The Cullin3-Rbx1-KLHL9 E3 ubiquitin ligase complex ubiquitinates Rheb and supports amino acid-induced mTORC1 activation

Mechanistic target of rapamycin complex 1 (mTORC1) is recruited to the lysosomal membrane by the active Rag heterodimer, where mTORC1 interacts with active Rheb for its activation. It has been shown that polyubiquitination of Rheb is crucial for enhancing its interaction with mTORC1 on the lysosome. However, the specific ubiquitin ligases for Rheb, which promotes mTORC1 activation, remain elusive. We report that the CUL3-RBX1-KLHL9 E3 ubiquitin ligase complex is translocated to the lysosome and ubiquitinates Rheb in response to amino acid stimulation. KLHL9 serves as an essential adaptor for CUL3-RBX1 to target Rheb on the lysosome. Deleting either CUL3, RBX1, or KLHL9 diminishes Rheb ubiquitination and reduces amino acid-induced mTORC1 activation without impacting lysosomal mTORC1 localization or Akt activity. Thus, the CUL3-RBX1-KLHL9 complex functions as a mTORC1 activator by acting as an E3 ubiquitin ligase for Rheb and supports amino acid-induced mTORC1 activation.