 μg/ml), respectively. The cDNA for human METTL20 (Thermo Scientific, Loughborough, UK) was amplified by PCR with the forward and reverse primers: 5′-CGCGGATCCGGAATGGCTTTGAGTCTAGGTTGGAAAG-3′ and 5′-ATAGTTTAGCGGCCGCCCAGGCTGAAAACCCCACACTGTGC-3′, respectively. It was cloned into the inducible expression vector pcDNA5/FRT/TO (Invitrogen) with sequences encoding C-terminal FLAG and Strep tags, and incorporated stably into HEK293T Flp-InTM T-RexTM cells. Plasmids were transfected into 143B cells with Lipofectamine 2000 (Invitrogen). After 24 h, the mitochondria were labeled with MitoTracker Orange (200 nm; Invitrogen), and the nuclei were stained with DAPI (0.4 μg/ml). The same cells were fixed with paraformaldehyde (4%, w/v) and permeabilized with Triton X-100 (0.5%, v/v). The FLAG-tagged METTL20 was detected with mouse M2 anti-FLAG antibody (Sigma) followed by Alexa Fluor 488 goat anti-mouse secondary antibody (Invitrogen). The fluorescent signal was visualized with a Zeiss 510 LSM confocal microscope (Zeiss, Cambridge, UK). Submitochondrial particles were prepared from bovine heart mitochondria (37.Pryde K.R. Hirst J. Superoxide is produced by the reduced flavin in mitochondrial complex I: a single, unified mechanism that applies during both forward and reverse electron transfer.J. Biol. Chem. 2011; 286: 18056-18065Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). They were centrifuged (37,000 × g for 20 min, and then at 110,000 × g for 30 min), and the supernatant was fractionated at room temperature by ammonium sulfate precipitation. The 60–90% fraction was re-solubilized in buffer A (pH 7.4) consisting of 25 mm HEPES and 0.1 mm EDTA, dialyzed against the same buffer, and fractionated by cation exchange chromatography on a HiTrap SP HP column (1 ml; GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The sample was loaded onto the column equilibrated in buffer A at a flow rate of 0.5 ml/min, and eluted with a linear gradient of 0–150 mm NaCl in buffer A over 60 min at room temperature. Fractions containing the partially purified ETF were analyzed by SDS-PAGE and mass spectrometry. Plasmid pcDNATM5/FRT/TO encoding METTL20 with C-terminal StrepII and FLAG tags was cotransfected in the presence of Lipofectamine 2000 (Invitrogen) with plasmid pOG44 into human HEK293T Flp-In T-Rex cells (total DNA, 1 μg; pOG44:pcDNA5/FRT/TO, 7:1 by weight) (Invitrogen) (38.He J. Cooper H.M. Reyes A. Di Re M. Kazak L. Wood S.R. Mao C.C. Fearnley I.M. Walker J.E. Holt I.J. Human C4orf14 interacts with the mitochondrial nucleoid and is involved in the biogenesis of the small mitochondrial ribosomal subunit.Nucleic Acids Res. 2012; 40: 6097-6108Crossref PubMed Scopus (61) Google Scholar). After 24 h, the medium was replaced with the selective medium containing blasticidin (15 μg/ml) and hygromycin (100 μg/ml) and inducible cell lines expressing the recombinant protein were selected. Expression of tagged METTL20 was induced for 24 h with doxycycline (20 ng/ml). Mitochondria were prepared (38.He J. Cooper H.M. Reyes A. Di Re M. Kazak L. Wood S.R. Mao C.C. Fearnley I.M. Walker J.E. Holt I.J. Human C4orf14 interacts with the mitochondrial nucleoid and is involved in the biogenesis of the small mitochondrial ribosomal subunit.Nucleic Acids Res. 2012; 40: 6097-6108Crossref PubMed Scopus (61) Google Scholar) and solubilized in 1% (w/v) n-dodecyl β-d-maltoside for affinity purification of tagged METTL20 and associated proteins. To prepare mitoplasts, cells were suspended at a protein concentration of 10 mg/ml in PBS-inhibitor (phosphate-buffered saline with complete EDTA-free protease inhibitor from Roche Applied Science) and enriched for mitoplasts by addition of an equal volume of digitonin (1 mg/ml) in PBS-inhibitor to give a detergent:protein ratio of 1:10 (w/w) (39.Klement P. Nijtmans L.G. Van den Bogert C. Houštěk J. Analysis of oxidative phosphorylation complexes in cultured human fibroblasts and amniocytes by blue-native-electrophoresis using mitoplasts isolated with the help of digitonin.Anal. Biochem. 1995; 231: 218-224Crossref PubMed Scopus (94) Google Scholar). The sample was centrifuged (11,000 × g, 5 min, 4 °C), and mitoplasts were solubilized from the pellet with 1% (w/v) n-dodecyl β-d-maltoside. The enriched mitochondrial protein preparation was loaded onto a StrepII tag gravity column (Pierce Spin Column containing Strep-Tactin-Sepharose) followed by 5 column volumes of wash buffer (20 mm HEPES, pH 7.6, 0.2 mm EDTA, 150 mm NaCl, 2 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, Roche protease inhibitor (1/50; v/v), and 0.05% (w/v) n-dodecyl β-d-maltoside). Bound protein was eluted with 6 portions of 0.5 column volumes of wash buffer containing 10 mm desthiobiotin. Eluates 1–3 were combined and analyzed by SDS-PAGE and mass spectrometry. Transcripts of METTL20 were suppressed transiently in human 143B cells with 30 nm siRNAs and transcripts of METTL20 or ETFβ in mouse C2C12 cells with 100 nm siRNAs (Sigma). Allstars negative control siRNA (Qiagen, Crawley, UK) was used at the same concentration as the specific siRNA. Two transfections were performed at 0 and 72 h. The levels of the transcripts (normalized to endogenous β-actin) were investigated at 48 and 120 h by quantitative real-time PCR performed with TaqMan gene expression assays (Invitrogen) on cDNA prepared with a Cells-to-CT kit (Invitrogen). Mitoplasts were prepared 48 h after each transfection, and the oxygen consumption rate (OCR) of C2C12 cells was assessed 48 h after the second transfection. Protein concentrations were estimated by bicinchoninic acid assay (Pierce, ThermoFisher). Proteins were fractionated by SDS-PAGE on Novex Tris glycine 10–20% acrylamide gradient gels (Invitrogen) and transferred electrophoretically to an Immobilon P membrane (Millipore, Billerica, MA) for immunodetection. The membrane was washed in PBS containing 0.01% (v/v) Tween 20 (PBST), and then treated with a solution of dried skimmed milk, or where anti-methyl antibodies were to be employed, with bovine serum albumin (3%, w/v) in the same buffer. Bound proteins were reacted with primary rabbit antibodies with specificities for dimethyllysine and trimethyllysine (Abcam, Cambridge, UK) for ETFβ and citrate synthase (Proteintech, Manchester, UK) and NDUFB8 (Sigma). Bound rabbit antibodies were peroxidase conjugated with goat anti-rabbit antiserum (ThermoFisher), and detected with enhanced chemiluminescence reagents (GE Healthcare). Bovine, human, and mouse proteins in extracts and column fractions were reduced, alkylated, and fractionated by SDS-PAGE in 12–22 or 10–20% polyacrylamide gradient gels, as described before (25.Rhein V.F. Carroll J. Ding S. Fearnley I.M. Walker J.E. NDUFAF7 methylates arginine 85 in the NDUFS2 subunit of human complex I.J. Biol. Chem. 2013; 288: 33016-33026Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Proteins were characterized by tandem-MS analysis of tryptic, chymotryptic, and AspN digests of Coomassie Blue-stained bands or sections of gel. Extracted peptides were analyzed in a MALDI-TOF-TOF mass spectrometer (model 4800; AB-Sciex, Warrington UK) with α-cyano-4-hydroxycinnamic acid as matrix, or in an LTQ OrbiTrap XL-ETD (electron transfer dissociation) mass spectrometer (Thermo Scientific) coupled to a Proxeon Easy-nLC (Thermo Scientific) with a C18 column (100 mm × 75 μm inner diameter; Nanoseparations, Nieukoop, The Netherlands) for reverse-phase fractionation of peptides using an acetonitrile gradient in 0.1% (v/v) formic acid with a solvent flow rate of 300 nl/min. Peptides were fragmented by collision-induced dissociation with either air (4800 instrument) or nitrogen (LTQ OrbiTrap XL-ETD), or by ETD with fluoranthene radical anions (LTQ OrbiTrap XL-ETD) and supplemental activation (40.Swaney D.L. McAlister G.C. Wirtala M. Schwartz J.C. Syka J.E. Coon J.J. Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors.Anal. Chem. 2007; 79: 477-485Crossref PubMed Scopus (311) Google Scholar). MALDI-TOF-TOF data were analyzed with Mascot (41.Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data.Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6771) Google Scholar) (version 2.4.0) using the following parameters: NCBInr database 20120611, taxonomy, Mammalia; precursor ion mass tolerance 70 ppm; fragment ion mass tolerance 0.8 Da; Met oxidation variable, Cys-carbamidomethyl fixed (iodoacetamide-treated samples) or Cys-propionamide (non-alkylated samples); trypsin 2-missed cleavages. A significance threshold of p < 0.05 was used for peptide identification. Orbitrap peptide fragmentation data were analyzed using Proteome Discoverer 1.3 (Thermo Fisher) with Mascot and Peptide Validator nodes. The following parameters were employed: UniProt_2013_04, taxonomy, Mammalia (for bovine and mouse)/Homo sapiens (for human); precursor ion mass tolerance 5 ppm; fragment ion mass tolerance 0.5 Da; variable modifications, Met oxidation, Lys/Arg-methyl, Lys/Arg-dimethyl, Lys-trimethyl; fixed modification Cys-carbamidomethyl; trypsin 2-missed, AspN_ambic 3-missed, chymotrypsin 4-missed cleavages; decoy database search (false discovery rate values 0.01 and 0.05). The fragmentation spectra of modified peptides were interpreted manually. ETD fragment ions were assigned as c and z ions (including related z+H ions) (42.Sun R-X. Dong M-Q. Song C-Q. Chi H. Yang B. Xiu L-Y. Tao L. Jing Z-Y. Liu C. Wang L-H. Fu Y. He S.M. Improved peptide identification for proteomic analysis based on comprehensive characterization of electron transfer dissociation spectra.J. Proteome Res. 2010; 9: 6354-6367Crossref PubMed Scopus (39) Google Scholar). The relative abundance of methylation of ETFβ was determined by analysis of an AspN-generated peptide (EPRYATLPNIMKAKKKKI) that contains both methylated lysine residues. Monoisotopic m/z values used are 710.4263 and 715.7579 (M + 3H)3+, plus 533.0717 and 537.0704 (M + 4H)4+ for the non-methylated and non-methylated/Met-oxidized forms of the peptide, respectively. 14.0156 Da were added for each methyl group. The peak areas of Gaussian smoothed extracted ion chromatograms, with a m/z tolerance of 5 ppm and overlapping peptide retention times, were obtained with the aid of Xcalibur software. The intact bovine ETF subunits were separated by reverse-phase chromatography and their molecular masses were determined by LC-MS (43.Carroll J. Fearnley I.M. Wang Q. Walker J.E. Measurement of the molecular masses of hydrophilic and hydrophobic subunits of ATP synthase and complex I in a single experiment.Anal. Biochem. 2009; 395: 249-255Crossref PubMed Scopus (30) Google Scholar). Inducible HEK293T cells expressing either tagged METTL20 or METTL12 were grown in “heavy” DMEM containing arginine and lysine isotopically labeled with 15N and 13C, and in “light” DMEM containing 14N- and 12C-arginine and -lysine (Sigma) (44.Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4576) Google Scholar). These media were supplemented with penicillin (100 units/ml) and streptomycin (0.1 mg/ml), and proline (200 mg/liter) to suppress the conversion of arginine to proline, and with dialyzed FBS (10% v/v) (Invitrogen) to prevent dilution of heavy isotopes. To ensure maximal incorporation, the cell populatio" @default.
• W2014976589 created "2016-06-24" @default.
• W2014976589 creator A5007387177 @default.
• W2014976589 creator A5024069094 @default.
• W2014976589 creator A5028904725 @default.
• W2014976589 creator A5029211607 @default.
• W2014976589 creator A5031090925 @default.
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• W2014976589 date "2014-08-01" @default.
• W2014976589 modified "2023-10-14" @default.
• W2014976589 title "Human METTL20 Methylates Lysine Residues Adjacent to the Recognition Loop of the Electron Transfer Flavoprotein in Mitochondria" @default.
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