The intracellular amino acid pool within the lysosome has been shown to activate the mTORC1 signaling pathway (Zoncu et al., 2011; Jewell et al., 2013). However, how the sequester of the signaling molecules within the lysosome occurs remains poorly understood. New research from Wu et al. (2016) shows that the suppression of FLCN, a tumour suppressor gene associated with the Birt-Hogg-Dubé (BHD) syndrome, controls mTORC1 activity by modulating the lysosomal leucine levels. FLCN exerts this new function by regulating the accumulation of the amino acid transporter PAT1 on the lysosome surface.
Previous work by the authors using a Drosophila model showed that fly FLCN mutants had a growth defects and did not reach adulthood. Interestingly, the growth defects were rescued by supplementation with high levels of leucine, but not other amino acids, in the medium. Adding an inhibitor of mTOR to the leucine-rich medium failed to rescue the slow growth phenotype (Liu et al., 2013). In mammalian cell systems, FLCN was necessary to anchor mTORC1 and this function was performed at the lysosome (Petit et al., 2013; Tsun et al., 2013). In the present study, Wu et al. (2016) show in human embryonic kidney (HEK) 293 cells, that depletion of FLCN suppresses mTORC1 activity. Addition of high amounts of leucine, but not other tested amino acids, to the medium reverses this down-regulation of mTORC1 in FLCN-depleted cells. Starving cells and then adding either basal or high levels of leucine to the medium shows that depletion of FLCN sensitizes cells to the available leucine for mTORC1 induction. The authors confirm work by previous groups (Petit et al., 2013; Tsun et al., 2013) showing that FLCN is localized on the lysosome in a nutrient-dependent manner. They show by western blotting that leucine is reduced in lysosomes from cells lacking FLCN and that this effect is reversed by supplementation with high levels of leucine in the medium. This suggests that FLCN plays an important role keeping the leucine level in lysosome but this can alternatively be achieved by increasing the amount of leucine supplied to the environment to keep the leucine signal strong enough to activate mTORC1. To test if ectopic FLCN would sequester more leucine to the lysosome, the authors generate a cell line overexpressing FLCN (FLCN-HA). In starving conditions, leucine levels and mTORC1 activation are decreased in wild-type cells. In FLCN-HA cells this reduction is much less pronounced suggesting that ectopic FLCN promotes the accumulation of leucine within the lysosome, enabling cells to resist to leucine shortages in the environment and facilitating mTORC1 activation. Similar results were shown in a Drosophila model. FLCN is located on the lysosome and it seems to regulate the efflux of leucine. This process requires it to be loaded onto membrane-anchored transporters. PAT1, a lysosome-associated membrane protein whose overexpression promotes the release of amino acids from lysosome and inhibits mTORC1 (Zoncu et al., 2011) seemed a good candidate to be involved in the FLCN-mediated mTORC1 signaling. After confirming that overexpression of PAT1 in HEK293 cells inhibits mTORC1 the authors show that both the addition of high levels of leucine to the medium and the overexpression of FLCN prevent this inhibitory effect. In addition, depletion of PAT1 using siRNA inhibits the starvation-induced decrease of mTORC1 activation and this effect is counteracted by co-depletion of FLCN. These results led to the conclusion that FLCN and PAT1 antagonize each other to control mTORC1 pathway. In starvation, cells recruit PAT1 to the lysosome to recycle the luminal storage, resulting in a decreased leucine level. However, authors show that this recruitment of PAT1 can be inhibited by ectopic FLCN resulting in the maintenance of the lysosome level. Immunoprecipitation experiments show that FLCN may regulate the localization of PAT1 through direct interactions.
In summary, this study shows a new role for FLCN in modulating the leucine level in the lysosome and how important this mechanism is to regulate mTORC1 signaling pathway. Previous work by Preston et al. (2011) on the FLCN deficient BHD patient-derived renal tumour cell line (UOK257) showed that these cells have a higher dependency on glucose metabolism demonstrating a loss of ‘metabolic flexibility’. The present work sheds light into how cells sense the available nutrients in their environment and helps to appreciate the context-dependent relations between FLCN and mTORC1, crucial for the advancement in BHD syndrome research where targeting metabolic pathways may be used therapeutically to treat BHD-associated kidney lesions.
Wu X, Zhao L, Chen Z, Ji X, Qiao X, Jin Y, & Liu W (2016). FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1. PloS one, 11 (6) PMID: 27280402