The METTL20 Homologue from Agrobacterium tumefaciens Is a Dual Specificity Protein-lysine Methyltransferase That Targets Ribosomal Protein L7/L12 and the β Subunit of Electron Transfer Flavoprotein (ETFβ)

Abstract

Human METTL20 is a mitochondrial, lysine-specific methyltransferase that methylates the ␤-subunit of electron transfer flavoprotein (ETF␤). Interestingly, putative METTL20 orthologues are found in a subset of ␣-proteobacteria, including Agrobacterium tumefaciens. Using an activity-based approach, we identified in bacterial extracts two substrates of recombinant METTL20 from A. tumefaciens (AtMETTL20), namely ETF␤ and the ribosomal protein RpL7/L12. We show that AtMETTL20, analogous to the human enzyme, methylates ETF␤ on Lys-193 and Lys-196 both in vitro and in vivo. ETF plays a key role in mediating electron transfer from various dehydrogenases, and we found that its electron transferring ability was diminished by AtMETTL20-mediated methylation of ETF␤. Somewhat surprisingly, AtMETTL20 also catalyzed monomethylation of RpL7/L12 on Lys-86, a common modification also found in many bacteria that lack METTL20. Thus, we here identify AtMETTL20 as the first enzyme catalyzing RpL7/ L12 methylation. In summary, here we have identified and characterized a novel bacterial lysine-specific methyltransferase with unprecedented dual substrate specificity within the seven ␤-strand class of lysine-specific methyltransferases, as it targets two apparently unrelated substrates, ETF␤ and RpL7/L12. Moreover, the present work establishes METTL20-mediated methylation of ETF␤ as the first lysine methylation event occurring in both bacteria and humans.

Uncovering human METTL12 as a mitochondrial methyltransferase that modulates citrate synthase activity through metabolite-sensitive lysine methylation

“…2C). In addition we detected after longer exposures weak automethylation of METTL12 similar to what has been observed for many other KMTs (12,17,20,33). In summary, these results show that METTL12 is a mitochondrial enzyme with protein MTase activity.…”

“…First, NaCl concentration was reduced to 50 mM by diluting cell lysates with dilution buffer (50 mM Tris-HCl, pH 7.4, 5% glycerol), and then extracts were applied onto S-column equilibrated in dilution buffer. Material bound to the S-column was eluted in 100-l aliquots by a step gradient of increasing NaCl concentrations prepared in dilution buffer, similarly as previously described (33).…”

Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

“…We have previously shown that several recombinant 7BS KMTs can methylate specific proteins when added to a cellular extract and that KMT substrates can be identified by fractionation of the extract ( 8 , 14 , 36 ). Moreover, such methylation is often strongly enhanced in extracts from cells that lack the relevant KMT, due to an increased level of unmethylated substrate ( 6 , 36 , 37 ). Thus, to investigate the potential protein MTase activity of eEF1A-KMT4, protein extracts from EEF1AKMT4 -deficient and WT HAP-1 cells were incubated with recombinant eEF1A-KMT4 and [ 3 H]AdoMet.…”

Approaches and Guidelines for the Identification of Novel Substrates of Protein Lysine Methyltransferases

“…One example of a method that falls into this category are peptide arrays that have been used over the years to map out substrate specificity profiles for SET enzyme family members and have revealed many non-histone substrates, some of which have been validated through complementary cell-based analysis (Dhayalan et al, 2011;Lanouette et al, 2015;Rathert et al, 2008;Schuhmacher et al, 2015;Weirich et al, 2016). Other successful approaches for the identification of PKMT substrates are protein interaction analyses aiming to identify novel binding substrates, which are then candidates for methylation studies (Ahmed et al, 2016;Cloutier et al, 2013;Davydova et al, 2014;Jakobsson et al, 2013;Kernstock et al, 2012;Lanouette et al, 2015), and biochemical purification of methylated proteins either based on enzyme activity (Malecki et al, 2015(Malecki et al, , 2016 or using methyl lysine-specific antibodies or binding domains (Cao et al, 2013;Moore et al, 2013). Moreover, cellular protein labeling studies with modified AdoMet have been employed to identify PKMT substrates (Shimazu et al, 2014), and SILAC studies with wild-type and PKMT knockout cell lines have provided the first proteome-wide collections of PKMT substrates (Carlson et al, 2015;Olsen et al, 2016).…”

“…Dot1L is a prominent member of the 7BS group known to methylate histone H3 at K79 (Feng et al, 2002;van Leeuwen et al, 2002). However, recent studies have identified 7BS methyltransferases that have non-histone protein substrates, and their initial candidate substrates were often identified by protein interaction studies and later validated biochemically (Davydova et al, 2014;Jakobsson et al, 2013;Kernstock et al, 2012;Malecki et al, 2015Malecki et al, , 2016.…”