Abstract
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, Lys(200) and Lys(203), 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.
Highlights
Many proteins are modified by lysine methylation
Human METTL20 Is an Evolutionarily Conserved Mitochondrial Protein Methyltransferase—As an initial step in our functional characterization of human METTL20, we performed a Basic Local Alignment Search Tool (BLAST) search, which revealed the presence of putative METTL20 orthologues in a wide range of organisms (Fig. 1A)
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)
Summary
Many proteins are modified by lysine methylation. Results: It is shown that the previously uncharacterized enzyme METTL20 methylates electron transfer flavoprotein  (ETF), thereby inhibiting its ability to mediate electron transfer from acyl-CoA dehydrogenases. DOT1L and METTL10, which are transfer flavoprotein; GCDH, glutaryl-CoA dehydrogenase; MTase, methyltransferase; KMT, lysine methyltransferase; MCAD, medium-chain acyl-CoA dehydrogenase; MTS, mitochondrial targeting sequence; MTF16, Methyltransferase Family 16; MS, mass spectrometry; SARDH, sarcosine dehydrogenase; DCIP, 2,6-dichloroindophenol. 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 isovalerylCoA dehydrogenase, are involved in the oxidation of amino acids [20, 21]. We demonstrate that methylation of ETF impairs its ability to extract electrons from two acyl-CoA dehydrogenases, namely MCAD and GCDH, suggesting an important functional role for METTL20-mediated methylation of ETF
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