Figure 3.

The putative FAM173B orthologue from C. elegans methylates Lys-43 in ATPSc. A, complementation of FAM173B KO cells with the putative FAM173B orthologue from C. elegans (Y39A1A.21) restores the methylation of ATPSc at Lys-43. The methylation status of ATPSc from HAP1 FAM173B KO, or KO cells expressing FLAG-tagged Y39A1A.21 protein was analyzed as in Fig. 2D. Error bars, range of values from three independent analyzes of KO cells and three different clones of complemented cells, as indicated in Fig. S3. B, in vivo methylation of ATPSc from C. elegans. Extracts enriched in mitochondrial membrane proteins were prepared from worms and resolved by SDS-PAGE. The portion of the gel corresponding to ATPSc was chymotrypsin-digested and analyzed by MS. Shown are representative, normalized extracted ion chromatograms gated for different methylation states of the indicated chymotrypsin-generated peptide from C. elegans ATPSc, with Lys-43 marked in red. Percentages indicate the area under each peak, relative to the total area of all peaks. A.U., arbitrary units. C, MS/MS fragmentation spectrum demonstrating trimethylation of Lys-43 in ATPSc from C. elegans.