Fig. 4: O-GlcNAcylation of LARS1 S1042 inhibits the interaction of LARS1 with RagD.

a–c A surface representation of the RBD-VC domain structure of “sensing-off” LARS1 (PDB: 6KR7), “sensing-on” LARS1 (PDB: 6KQY), and O-GlcNAcylated LARS1 (modeled by Vienna PTM) were colored gray. S1042 is colored cyan, and H958, E960, and K970 are colored magenta. d Superimposition of the RBD-VC domain structures of “sensing-off” LARS1, “sensing-on” LARS1, and O-GlcNAcylated LARS1. “Sensing-off” LARS1, “sensing-on” LARS1, and O-GlcNAcylated LARS1 are represented as green, pink, and orange cartoon models, respectively. e Near-UV CD spectra of WT LARS1 and S1042A mutant LARS1 with or without OGT1. f His-tagged WT and S1042A mutant LARS1 were purified from E. coli with or without WT or N567K OGT1 expression. Each LARS1 protein was incubated with proteinase K for the indicated duration. g His-tagged WT and S1042A mutant LARS1 were purified with Ni-NTA beads from E. coli with or without WT or N567K OGT1 expression. Each LARS1 protein sample was incubated with the WT RagD or WT RagC in the presence or absence of 200 μM ATP/2 mM leucine. For in vitro GTPase assay, His-tagged WT or S1042A LARS1 proteins purified from E. coli expressing WT or N567K OGT1 were incubated with WT RagD or WT RagC proteins in the presence or absence of 200 μM ATP/2 mM leucine. After 1 h, samples were precipitated with GTP-conjugated beads. h, j SW620 cells were transfected with the indicated LARS1 constructs. After 24 h, the cells were starved of glucose for 4 h and supplemented with 11 mM glucose for 30 min. i The LARS1-RagD interaction and S6K T389 phosphorylation of h were quantified and are indicated as the percentage of the control without glucose or OGT1 (mean ± SEM, n = 3 independent experiments). k GTP-bound RagD and S6K T389 phosphorylation of j were quantified and are indicated as the percentage of the control without glucose or GTP-bound RagD (Q121L) (mean ± SEM, n = 3 independent experiments). P-value was determined by two-tailed unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Representative data of three experiments with similar results. Source data are provided as a Source Data file.