Human METTL20 is a mitochondrial lysine methyltransferase that targets the β subunit of electron transfer flavoprotein (ETFβ) and modulates its activity

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.

Keywords: Electron Transfer Flavoprotein; Enzyme Catalysis; Mitochondrial Metabolism; Post-translational Modification (PTM); Protein Methylation; Protein Targeting.

FIGURE 1.

Human METTL20 is an evolutionarily conserved protein methyltransferase localized to mitochondria. A, alignment of METTL20 orthologues from Homo sapiens (Hs; NP_776163.1), Xenopus laevis (Xl; NP_001090037.1), Danio rerio (Dr; NP_001154967.1), Anopheles gambiae (Ag; XP_308218.4), Caenorhabditis elegans (Ce; NP_491943.2), Rhizobium etli (Re; YP_471372.1), and Agrobacterium tumefaciens (At; NP_355584.1). Predicted α-helices (rectangles) and β-strands (arrows) are indicated. Hallmark motifs of 7BS (Motif 1, Post 1, and Motif 2) and MTF16 (DXXY) MTases are boxed. The vertical arrow shows the predicted position of cleavage of putative MTS in human METTL20. B, in vivo confocal fluorescence microscopy images of HeLa cells after 24 h of transient transfection, expressing either GFP, METTL20-GFP, or (1–42)-METTL20-GFP, in the presence of MitoTracker and Hoechst dyes. Data were acquired through green (GFP), red (MitoTracker), and blue (Hoechst) channels and merged. C, recombinant human METTL20 catalyzes methylation of a ∼28-kDa protein in human cell extracts. Extracts from HEK293 or HeLa cells were incubated with [³H]SAM in the absence (lanes 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 ∼28-kDa substrate and molecular mass markers are indicated.

FIGURE 2.

Identification of ETFβ as substrate of recombinant human METTL20. A, partial purification of the ∼28-kDa METTL20 substrate from HEK293 extracts by ion exchange chromatography. Schematic representation of ion exchange-based fractionation procedure (left panel). The various fractions were incubated with [³H]SAM in the presence of METTL20, and methylation was detected by fluorography (right panel). B, ETFβ present in extracts from HEK293 cells is dimethylated at Lys²⁰⁰. The 0.15 S fraction was resolved by SDS-PAGE and Coomassie-stained. The region of gel containing ETFβ was excised and subjected to trypsin digestion, and the resulting tryptic peptides were analyzed by MS. The figure shows fragmentation mass spectra of ETFβ-derived peptide with series of b-ions (red) and y-ions (blue) mapped onto the sequence of the peptide, supporting Lys²⁰⁰ of ETFβ as target of dimethylation. C, METTL20 methylates Lys²⁰⁰ in ETFβ in extracts from HEK293 cells. The 0.15 S fraction was incubated in the absence or presence of METTL20 and nonradioactive SAM (1 mm) and analyzed as in B. The relative intensities of MS signals representing different ETFβ methylation states at Lys²⁰⁰, in samples incubated with or without METTL20, are shown.

FIGURE 3.

Recombinant human METTL20 methylates ETFβ on Lys²⁰⁰ and Lys²⁰³in vitro. A, METTL20 methylates the β-subunit of the ETFα/β heterodimer. ETFα/β containing ETFβ WT or mutant as indicated was incubated with [³H]SAM in the absence or presence of METTL20. Proteins were resolved by SDS-PAGE and then transferred to a PVDF membrane that was stained with Ponceau S (bottom panel). The membrane was subsequently subjected to fluorography (top panel). B, ETFβ mutant (K200A/K202A) is methylated on Lys²⁰³ by METTL20. ETFα/β with mutant (K200A/K202A) β-subunit was treated with METTL20 in the presence of nonradioactive SAM. Proteins were analyzed as in Fig. 2B. The figure shows fragmentation mass spectra of ETFβ-derived peptides with series of b-ions (red) and y-ions (blue) mapped onto the sequence of the peptide, supporting Lys²⁰³ as target of di- and trimethylation. C, METTL20 targets Lys²⁰⁰ and Lys²⁰³ in ETFβ. ETFα/β with WT or mutant (K200R, K203R, and K200R/K203R) β-subunit was incubated with [³H]SAM in the absence or presence of METTL20, and methylation was analyzed as in A. D, Lys²⁰⁰ and Lys²⁰³ of ETFβ are independently methylated by METTL20. ETFα/β heterodimer (∼1.5 μm) containing either WT or mutant β-subunit was incubated with various concentrations (0–25 μm) of METTL20, in the presence of [³H]SAM ([SAM]_total = 32.6 μm). Proteins were precipitated with 10% TCA and subjected to scintillation counting. E, METTL20-catalyzed methylation introduces up to three methyl groups at each of the sites Lys²⁰⁰ and Lys²⁰³ in ETFβ. Mutant or WT ETFα/β was incubated with nonradioactive SAM in the presence or absence of METTL20. Proteins were then resolved by SDS-PAGE and subjected to Coomassie staining. The ETFβ-containing region of the gel was excised and subjected to AspN digestion, and the resulting proteolytic peptides were analyzed by MS. The figure shows the relative intensities of signals corresponding to the different methylation states of the AspN-digested peptide encompassing residues 184–212 (red color indicates the location of the two methylation sites Lys²⁰⁰ and Lys²⁰³ in the peptide sequence). F, METTL20-mediated lysine methylation of ETFβ occurs specifically at residues Lys²⁰⁰ and Lys²⁰³. ETFα/β with β-subunit WT or mutant (with indicated sequence at residues 200–205) was incubated with [³H]SAM in the absence or presence of METTL20, and methylation was assessed by fluorography, as in A.

FIGURE 4.

METTL20 methylates ETFβ in vivo. A, confocal fluorescence microscopy images of Flp-In T-REx-293 cells with stably inserted METTL20-GFP fusion gene, in the absence or presence of 1 μg/ml Dox to induce expression of the fusion protein for 48 h, in the presence of MitoTracker and Hoechst dyes. The data were acquired as in Fig. 1B. B, overexpression of a METTL20-GFP fusion protein results in complete trimethylation of Lys²⁰⁰ in ETFβ. Cells as in A were either untreated or treated with Dox for 48 h. ETFβ was partially purified from cell extracts by anion exchange chromatography on a Q column. The ETFβ-containing flow-through was resolved by SDS-PAGE, and the ETFβ-containing region was excised from the Coomassie-stained gel and then subjected to trypsin digestion and MS analysis. The relative intensities of MS signals representing different ETFβ methylation states at Lys²⁰⁰, as in Fig. 2B, are shown. C, overexpression of a METTL20-GFP fusion protein abolishes hypomethylation of ETFβ. Cells, as in A, were incubated for 48 h in the absence or presence of Dox. ETFβ was partially purified as in B and incubated with [³H]SAM in the absence or presence of recombinant METTL20, resolved by SDS-PAGE, transferred to PVDF membrane, and stained with Ponceau S (bottom panel). The membrane was probed with anti-ETFβ antibody as loading control (top panel). The membrane was then stripped, dried, and subjected to fluorography (middle panel). Arrows show the positions of methylated ETFβ and automethylated METTL20 (middle panel), as well as the position of METTL20 on Ponceau S stained membrane (bottom panel).

FIGURE 5.

METTL20-mediated methylation of ETFβ impairs its ability to mediate electron transfer from acyl-CoA dehydrogenases. A, methylation of ETFβ inhibits the rate of ETFα/β-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 μm), either nonmethylated (ETFα/β) or methylated (ETFα/β^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 ETFα/β or ETFα/β^met on the oxidation of octanoyl-CoA by MCAD. Reactions containing MCAD in the presence of increasing ETF, either nonmethylated (ETFα/β) or methylated (ETFα/β^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 ETFβ. Recombinant ETFα/β was incubated with [³H]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 ETFβ to support oxidation of octanoyl-CoA by MCAD. ETFα/β containing either WT or mutant (K200R, K203R, or K200R/K203R) β-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 ETFα/β WT or ETFα/β mutants, measured in the absence of METTL20. Sample variation is expressed as standard deviation (n = 10). *, p value < 0.01; n.s., not significant. E, structure of the region of interaction between ETFβ (green) and MCAD (cyan) (Protein Data Bank code 1T9G) (18). The recognition loop followed by Lys²⁰⁰ and Lys²⁰³ (magenta) of ETFβ is indicated. The ϵ-nitrogen (site of methylation) in Lys residues is indicated in dark blue.