Sestrin2 is a leucine sensor for the mTORC1 pathway. Leucine, but not arginine, disrupts the Sestrin2-GATOR2 interaction by binding to Sestrin2 with a Kd of 20 μM, which is the leucine concentration that half-maximally activates mTORC1. This leucine-binding capacity is required for leucine to activate mTORC1 in cells. Sestrin2 is a GATOR2-interacting protein that inhibits mTORC1 signaling. The results indicate that Sestrin2 is a leucine sensor for the mTORC1 pathway. Leucine directly regulates the Sestrin2-GATOR2 interaction. Activation of mTORC1 by amino acids requires the pentameric GATOR2 complex. Sestrin2 binds to GATOR2 in an amino acid-sensitive manner. Leucine depletion causes Sestrin2 to bind to GATOR2, inducing the interaction as effectively as complete amino acid starvation. Leucine re-addition rapidly reverses the binding. Sestrin2 is homologous to Sestrin1 and Sestrin3. When overexpressed, all three can interact with GATOR2. Leucine starvation and stimulation strongly regulate the interaction of endogenous Sestrin1 with GATOR2. In contrast, endogenous Sestrin3 binds to GATOR2 irrespective of leucine concentrations. Leucine disrupts the Sestrin2-GATOR2 interaction in vitro. When added to purified complexes, leucine dose-dependently disrupts the Sestrin2-GATOR2 complex, with the half maximal effect at about 1 μM. Methionine and isoleucine are considerably less potent. Sestrin2 binds leucine with a Kd of 20 μM. Radiolabeled leucine binds to Sestrin2, but not WDR24 or Rap2A. Leucine binds to Sestrin2, but not to Rap2A. Sestrin1 binds leucine to a similar extent as Sestrin2, whereas Sestrin3 binds very weakly. Sestrin2 binds leucine directly. Leucine, but not arginine, shifts the melting temperature of bacterially-produced Sestrin2. Leucine has a Kd of 20 ± 5 μM for Sestrin2. Methionine and isoleucine compete leucine binding with Ki of 354 ± 118 μM and 616 ± 273 μM, respectively. These values correlate with the relative potencies of leucine, methionine, and isoleucine in disrupting the Sestrin2-GATOR2 interaction. Sestrin2 regulates mTORC1Sestrin2 is a leucine sensor for the mTORC1 pathway. Leucine, but not arginine, disrupts the Sestrin2-GATOR2 interaction by binding to Sestrin2 with a Kd of 20 μM, which is the leucine concentration that half-maximally activates mTORC1. This leucine-binding capacity is required for leucine to activate mTORC1 in cells. Sestrin2 is a GATOR2-interacting protein that inhibits mTORC1 signaling. The results indicate that Sestrin2 is a leucine sensor for the mTORC1 pathway. Leucine directly regulates the Sestrin2-GATOR2 interaction. Activation of mTORC1 by amino acids requires the pentameric GATOR2 complex. Sestrin2 binds to GATOR2 in an amino acid-sensitive manner. Leucine depletion causes Sestrin2 to bind to GATOR2, inducing the interaction as effectively as complete amino acid starvation. Leucine re-addition rapidly reverses the binding. Sestrin2 is homologous to Sestrin1 and Sestrin3. When overexpressed, all three can interact with GATOR2. Leucine starvation and stimulation strongly regulate the interaction of endogenous Sestrin1 with GATOR2. In contrast, endogenous Sestrin3 binds to GATOR2 irrespective of leucine concentrations. Leucine disrupts the Sestrin2-GATOR2 interaction in vitro. When added to purified complexes, leucine dose-dependently disrupts the Sestrin2-GATOR2 complex, with the half maximal effect at about 1 μM. Methionine and isoleucine are considerably less potent. Sestrin2 binds leucine with a Kd of 20 μM. Radiolabeled leucine binds to Sestrin2, but not WDR24 or Rap2A. Leucine binds to Sestrin2, but not to Rap2A. Sestrin1 binds leucine to a similar extent as Sestrin2, whereas Sestrin3 binds very weakly. Sestrin2 binds leucine directly. Leucine, but not arginine, shifts the melting temperature of bacterially-produced Sestrin2. Leucine has a Kd of 20 ± 5 μM for Sestrin2. Methionine and isoleucine compete leucine binding with Ki of 354 ± 118 μM and 616 ± 273 μM, respectively. These values correlate with the relative potencies of leucine, methionine, and isoleucine in disrupting the Sestrin2-GATOR2 interaction. Sestrin2 regulates mTORC1