Weak Membrane Interactions Allow Rheb to Activate mTORC1 Signaling Without Major Lysosome Enrichment

Stable localization of the Rheb GTPase to lysosomes is thought to be required for activation of mTORC1 signaling. However, the lysosome targeting mechanisms for Rheb remain unclear. We therefore investigated the relationship between Rheb subcellular localization and mTORC1 activation. Surprisingly, we found that although Rheb was prominently enriched at the endoplasmic reticulum (ER), Rheb was undetectable at lysosomes. Functional assays in knockout human cells revealed that farnesylation of the C-terminal CaaX motif on Rheb was essential for both Rheb enrichment on ER membranes and mTORC1 activation. However, constitutively targeting Rheb to ER membranes did not support mTORC1 activation. Further systematic analysis of the Rheb hypervariable region revealed that weak, non-selective, farnesylation-dependent, membrane interactions confer Rheb function without the need for a specific lysosome targeting motif. Collectively, these results argue against stable interactions of Rheb with lysosomes and instead that transient membrane interactions optimally allow Rheb to activate mTORC1 signaling.