Regulation of the Rheb GTPase via GATOR1 Complex

ABSTRACT mTORC1 coordinates cellular growth and metabolism by integrating inputs from both amino acids and growth factors, and its activation requires two upstream branches involving the Rag GTPases and the Rheb GTPase. These branches are regulated by distinct GAP complexes: GATOR1 (Depdc5-Nprl2-Nprl3) inhibits RagA/B, and TSC (TSC1-TSC2-TBC1D7) inhibits Rheb. Despite the prevailing view that these pathways converge only at mTORC1 itself, several observations suggest upstream crosstalk. This gap is especially striking in organisms like C. elegans and S. cerevisiae that lack the TSC complex yet maintain fully responsive mTORC1 signaling. How these inputs are dynamically coordinated under complex physiological conditions and in organisms lacking the key components remain unknown. We performed unbiased quantitative proteomics in C. elegans and identified the GATOR1 complex as a previously unrecognized RHEB-1 ( C. elegans ortholog of Rheb) interactor. Through biochemical validation in human cells, we show that nucleotide-free Rheb associates with the Nprl2–Nprl3 subunits of GATOR1, whereas GTP-bound or membrane-detached Rheb mutants fail to bind. Nutrient stress, but not direct pharmacologic inhibition of mTORC1, robustly induced this interaction. In TSC2-null cells, where Rheb is constitutively GTP-loaded, Rheb-Nprl2/3 binding was strongly diminished and was restored by expressing the nucleotide-free Rheb S20N mutant, demonstrating that Rheb’s nucleotide state governs this interaction. Pulldown assays confirmed that the Nprl2/3 heterodimer is sufficient for binding nucleotide-free Rheb. Structural modeling using AlphaFold3 consistently positioned Rheb at a conserved site on Nprl3 distinct from the RagA/B GAP-active surface of Nprl2, supporting a non-catalytic mode of association. Together, these findings identify a conserved, nutrient-regulated physical interaction between Rheb and the Nprl2/3 subunits of GATOR1, revealing a previously unrecognized point of convergence between the growth factor and amino acid branches of the mTORC1 pathway. This model provides a direct molecular link between the Rag and Rheb branches, furthering our understanding of how nutrient stress fine-tunes mTORC1 signaling.