Structural mechanism of a Rag GTPase activation checkpoint by the lysosomal folliculin complex

Abstract

The tumor suppressor folliculin (FLCN) enables nutrient-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase via its guanosine triphosphatase (GTPase) activating protein (GAP) activity toward the GTPase RagC. Concomitant with mTORC1 inactivation by starvation, FLCN relocalizes from the cytosol to lysosomes. To determine the lysosomal function of FLCN, we reconstituted the human lysosomal FLCN complex (LFC) containing FLCN, its partner FLCN-interacting protein 2 (FNIP2), and the RagAGDP:RagCGTP GTPases as they exist in the starved state with their lysosomal anchor Ragulator complex and determined its cryo–electron microscopy structure to 3.6 angstroms. The RagC-GAP activity of FLCN was inhibited within the LFC, owing to displacement of a catalytically required arginine in FLCN from the RagC nucleotide. Disassembly of the LFC and release of the RagC-GAP activity of FLCN enabled mTORC1-dependent regulation of the master regulator of lysosomal biogenesis, transcription factor E3, implicating the LFC as a checkpoint in mTORC1 signaling.

Structure of the lysosomal SCARF (L-SCARF) complex, an Arf GAP haploinsufficient in ALS and FTD

“…Cells in the -AA condition were transferred to imaging buffer (10 mM HEPES, pH7.4, 136 mM NaCl, 2.5 mM KCl, 2 mM CaCl 2 , 1.2 mM MgCl 2 ) and cells in the +AA condition were transferred to imaging buffer supplemented with amino acids, 5 mM glucose, and 1% dialyzed FBS (+AA) and imaged by spinning-disk confocal microscopy. Lysosomal enrichment was scored as described 31 using a home-built Matlab script to determine the lysosomal enrichment of GFP SMCR8. The score was analyzed for at least ten cells for each condition.…”

“…The structure of the complex places it in the same class of heterodimeric longin-DENN domain protein GAP complexes as FLCN-FNIP 31,32 . FLCN-FNIP is a major node for communicating lysosomal nutrient status to the nucleus by virtue of its regulation of the RagC GTPase 34 and the phosphorylation of transcription factors regulating lysosome biogenesis and autophagy 31 . These data show how C9orf72 is stabilized on the lysosome in amino acid starvation by bridging contacts made by SMCR8 to WDR41.…”

Nutrient Signaling and Lysosome Positioning Crosstalk Through a Multifunctional Protein, Folliculin

“…The loss of FLCN inhibits the phosphorylation of transcription factors TFEB and TFE3, promoting their nuclear translocation and activating lysosome biogenesis (Hong et al, 2010;Martina et al, 2014). Supporting this notion, structure-function analysis have suggested that the role of FLCN in regulating TFEB and TFE3 localization is dependent on the GAP activity of FLCN toward RagC (Lawrence et al, 2019), whereas immunoblotting assays in mammalian and nematode cells suggested that the loss of FLCN drives TFEB and TFE3 nuclear localization independently from the canonical mTORC1 pathway (El-Houjeiri et al, 2019). On the other hand, FLCN-deficient human cells appear to have impaired autophagosome maturation , suggesting a role for FLCN in autophagy regulation, which has also been reported during studies of the AMPK-FLCN functional relationship (see Section "A Potential Interaction of FLCN-FNIP With AMPK").…”

“…A structural comparison between the GATOR1 and FLCN-FNIP complexes, combined with GTPase assays, led to the identification of FLCN-Arg164 as the catalytic residue exerting the GAP activity toward RagC, as mutations affecting this residue reduced the nucleotide exchange rate 100-fold (Lawrence et al, 2019;Shen et al, 2019). However, this residue is pointing away from the RagC nucleotide binding site, thus explaining the lack of GAP activity when FLCN is recruited to the lysosome under nutrient-starved conditions (Figure 4B, inset).…”

“…Additionally, frameshift mutations that would cause premature stop codons in both FNIP1 and FNIP2 have been reported in gastric and colorectal malignancies, supporting a role for FNIP1 and FNIP2 in the development of these cancers (Mo et al, 2019), however further studies are required to clarify the roles of FLCN interacting partners in tumorigenesis. Recent structural studies have shown that the FLCN and FNIP proteins each contain both a longin and a differentially expressed in normal versus neoplastic cells (DENN) domain (Nookala et al, 2012;Zhang et al, 2012;Lawrence et al, 2019;Shen et al, 2019), which are protein folds that have been variously implicated in the regulation of small GTPases and membrane trafficking. Accordingly, functional studies support the notion that FLCN-FNIP complex regulates both the Rag and Rab GTPase families (Dodding, 2017;Schmidt and Linehan, 2018), which in turn modulate the key mTORC1 signaling pathway and lysosomal distribution respectively, in a manner dependent on amino acid availability.…”