Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here we show that during single membrane ATG8 conjugation (SMAC), Parkin-dependent mitophagy, and Salmonella -induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal homeostasis and capacity. GABARAP directly binds to a novel LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex with picomolar affinity and regulates its relocalization to these GABARAP-conjugated membrane compartments. This disrupts the regulation of RagC/D by the FLCN/FNIP GAP complex, resulting in impaired mTOR-dependent phosphorylation of TFEB without changing mTOR activity towards other substrates. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a universal molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.

GABARAP membrane conjugation sequesters the FLCN-FNIP tumor suppressor complex to activate TFEB and lysosomal biogenesis