Human METTL20 Is a Mitochondrial Lysine Methyltransferase That Targets the β Subunit of Electron Transfer Flavoprotein (ETFβ) and Modulates Its Activity*

Background: Many proteins are modified by lysine methylation. Results: It is shown that the previously uncharacterized enzyme METTL20 methylates electron transfer flavoprotein β (ETFβ), thereby inhibiting its ability to mediate electron transfer from acyl-CoA dehydrogenases. Conclusion: METTL20-mediated methylation modulates the function of ETFβ. Significance: The first mitochondrial lysine-specific protein methyltransferase in animals is reported, and the resulting methylation is shown to have functional consequences. Proteins are frequently modified by post-translational methylation of lysine residues, catalyzed by S-adenosylmethionine-dependent lysine methyltransferases (KMTs). Lysine methylation of histone proteins has been extensively studied, but it has recently become evident that methylation of non-histone proteins is also abundant and important. The human methyltransferase METTL20 belongs to a group of 10 established and putative human KMTs. We here found METTL20 to be associated with mitochondria and determined that recombinant METTL20 methylated a single protein in extracts from human cells. Using an methyltransferase activity-based purification scheme, we identified the β-subunit of the mitochondrially localized electron transfer flavoprotein (ETFβ) as the substrate of METTL20. Furthermore, METTL20 was found to specifically methylate two adjacent lysine residues, Lys200 and Lys203, in ETFβ both in vitro and in cells. Interestingly, the residues methylated by METTL20 partially overlap with the so-called “recognition loop” in ETFβ, which has been shown to mediate its interaction with various dehydrogenases. Accordingly, we found that METTL20-mediated methylation of ETFβ in vitro reduced its ability to receive electrons from the medium chain acyl-CoA dehydrogenase and the glutaryl-CoA dehydrogenase. In conclusion, the present study establishes METTL20 as the first human KMT localized to mitochondria and suggests that it may regulate cellular metabolism through modulating the interaction between its substrate ETFβ and dehydrogenases. Based on the previous naming of similar enzymes, we suggest the renaming of human METTL20 to ETFβ-KMT.

Electron transfer flavoprotein (ETF) is a FAD-containing, mitochondrial protein composed of two subunits: ␣ and ␤ (ETF␣ and ETF␤, respectively) (13)(14)(15)(16). ETF acts as a mobile electron carrier that shuttles electrons between several FADcontaining dehydrogenases present in the mitochondrial matrix and the membrane-bound ETF:quinone oxidoreductase (17). Electrons are then passed on to the ubiquinone pool of the mitochondrial respiratory chain. Altogether, there are 13 human dehydrogenases that interact with ETF, and it has been proposed that a "recognition loop" within the ETF␤ chain, composed of residues 191-200, is responsible for interaction with the dehydrogenases (18). Some of these dehydrogenases, e.g. medium chain acyl-CoA dehydrogenase (MCAD), are involved in ␤-oxidation of fatty acids (19), whereas others, including glutaryl-CoA dehydrogenase (GCDH) and isovaleryl-CoA dehydrogenase, are involved in the oxidation of amino acids (20,21). Yet another group is involved in oxidative reactions in the pathway for degradation of choline to glycine, resulting in the transfer of one-carbon moieties. These are dimethylglycine dehydrogenase (DMGDH) and sarcosine dehydrogenase (SARDH), which demethylate dimethylglycine and sarcosine, respectively (22,23). Both DMGDH and SARDH also contain tetrahydrofolate as a co-factor, which serves to accept formaldehyde released during removal of methyl group from dimethylglycine and sarcosine, thus producing 5,10-methylene-tetrahydrofolate (23). In summary, ETF is involved in oxidation of several types of metabolites, and, in addition to complexes I and II, it is the third major provider of electrons to the ubiquinone pool of the mitochondrial respiratory chain.
Human METTL20 is a MTF16 member, suggesting its involvement in protein methylation, and we set out to investigate the function of this uncharacterized protein. Thus, in the present report, we provide evidence that human METTL20 is a mitochondrial protein containing a functional N-terminal mitochondrial targeting sequence (MTS). Furthermore, by using an activity-based approach, we identified METTL20 as a highly specific MTase responsible for methylation of ETF␤ and provide direct biochemical evidence that METTL20 is targeting ETF␤ at two residues, namely Lys 200 and Lys 203 . We also show that METTL20 can methylate ETF␤ in vivo. Finally, we demonstrate that methylation of ETF impairs its ability to extract electrons from two acyl-CoA dehydrogenases, namely MCAD and GCDH, thus suggesting an important functional role for METTL20-mediated methylation of ETF␤.
Cloning and Mutagenesis-ORFs for human METTL20, ETF␣, ETF␤, MCAD, and GCDH were cloned from cDNA generated from HEK293 or HeLa cells, using Phusion DNA Polymerase HF (Thermo Fisher Scientific, Waltham, MA). PrimerX program was used to design mutagenesis primers. All constructs were sequence-verified, including tags.
Bioinformatics Analysis-The NCBI Basic Local Alignment Search Tool (BLAST) was used to identify sequence homologues of human METTL20 (24). Multiple protein sequence alignments and secondary structure predictions were performed using algorithms embedded in the Jalview (v2.8) interface (25). Prediction of the N-terminal MTS and the location of the putative cleavage site were performed using the MitoProt algorithm (26) available at the ExPASy Bioinformatics Resource Portal.
Transient Transfection and Generation of Stable Cell Lines-HeLa cells were transiently transfected with pEGFP-N1 plasmid (Clontech), either empty or containing cDNA encoding human METTL20 or its putative MTS (amino acids 1-42). Cells were analyzed by confocal fluorescence microscopy 24 h after transfection. Plasmid pcDNA5/FRT/TO (Invitrogen, Thermo Fisher Scientific) was used for stable incorporation of METTL20-GFP fusion gene into Flp-In T-REx-293 cells (Invitrogen), according to the manufacturer's protocol, and the resulting cells were designated as TRex-METTL20-GFP cells. Overexpression of METTL20-GFP was induced for 48 h with 1 g/ml doxycycline (Dox), and cells were subjected to confocal fluorescence microscopy or collected for biochemical analysis by scraping.
Confocal Fluorescence Microscopy-Living cells were stained with 50 nM MitoTracker Deep Red FM (Invitrogen) and 0.5 g/ml Hoechst 33258 (Sigma-Aldrich) to visualize the mitochondria and the nuclei, respectively. Cells were imaged using an Olympus FluoView 1000 (Ix81) confocal fluorescence microscopy system with a PlanApo 60ϫ NA 1.1 oil objective (Olympus, Hamburg, Germany). The different fluorophores were excited at 405 nm (Hoechst), 488 nm (GFP), and 635 nm (MitoTracker), and Kalman averaging (n ϭ 3) was used to record multichannel images. The fluorescent signals emitted from GFP, MitoTracker, and Hoechst were acquired through green, red, and blue channels, respectively, and merged.
Preparation and Fractionation of Cell Extracts-Human cells were lysed for 10 min at 4°C in lysis buffer 3 (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Triton X-100, 5% glycerol, 0.5 mM DTT, and 1ϫ Complete (EDTA-free) protease inhibitor mixture), and the resulting lysates were sonicated and cleared by centrifugation. Cell lysates were fractionated at 4°C by ion exchange chromatography using either Pierce Strong Anion Exchange Q or Pierce Strong Cation Exchange S Spin Columns (Thermo Fisher Scientific). In short, lysates were diluted with dilution buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, and 5% glycerol), to bring NaCl concentration to 50 mM and then applied onto the Q column equilibrated in dilution buffer. Material bound to the Q column was eluted by a step gradient of increasing NaCl concentrations prepared in dilution buffer, in 100-l fractions: 0.15Q (eluted between 0.05 and 0.15 M NaCl), 0.3Q (eluted between 0.15 and 0.3 M NaCl), and 0.5Q (eluted between 0.3 and 0.5 M NaCl). Unbound material, which was released in the Q column flow-through (FTQ) was reapplied onto the S column equilibrated in dilution buffer. The unbound material, present in the S column flow-through, was designated FTS, whereas material bound to the S column was eluted in 100-l fractions by a step gradient of increasing NaCl concentrations prepared in dilution buffer, analogous to the elution from the Q column. To partially purify ETF␤ from TRex-METTL20-GFP cells, cell extracts were loaded on the Q column, and ETF␤ was recovered in FTQ.
Preparation of Samples for MS Analysis-In vitro methylation of recombinant or cellular (in extract) ETF␤ for the purpose of MS analysis was performed as in the above section, except that radiolabeled SAM was replaced by 1 mM nonradioactive SAM. Proteins were resolved by SDS-PAGE and stained with Coomassie, and the portion of gel containing the protein of interest was cut out and subjected to in-gel trypsin (Sigma-Aldrich) or AspN (Roche) digestion, followed by MS analysis of the resulting proteolytic fragments, as previously described (8). MS data were analyzed with an in-house maintained human protein sequence database using SEQUEST TM and Proteome Discoverer TM (Thermo Fisher Scientific). The mass tolerances of a fragment ion and a parent ion were set as 0.5 Da and 10 ppm, respectively. Methionine oxidation and cysteine carbamido-methylation were selected as variable modifications. ETF␤ methylated peptide MS/MS spectra were manually searched by Qual Browser (v2.0.7).
Large Scale Preparation of Methylated Recombinant ETF␣/ ␤-Large scale preparation of methylated ETF␣/␤ (ETF␣/␤ met ) was made by overnight incubation at 23°C of 40 M ETF␣/␤ and 40 M ⌬38-METTL20, in MTase assay buffer containing 3 mM SAM and 50 M FAD. For the control reactions, METTL20 and SAM were omitted. Both reactions were subject to ion exchange purification, by loading onto Pierce strong anion exchange Q spin columns, equilibrated in MTase assay buffer, and the flow-through (containing ETF␣/␤) was collected and rebuffered to storage buffer 3, using centrifugal concentrators with a 50-kDa cutoff. Protein purity and concentration was determined as in previous sections. Both ETF␣/␤ and ETF␣/ ␤ met had equal A 280 /A 450 ratios.
DCIP Reduction Assay-The ability of ETF␣/␤ to mediate electron transfer from acyl-CoA dehydrogenases was tested using the previously published DCIP reduction method (19), with some modifications. Typically, 250 -350-l mixtures were prepared, containing ETF␣/␤ (0. . Reaction mixtures were allowed to equilibrate at room temperature for 15 min, and then the reaction was started by adding, through manual mixing, glutaryl-CoA (50 M) to assay GCDH or octanoyl-CoA (50 M) to assay MCAD. The kinetics of DCIP reduction (bleaching) was monitored continuously at 600 nm for 3-5 min, using a Shimadzu UV-1601 spectrophotometer. The rate of DCIP reduction was calculated from the slope of the linear part of the kinetic curve, using molecular extinction coefficient of DCIP: ⑀ ϭ 21,700 M Ϫ1 cm Ϫ1 and expressed in M of DCIP reduced per minute. Student's t test was used to evaluate the probability (p values) that two populations are the same.

RESULTS
Human METTL20 Is an Evolutionarily Conserved Mitochondrial Protein Methyltransferase-As an initial step in our functional characterization of human METTL20, we performed a BLAST search, which revealed the presence of putative METTL20 orthologues in a wide range of organisms (Fig. 1A). Interestingly, METTL20 shows a rather scattered and unusual distribution across the tree of life. In eukaryotes, METLL20 is found exclusively in multicellular animals and is apparently present in all chordates (animals with a dorsal nerve chord), while showing a more scattered distribution among other animals, such as insects and nematodes. Interestingly, METTL20 orthologues are also found in a small subset of bacteria, primarily those belonging to the Rhizobiales order of ␣-proteobacteria.
Sequence alignment of putative METTL20 orthologues from various organisms revealed the presence of conserved sequence segments, encompassing the hallmark motifs of the 7BS-MTases, as well as the characteristic (D/E)XX(Y/F) motif shared by the MTF16 members (Fig. 1A). Compared with its prokaryotic counterparts, human METLL20 contains a ϳ40-amino acid N-terminal extension, which, by using the MitoProt algorithm (26), was predicted to represent a MTS. Indeed, both full-length METTL20 and the isolated putative MTS were, when expressed as N-terminal fusions with GFP, able to target GFP to mitochondria, indicating that METTL20 is a mitochondrial protein (Fig. 1B).
Previously, we have shown that recombinant MTF16 enzymes with KMT activity can methylate specific proteins in cellular extracts (8,10), and we investigated METTL20 for such activity. In these assays, we used a METTL20 deletion mutant lacking the MTS, which we reasoned was likely dispensable for enzymatic activity. Methylation reactions with cellular extracts were performed in the presence of [ 3 H]SAM and then subjected to SDS-PAGE and fluorography, enabling the visualization of methylated proteins as distinct bands. Indeed, human METTL20 methylated a protein of ϳ28 kDa in extracts from both HeLa and HEK293 cells (Fig. 1C). Several additional labeled proteins were observed, but their labeling was not METTL20-dependent, indicating that they reflect methylations performed by endogenous MTases in the extract.
Identification of ETF␤ as the Substrate of Recombinant Human METTL20 -To reveal the identity of the ϳ28-kDa substrate by MS, we applied an activity-based purification strategy to reduce the complexity of the sample. Thus, a HEK293 cell lysate was fractionated using ion exchange chromatography, and the fractions were assayed for METTL20-mediated methylation ( Fig. 2A). We found that the ϳ28-kDa substrate bound poorly to an anion exchange (Q) column and was found mostly in the flow-through (FTQ) (Fig. 2A). When the FTQ fraction was applied to a cation exchange (S) column, the ϳ28-kDa protein was found to partially bind to the S column and was completely eluted with 0.15 M NaCl (0.15 S) ( Fig. 2A). To identify the METTL20 substrate, the 0.15 S fraction was resolved by SDS-PAGE, and the region corresponding to the substrate was excised and subjected to trypsin digestion, followed by protein identification through MS. The analysis identified 10 different proteins, and interestingly, and in agreement with the observed mitochondrial localization of METTL20, four of these were mitochondrial proteins, i.e. 3-hydroxyacyl-CoA dehydrogenase type 2, ETF␤, adenylate kinase 2, and malate dehydrogenase (Table 1). To directly identify the METTL20 target, as well as the residue(s) being methylated, a parallel sample had been generated, where the 0.15 S fraction had been treated with METTL20 in the presence of nonradioactive SAM. Intriguingly, only a single peptide was found to display a different degree of methylation between the untreated and the METTL20-treated samples, namely the ETF␤-derived YATLPNIMKAK peptide (residues 192-202), where the methylation level of Lys 200 was substantially increased in the METTL20-treated sample (Fig. 2, B and C). In the untreated sample, Lys 200 was found to be mostly unmethylated and dimethylated with some monomethylation and trimethylation. In contrast, in the METTL20-treated sample Lys 200 was exclusively found in the dimethylated and trimethylated states. Notably, among the four potential METTL20 substrates localized to mitochondria, ETF␤ was the one showing the highest sequence coverage and also displayed the second highest score (Table 1). Thus, these results indicate that METTL20 can catalyze the methylation of Lys 200 in ETF␤ and suggest that this enzyme mediates this reaction also in vivo.
Recombinant METTL20 Catalyzes Methylation of Lys 200 and Lys 203 in ETF␤ in Vitro-To directly demonstrate that METTL20 can methylate ETF␤, we expressed and purified His 6 -tagged human ETF␤ and found recombinant METTL20 to methylate this substrate in vitro (data not shown). However, because the isolated ETF␤ subunit displayed stability problems, we generated ETF␣/␤ heterodimers through co-expression and co-purification of untagged ETF␣ with His 6 -tagged ETF␤ in E. coli. Indeed, the ␤-subunit of recombinant ETF␣/␤ was subject to methylation by METTL20 in vitro (Fig. 3A).
Our previous results (Fig. 2, B and C) indicated that Lys 200 is target of METTL20-mediated methylation, and importantly, this residue has already been annotated as methylated in vivo, according to the Phosphosite database (27). Interestingly, Phosphosite also reports another methylation site in ETF␤, namely the neighboring Lys 202 . To further investigate the potential methylation at these two sites, we generated K200A, K202A, and K200A/K202A mutants, which were tested (in the context of ETF␣/␤) for METTL20-mediated methylation. Somewhat surprisingly, mutating both these residues did not abolish methylation, suggesting the presence of other methylation sites (Fig. 3A). Indeed, when the K200A/K202A mutated ETF␣/␤ was methylated in vitro and subjected to MS analysis, di-and trimethylation at a neighboring site, Lys 203 , was observed (Fig.  3B). Thus, we next tested a K200R/K203R double mutant and found that METTL20 mediated methylation was completely abolished (Fig. 3C). In contrast, the K200R and K203R single mutants were methylated, albeit at reduced levels. Furthermore, when the WT and mutant ETF␣/␤ substrates were treated with different amounts of METTL20, the titration curves for K200R and K203R were similar and plateaued off at a level corresponding to ϳ50% of that observed with the WT protein (Fig. 3D). This suggested that Lys 200 and Lys 203 are methylated at similar efficiencies and that their methylation together account for all the observed METTL20-mediated methylation of ETF␤.
To specifically investigate the methylation state of Lys 200 and Lys 203 after METTL20-mediated methylation, mutant or WT ETF␣/␤ was incubated with recombinant METTL20, and an AspN generated peptide encompassing both methylation sites was analyzed by MS. We found the peptide derived from WT  1 and 3) or presence (lanes 2 and 4) of METTL20. Proteins were resolved by SDS-PAGE and transferred onto a PVDF membrane, which was subjected to fluorography. The positions of the ϳ28-kDa substrate and molecular mass markers are indicated.
ETF␤ to be primarily tetra-, penta-, or hexamethylated after METTL20 treatment, whereas corresponding peptides from the K200R and K203R mutants were mostly di-or trimethylated (Fig. 3E). As expected, no methylation was observed for the K200R/K203R double mutant. These results show that METTL20 is a nonprocessive enzyme capable of generating several different methylation states.
Clearly, the above results show that Lys 200 and Lys 203 are targets of METTL20, but, because we were unable to obtain sequence information for the AspN-generated peptide by MS, we set out to obtain independent evidence that these are the only methylation sites. Lys 200 and Lys 203 are found as part of the sequence KAKKKK (residues 200 -205), i.e. in the context of several other lysines, and to individually investigate the potential methylation at these residues, the KAKKKK segment was mutated into RARRRR, and the individual Lys residues were reintroduced. When these mutants were tested in MTase assays with METTL20, only the ones with Lys in position 200 or 203, i.e. the sequences KARRRR or RARKRR, respectively, were methylated (Fig. 3F). In summary, the data presented above show that METTL20 is a highly specific methyltransferase, capable of independently methylating the two sites Lys 200 and Lys 203 in ETF␤ in vitro.
METTL20-mediated Methylation of ETF␤ in Cells-Having demonstrated that cellular ETF␤ is methylated and that recombinant METTL20 efficiently methylates ETF␤ in vitro, we set out to show that METTL20-mediated methylation of ETF␤ also can occur in a cellular context. To study this, we generated a human cell line expressing a fusion protein between GFP and METTL20 (METTL20-GFP) under a tetracycline inducible promoter. To verify the inducible expression of METTL20-GFP, cells were treated with the tetracycline analogue Dox, and, indeed, a strong GFP signal was observed in mitochondria (Fig. 4A).
To address whether ectopically expressed METTL20 methylates cellular ETF␤, we analyzed the methylation status of the tryptic peptide encompassing Lys 200 by MS (unfortunately, we were not able to detect the AspN-generated peptide that encompassed both methylation sites, shown in Fig. 3E, in these cellular samples). In the absence of Dox treatment, Lys 200 was found predominantly in the dimethylated state but also showed some mono-or trimethylation. However, after induction of METTL20-GFP expression through Dox treatment, Lys 200 was shifted to nearly complete (Ͼ99.5%) trimethylation (Fig. 4B). Because ETF␤ from cells with ectopically expressed METTL20 is completely trimethylated, one would predict that ETF␤ from such cells is not substrate for further methylation by METTL20. To test this, thereby providing independent evidence for METTL20-mediated methylation of ETF␤ in cells, we subjected an extract from the Dox-treated cells to in vitro methylation by METTL20 in the presence of [ 3 H]SAM. Indeed, extracts from the Dox-induced cells showed no radiolabeled band corresponding to ETF␤ in fluorography, whereas the noninduced cells showed a strong band (Fig. 4C). Taken together, the results described above show that METTL20 is capable of methylating ETF␤ also in vivo.
ETF␣/␤ Methylation Reduces Electron Transfer from Acyl-CoA Dehydrogenases-ETF␣/␤ acts as a mobile electron carrier that shuttles electrons from 13 different mitochondrial dehydrogenases to ETF:quinone oxidoreductase (16). Apparently, the interaction between ETF␣/␤ and these dehydrogenases is mediated by a recognition loop in ETF␤ encompassing residues 191-200 (18), i.e. a region that partially overlaps with the segment methylated by METTL20. Thus, to investigate the interesting possibility that METTL20-induced methylation has a modulatory/regulatory function, we assessed the effect of methylation on the ability of ETF␣/␤ to receive electrons from dehydrogenases in vitro. We found that some dehydrogenases were difficult to express and purify or failed to show ETF-dependent activity in vitro (data not shown), However, we were able to express and purify recombinant MCAD and GCDH, and both these enzymes displayed ETF␣/␤-dependent reduction of their respective substrates, octanoyl-CoA and glutaryl-CoA (Fig. 5A). In these experiments, DCIP was used as the final electron acceptor, and ETF-dependent dehydrogenase activity was monitored as bleaching of DCIP, resulting from its reduction. Interestingly, when we compared methylated (ETF␣/␤ met ) and unmethylated ETF␣/␤ for their ability to transfer electrons from the dehydrogenases to DCIP, we found that methylation substantially reduced the extent of electron transfer from both MCAD and GCDH, i.e. by ϳ30 and 40%, respectively (Fig. 5A). To exclude the possibility that the observed differences between ETF␣/␤ and ETF␣/␤ met originated from small errors in protein concentration determination, we also performed an experiment where the concentration of ETF␣/␤ or ETF␣/␤ met was varied, whereas that of MCAD was kept constant. The resulting titration curves show that the observed difference between ETF␣/␤ and ETF␣/␤ met would approximately correspond to a 2-3-fold difference in ETF␣/␤ concentration, i.e. far more than what would expectedly be caused by inaccurate protein concentration measurements (Fig. 5B).
To further establish that the observed effects of METTL20 on the electron transferring ability of ETF reflect METTL20mediated methylation, we performed various control experiments. First, we generated a catalytically inactive METTL20 enzyme by mutating a key conserved residue in motif I (D121A) (Fig. 5C), and we found that the mutant METTL20, unlike its wild-type counterpart, had no significant effect on ETF␤-mediated electron transfer (Fig. 5D). Second, we also tested the effect of METTL20 on ETF␤ mutants where the methylated Lys residues had been individually or collectively replaced by Arg, i.e. K200R, K203R, and K200R/K203R. Notably, the mutants K203R and K200R/K203R ETF␤ were capable of mediating electron transfer, but this was not influenced by neither wildtype nor mutant METTL20 (Fig. 5D). Electron transfer by the K200R mutant was significantly reduced by enzymatically active METTL20, but the effect was substantially weaker than for WT ETF␤. In summary, the above experiments strongly indicate that METTL20-mediated methylation of ETF␤ has an inhibitory effect on the transfer of electrons from MCAD and

DISCUSSION
We have here identified the biochemical function of the human lysine-specific MTase METTL20 using both in vitro and in vivo approaches. We found METTL20 to be associated with mitochondria and to specifically methylate ETF␤ on two adjacent lysine residues, thereby establishing METTL20 as the first mitochondrial KMT in multicellular organisms. Moreover, methylation reduced the ability of ETF␤ to receive electrons from the dehydrogenases MCAD

Identification of Human METTL20 as Mitochondrial KMT
430 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 1 • JANUARY 2, 2015 and GCDH, demonstrating its functional significance and suggesting a regulatory role.
Previously, we reported, based on bioinformatics analysis, that METTL20 belonged to a family of 10 putative and established human KMTs (6). Other characterized members are, similarly to METTL20, highly specific KMTs that methylate unique cellular substrates, i.e. the chaperones VCP (METTL21D/VCP-KMT) and Hsp70/HSPA (METTL21A/HSPA-KMT), calmodulin (C2orf34/ CaM-KMT), the DNA repair protein KIN17 (METTL22), and the eukaryotic translation elongation factor 2 (FAM86A/eEF2-KMT) (6 -10). Based on the previous nomenclature for naming these enzymes, we suggest that METTL20 is redubbed ETF␤-KMT. The  substrates of these enzymes have been mostly identified in screens for interacting proteins, but we have here used an activity-based purification scheme for identifying ETF␤ as the substrate of METTL20, thus demonstrating this as an alternative and viable approach for identifying the substrates of uncharacterized KMTs.
Very recently, while the present manuscript was in preparation, Rhein et al. (28) actually implicated METTL20 in methylation of ETF␤. Tandem affinity purification tagging experiments revealed ETF␤ as a binding partner of METTL20, and ETF␤ was found to be methylated at Lys 200 and Lys 203 . METTL20 was suggested to be responsible for ETF␤ methylation, because modulation of cellular METTL20 levels through ectopic overexpression or siRNA mediated knockdown caused increased or decreased ETF␤ methylation, respectively. The study by Rhein et al. (28) nicely complements the present work, in that it demonstrated methylation of ETF␤ on both Lys 200 and Lys 203 in cellular samples, whereas we, for technical reasons, were unable to show methylation at Lys 203 in vivo. However, unlike the present study, which includes extensive in vitro characterization of the activity of recombinant METTL20 on various ETF␤-derived substrates, the paper by Rhein et al. (28) did not directly demonstrate the ability of METTL20 to methylate ETF␤, thus leaving the possibility that the effect of METTL20 on ETF␤ methylation levels could be indirect. The relevance of such a concern is illustrated by a recent study that concluded, from similar overexpression and knockdown experiments, that FIGURE 5. METTL20-mediated methylation of ETF␤ impairs its ability to mediate electron transfer from acyl-CoA dehydrogenases. A, methylation of ETF␤ inhibits the rate of ETF␣/␤-dependent oxidation of glutaryl-CoA by GCDH and oxidation of octanoyl-CoA by MCAD. Reactions containing GCDH or MCAD (as indicated), in the absence or presence of ETF (0.2 M), either nonmethylated (ETF␣/␤) or methylated (ETF␣/␤ met ), were started by the addition of glutaryl-CoA or octanoyl-CoA, respectively, and their oxidation was followed by measuring the rate of DCIP reduction. B, concentration dependence of the effect of ETF␣/␤ or ETF␣/␤ met on the oxidation of octanoyl-CoA by MCAD. Reactions containing MCAD in the presence of increasing ETF, either nonmethylated (ETF␣/␤) or methylated (ETF␣/␤ met ), were started by the addition of octanoyl-CoA, and its oxidation was followed as in A. C, D121A mutated METTL20 is unable to methylate ETF␤. Recombinant ETF␣/␤ was incubated with [ 3 H]SAM in the absence or presence of recombinant METTL20, either WT or mutant (D121A). Material was analyzed as in Fig. 3A. The positions of protein bands are indicated by arrows. D, enzymatically active METTL20 inhibits the ability of ETF␤ to support oxidation of octanoyl-CoA by MCAD. ETF␣/␤ containing either WT or mutant (K200R, K203R, or K200R/K203R) ␤-subunit was incubated with nonradioactive SAM, in the absence or presence of recombinant human METTL20, either WT or mutant (D121A). Reactions were then diluted with DCIP assay buffer containing MCAD, octanoyl-CoA was added, and its oxidation was followed as in A. Results from individual experiments were normalized for the activity of either ETF␣/␤ WT or ETF␣/␤ mutants, measured in the absence of METTL20. Sample variation is expressed as standard deviation (n ϭ 10). *, p value Ͻ 0.01; n.s., not significant. E, structure of the region of interaction between ETF␤ (green) and MCAD (cyan) (Protein Data Bank code 1T9G) (18). The recognition loop followed by Lys 200 and Lys 203 (magenta) of ETF␤ is indicated. The ⑀-nitrogen (site of methylation) in Lys residues is indicated in dark blue.
the MTase SETD1A was responsible for methylation of Lys 561 in Hsp70 (HSPA1) (29). However, two subsequent, independent studies have established that this methylation reaction is catalyzed by METTL21A (8,9). Furthermore, the study by Rhein et al. (28) exclusively used peptide sequencing by MS/MS to identify Lys 200 and Lys 203 as methylation sites, and other similar analyses had actually identified Lys 200 and Lys 202 as methylation sites (27). Thus, an additional contribution of the present study is the firm establishment of Lys 200 and Lys 203 as the sites of METTL20-mediated methylation.
Lysine methylation of nuclear proteins, in particular histones, has been subject to extensive study, both with respect to the responsible enzymes and functional consequences. In recent years, it has become clear that Lys methylation of cytosolic proteins is also important, because several highly specific KMTs with cytosolic targets have been identified. The knowledge on Lys methylation of mitochondrial proteins is, however, very limited. Until the discovery of METTL20, only a single eukaryotic mitochondrial KMT had been identified, namely the Saccharomyces cerevisiae MTase Ctm1p, which methylates Lys 72 in cytochrome c (30). However, this methylation is not conserved in mammals, which lack a Ctm1p sequence homologue. Notably, several additional mitochondrial proteins have been identified as targets of lysine methylation, e.g. ATP synthase, citrate synthase, and large ribosomal protein 39 (MRPL39), as well as several members of solute carrier family 25 (SLC25) (27,31). This predicts the existence of additional, yet undiscovered, mitochondria-associated KMTs in humans.
Sequence homologues of METTL20 show a rather unusual distribution on the tree of life, because these proteins are limited to animals in the eukaryotes but also are found in a small subset of bacteria. Interestingly, most bacteria that have METTL20-like proteins belong to the alpha class of the phylum proteobacteria, and this group of bacteria has been indicated as the likely evolutionary precursor of mitochondria (32). This may suggest that methylation of ETF is a primordial function, acquired before the appearance of mitochondria. Indeed, ETF␤ from METTL20-containing bacteria has strong sequence homology to mammalian ETF␤, but future studies are required to address the possible role of ETF␤ methylation in bacteria.
Structural and biochemical studies indicated residues 191-200 in ETF␤ to form a recognition loop involved in the interaction between ETF and some of the mitochondrial dehydrogenases (16,18). Because Lys 200 and Lys 203 are located adjacent to this ETF␤ recognition loop, one may speculate that ETF␤ methylation may differentially affect its interaction with the various mitochondrial dehydrogenases, thus providing a regulatory mechanism for modulating the metabolism of fatty acids, certain amino acids, and choline. The three-dimensional structure of a complex between human ETF␣/␤ and one of its cognate dehydrogenases is available only in the case of MCAD (18), and in this structure, the side chains of Lys 200 and Lys 203 , which are part of the same ␣-helix, are localized in the vicinity of MCAD and point in the direction of the MCAD surface (Fig.  5E). In support of the notion that methylation of Lys 200 and Lys 203 can modulate the interaction of ETF␣/␤ with dehydrogenases, we showed that methylated ETF␣/␤ has reduced capability in mediating electron transfer from MCAD and GCDH to the artificial electron acceptor DCIP. We also found that mutation of Lys 200 or Lys 203 in ETF␤ partially or completely, respectively, abolished the observed effect of METTL20 on ETF␤mediated electron transfer, suggesting that methylation of both these sites is functionally important. In the recent study by Rhein et al. (28), siRNA-mediated knockdown of METTL20 was shown to cause a moderate decrease in the rate of oxygen consumption in permeabilized cells supplied with palmitoyl-Lcarnitine, further supporting the notion that ETF␤ methylation influences metabolism. The ETF-dependent dehydrogenases involved in one-carbon metabolism, DMGDH and SARDH, are structurally unrelated to those acting on acyl-CoAs, and their interaction with ETF has not been characterized. Thus, the interesting possibility exists that the interaction of these enzymes with ETF is more sensitive to ETF␤ methylation status than what is the case for the enzymes tested here. Recent studies have indicated that cellular SAM levels are subject to tight regulation, and the one-carbon units generated by DMGDH and SARDH can actually be used for replenishment of the cellular SAM pool (33). Thus, the interesting, though speculative, possibility exists that METTL20 acts as SAM sensor, which in a SAM-dependent manner regulates the flow of electrons from DMGDH and/or SARDH to ETF, thereby regulating the level of one-carbon units available for SAM synthesis. Further studies addressing the metabolic consequences of METTL20 inactivation will likely shed light on the very interesting potential role of METTL20 in regulating metabolism.