Fig. 5

SIRT4 regulates methionine metabolism through mediating MAT2A ADP ribosylation. A Volcano plot showing differential gene expression in mice’s livers upon methionine restriction. Red circles indicates genes increased in the Ctrl set while blue ones means genes decreased in the MR set. B Western blot analysis of MAT2A mono-ADP-ribosylation (MARylation) in HEK293 and SNU449 cells in which SIRT4 was silenced (shRNA). C The MARylation of MAT2A was measured by western blot in HEK293 or SNU449 cells stably expressing HA tagged SIRT4. D Western blot analysis of MAT2A MARylation in the FLAG immunoprecipitated samples from HEK293 or SNU449 cells treated with nicotinamide (NAM) for indicated times. E Western blot analysis of MAT2A MARylation in the FLAG immunoprecipitated samples from HEK293 or SNU449 cells treated with Sirtinol for indicated times. F Relative activity of MAT2A was shown in the SNU449 cells in which either c-Myc or SIRT4 was knockdown or overexpressed or c-Myc and SIRT4 overexpressed together. G Abundances of intracellular primary methionine cycle metabolites were compared between the control cells and the SNU449 cells where either c-Myc or SIRT4 was knockdown or stably overexpressed or c-Myc and SIRT4 overexpressed together. H MARylation level of mutant FLAG-MAT2A-E111A ectopically expressed in MAT2A-knockout HEK293 or SNU449 cells was measured by using anti-ADP ribosylation antibody when SIRT4 was silenced. I MARylation level of mutant FLAG-MAT2A-E111A ectopically expressed in MAT2A-knockout HEK293 or SNU449 cells was measured by using anti-ADP ribosylation antibody when SIRT4 was overexpressed. J MAT2A dimer are shown in blue or lightblue (PDB ID: 4NDN). The active site was shown in red circle. K E111 in MAT2A is conserved. Protein sequence alignment surrounding E111 colored in light blue from indicated species. Data are means ± SEM. Group differences were analyzed by two-tailed Student’s multiple comparison test (F, G) (**p < 0.01)