HOPS is required for non-canonical mTORC1 signaling by recruiting Rags and FLCN to lysosomal membranes

The mechanistic target of Rapamycin complex 1 (mTORC1) regulates cell growth and metabolism in response to nutrient availability. mTORC1 recruitment to lysosomes by the Rag dimer complex (RagA/B and RagC/D) is a crucial step in mTORC1 signaling. Substrates of the MiT/TFE transcription factor family, like TFEB and TFE3, directly interact with the Rag complex when activated by folliculin (FLCN). This selective recruitment and subsequent phosphorylation of MiT/TFE substrates leads to a non-canonical mTORC1 signaling pathway regulating lysosomal biogenesis and autophagy. Recently, we showed that compound heterozygous mutations in VPS41 specifically impair non-canonical mTORC1 signaling. VPS41, as part of the HOPS complex, is required for fusion of lysosomes with endosomes and autophagosomes. Here we addressed the mechanism by which VPS41/HOPS complex regulates mTORC1 activity. We show that multiple HOPS subunits interact with the Rag dimers as well as with FLCN. Depletion of HOPS subunits results in reduced lysosomal localization of both Rags and FLCN, and the nuclear translocation of TFE3/TFEB. The VPS41-Rag interactions required the RING domain of VPS41, but were independent of RHEB, Rag activity or presence of other HOPS components. We conclude that HOPS is necessary for the recruitment of crucial components of the non-canonical mTORC1 signaling pathway onto lysosomal membranes. These data further our molecular understanding of disease-causing VPS41/HOPS mutations and indicate a crucial role for HOPS in connecting lysosomal trafficking to lysosomal signaling.