Fig. 3. RagB^short inhibits GATOR1 through interaction with DEPDC5.

a–d, Transient expression of increasing amounts (5 ng, 25 ng or 100 ng DNA) of a non-Rag binding mutant of DEPDC5 (Y775A) suppresses mTORC1 more strongly than wild-type DEPDC5 in RagB^short-expressing cells (c and d) but not in RagA-expressing cells (a and b). Cells were subjected to amino-acid starvation (amino-acid-free DMEM + 10% dFBS) for 30 min to activate GATOR1: representative examples (a and c) and quantifications of three independent experiments, with metap2-transfected cells set to 1 (b and d). Circle indicates average, and error bars represent standard deviation; n = 3 biological replicates. Two-way ANOVA and Sidak’s post-hoc test. e–h, Transient expression of increasing amounts (5 ng, 25 ng or 100 ng DNA) of wild-type DEPDC5 (e and f) inhibits mTORC1 activity more strongly in RagA-expressing cells than in RagB^short-expressing cells, whereas expression of a non-Rag binding mutant of DEPDC5 (Y775A) (g and h) inhibits mTORC1 activity equally well in the presence of RagA or RagB^short. Cells were subjected to amino-acid starvation (amino-acid-free DMEM + 10% dFBS) for 30 min to activate GATOR1: representative examples (e and g) and quantification of three independent experiments, with RagA-expressing cells transfected with metap2 set to 1 (f and h). Circle indicates average, and error bars represent standard deviation; n = 3 biological replicates. Two-way ANOVA and Sidak’s post-hoc test. i, Schematic representation of GATOR1 binding to RagA or RagB^short via DEPDC5. DEPDC5 binding to RagB^short but not to RagA inhibits GATOR1 activity. Exact P values are shown in the graphs. NS, not significant. Source numerical data and unprocessed blots are available in source data.

Source data