FIGURE 2.

FAM86A and its yeast homologue Yjr129c are eEF2 methyltransferases. A, eEF2 co-purifies with FAM86A in TAP. Shown is a Coomassie Blue-stained SDS-polyacrylamide gel of TAP eluates from a Flp-In T-REx HEK293-derived cell line treated with doxycycline to induce expression of SBP- and CBP-tagged FAM86A (TAP-FAM86A) or the CBP-SBP tag alone (TAP). Purification buffers were supplemented with AdoMet (SAM) or AdoHcy (SAH) where indicated. The identity of the bait and co-purifying proteins was determined by peptide mass fingerprinting; asterisks indicate β-tubulin (50 kDa), pyruvate carboxylase (125 kDa), and acetyl-CoA carboxylase 1 (265 kDa). Molecular weight markers (in kilodaltons) are shown on the right. B, eEF2 from yjr129cΔ yeast extracts is efficiently methylated by FAM86A and Yjr129c. Top, MTase reactions with recombinant FAM86A or Yjr129c, [³H]AdoMet, and WT or yjr129cΔ yeast extracts separated by SDS-PAGE and subjected to fluorography; bottom, Ponceau S stain of the 100-kDa region of the same membrane. C, trimethylation of Lys-509 in eEF2. Shown are MS/MS fragmentation patterns of representative AspN-generated trimethylated peptides from the WT strain (top) and unmethylated peptides from the yjr129cΔ strain (bottom). D, Lys-509 in eEF2 is the target of methylation by the MTases Yjr129c and FAM86A. Shown are MS chromatograms of AspN-generated Lys-509-containing peptides from partially purified eEF2 from WT or yjr129cΔ yeast extracts. When indicated, the extracts had been incubated with recombinant Yjr129c or FAM86A in the presence of AdoMet. *, an unrelated peptide. A.u., arbitrary units. E, mutagenesis of Lys-509 in eEF2 abolishes FAM86A and Yjr129c-mediated methylation. Recombinant FAM86A or Yjr129c was incubated in the presence of [³H]AdoMet with WT or K509A His₆-eEF2, expressed, and purified from yjr129cΔ mutant yeast. The MTase reactions were separated by SDS-PAGE and subjected to fluorography (top) and Ponceau S staining (bottom).