GABARAP sequesters the FLCN-FNIP tumor suppressor complex to couple autophagy with lysosomal biogenesis

Abstract

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 the conjugation of ATG8 to single membranes (CASM), 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 capacity. GABARAP directly binds to a previously unidentified LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex and mediates sequestration to GABARAP-conjugated membrane compartments. This disrupts FLCN/FNIP GAP function toward RagC/D, resulting in impaired substrate-specific mTOR-dependent phosphorylation of TFEB. Thus, the GABARAP-FLCN/ FNIP-TFEB axis serves as a molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.

Non-canonical mTORC1 signaling at the lysosome

“…Lysosomal sequestration of FLCN by GABARAP Similar to lysosomal damage, other conditions promoting selective autophagy (e.g., mitochondrial damage, pathogen infections), as well as the use of lysosomal ionophores such as TRPML1 activators, have also been shown to promote TFEB nuclear translocation without affecting the phosphorylation of mTORC1 substrates S6K and 4E-BP1 [87]. Also in this case, the mechanism for TFEB activation has been shown to rely on the specific inactivation of RagC/D and on the integrity of the ATG conjugation system, independently of canonical autophagy (Figure 4).…”

Structural basis for FLCN RagC GAP activation in TFEB substrate-selective mTORC1 regulation

“…The past few years have seen a paradigm shift in understanding how the MiTF family of transcription factors is regulated downstream of mTORC1 in a uniquely FLCN and RagC GDPdependent manner (10,11). Numerous studies corroborate the discovery of a "non-canonical" mTORC1 pathway that specifically regulates MiT-TFE transcription factors (12,21,(35)(36)(37)(38). The new knowledge of FLCN structural mechanism provided by the AFC structure can potentially be exploited for therapeutic benefit.…”

“…The past few years have seen a paradigm shift in understanding how the MiTF family of transcription factors is regulated downstream of mTORC1 in a uniquely FLCN and RagC GDP -dependent manner ( 8, 9, 18, 19, 21, 32, 33 ). The new knowledge of FLCN structural mechanism provided by the AFC structure can potentially be exploited for therapeutic benefit.…”

“…The regulation of TOS motif-containing mTORC1 substrates (3), including regulators of mRNA translation and cell proliferation, such as 4E-BP1 and S6K1 is unaffected (9,18). Taken together, FLCN directly and selectively controls mTORC1 regulation of TFEB/TFE3 but no other substrates (19)(20)(21). These findings support the existence of separate mechanisms for control of mTORC1-dependent anabolic versus catabolic programs, and highlight a therapeutic potential for FLCN to be targeted as a means of mTOR inhibition for treating LSDs and neurodegenerative diseases, with fewer toxic effects on cell proliferation and translation.…”

“…We sought to further explore FLCN specificity for RagC by understanding why GATOR1 does not function as a GAP for RagC. We modelled RagC with GATOR1 and observed residues in GATOR1 (Thr 18 and Pro 19 ) that clashed with RagC at the interface (Fig. S6B).…”