Fig 1: The O-GlcNAcylation of LARS1 suppresses leucine-induced mTORC1 activation.a SW620 cells were transfected with the indicated LARS1 constructs. After 24 h, the cells were starved of leucine for 1.5 h and supplemented with 0.4 mM leucine for the indicated durations. Each cell lysate was immunoblotted with the indicated antibodies. b Quantification of S6K T389 phosphorylation of a. The value for the control sample at 0 min was set to 100% (mean ± SEM, n = 3 independent experiments). c, d SW620 cells were transfected with the indicated LARS1 constructs. After 24 h, the cells were starved of 0.4 mM leucine for 1.5 h and supplemented with 0.4 mM leucine for 15 min with or without 11 mM glucose. c Each cell lysate was used for lysosomal fractionation and immunoblotted with the indicated antibodies. d Each cell lysate was precipitated with anti-LARS1 antibody-conjugated agarose beads or GTP-conjugated agarose beads and analyzed by immunoblotting with the indicated antibodies. e A schematic of the LARS1 genomic location and the selected sgRNA targeting site. f Sequencing result of targeted genomic regions of LARS1. Red arrows point to overlapping peaks. g SW620 control or S1042A knock-in cells were starved of glucose for 4 h and supplemented with 11 mM glucose for 30 min. Each cell lysate was precipitated with sWGA-conjugated agarose beads or anti-LARS1 antibody-conjugated agarose beads and then immunoblotted with the indicated antibodies. h SW620 control or S1042A knock-in cells were starved of 0.4 mM leucine for 1.5 h and supplemented with 0.4 mM leucine for 15 min with or without 11 mM glucose. Each cell lysate was used for lysosomal fractionation and immunoblotted with the indicated antibodies. i SW620 control or S1042A knock-in cells were transfected with the indicated siRNAs. After 48 h, cells were starved of 0.4 mM leucine for 1.5 h and supplemented with 0.4 mM leucine for 15 min with or without 11 mM glucose. Each cell lysate was precipitated with anti-LARS1 antibody-conjugated agarose beads or GTP-conjugated agarose beads and analyzed by immunoblotting with the indicated antibodies. Representative data of three experiments with similar results. Source data are provided as a Source Data file.
Fig 2: The O-GlcNAcylation of LARS1 regulates ULK1-mediated LARS1 phosphorylation.a SW620 cells were starved and stimulated with 11 mM glucose for the indicated durations. b Quantification of LARS1 S720 phosphorylation and O-GlcNAcylation of a (mean ± SEM, n = 3 independent experiments). c, d SW620 cells were transfected with the indicated siRNAs. After 48 h, the cells were starved of glucose for 4 h, pre-incubated with streptolysin O (SLO) for 5 min, and supplemented with 11 mM glucose or 200µM indicated metabolites for 30 min. e SW620 cells were transfected with the indicated siRNAs. After 48 h, the cells were starved of glucose for 4 h with vehicle or indicated compounds (10µM Compound C, 20 µM SBI-0206965, 1 µM LYN1604, 1 mM AICAR) f 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. g SW620 cells were transfected with control or siRNA targeting OGT1. After 48 h, the cells were starved of glucose at the indicated durations. h SW620 cells were transfected with siRNA targeting ULK1. After 24 h, the cells were transfected with the indicated expression construct. After 24 h, the cells were starved of glucose for 4 h. Each sample was analyzed by immunoblotting with the indicated antibodies. i SW620 control or S1042A knock-in cells were starved of glucose for 4 h and supplemented with glucose for 30 min. a, c, d, e, f, g, i Each sample was subjected to immunoprecipitation with sWGA-conjugated agarose beads, anti-LARS1 antibody-conjugated beads or anti-myc antibody-conjugated agarose beads and analyzed by immunoblotting with the indicated antibodies. Representative data of three experiments with similar results. Source data are provided as a Source Data file.
Fig 3: 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.
Fig 4: LARS1 is O-GlcNAcylated at S1042 by OGT1 under glucose starvation.a SW620 cells were incubated with or without 11 mM glucose for 4 h. b SW620 cells were transfected with the indicated siRNAs. After 48 h, the cells were starved of glucose for 4 h, then permeabilized with streptolysin O for 5 min, and 200 µM of UDP-GlcNAc, as indicated, was added for 30 min. c SW620 cells were starved of glucose and supplemented with 11 mM glucose for the indicated durations. d SW620 cells were incubated with vehicle or 20 µM ST045849 for 24 h and starved of glucose for 4 h. a–d Each cell lysate was precipitated with succinylated wheat germ agglutinin (sWGA)-conjugated agarose beads or anti-LARS1 antibody-conjugated agarose beads and then immunoblotted with the indicated antibodies. e The site of LARS1 O-GlcNAcylation was mapped using mass spectrometry. f LARS1 protein sequences, including S1042, the O-GlcNAcylation site, among species were aligned using BLAST. Red alphabet indicates a conserved serine residue. g SW620 cells were transfected with the indicated LARS1 constructs. After 24 h, the cells were starved of glucose for 4 h. Each sample was precipitated with sWGA-conjugated agarose beads or anti-LARS1 antibody-conjugated agarose beads and analyzed by immunoblotting with the indicated antibodies. Representative data of three experiments with similar results. Source data are provided as a Source Data file.
Fig 5: The O-GlcNAcylation of LARS1 controls leucine-derived ATP production and protects cells under glucose starvation.a, c, d, e SW620 cells were transfected with the indicated LARS1 constructs. After 24 h, the cells were starved of glucose and leucine for 4 h then leucine was added for 4 h. a cells were exposed to 2 µM oligomycin, 0.5 µM FCCP, and 0.5 µM/0.5 µM rotenone/antimycin, and the OCR was measured. Left: OCR over time; right: bar graph of basal OCR and FCCP-treated maximal OCR from left (mean ± SEM, n = 3 independent experiments). b Summary of the leucine catabolism pathway. c, d, e cells were harvested and analyzed with c NADH/NAD+ assay kit and d ATP assay kit. e cells were incubated with CellToxTM Green dye and dead cells were detected by a live-cell imaging analyzer. (mean ± SEM, n = 3 independent experiments). f SW620 cells were transfected with the indicated LARS1 constructs. After 24 h, the cells were starved of glucose and leucine for 4 h then 0.4 mM leucine was added for 4 h with vehicle or indicated compounds: KIC, 200 µM a-ketoisocaproic acid; Rapa, 10 nM rapamycin; CHX, 20 µM cycloheximide. g SW620 control or S1042A knock-in cells were starved of glucose and leucine for 4 h then 0.4 mM leucine was added for 4 h. f, g 1 µM puromycin was added to the medium of the cells for 30 min. h, i, j SW620 cells were transfected with the indicated LARS1 constructs. After 24 h, the cells were starved of glucose and leucine for 4 h then leucine was added for 4 h. Each sample was harvested and analyzed with h ATP assay kit and i CellToxTM Green dye signals (mean ± SEM, n = 3 independent experiments). j During leucine re-supplementation, vehicle or 1 µM puromycin was also added and incubated for 30 min. 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.
from Cell Signaling Technology for LARS Antibody