FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1

Abstract

The intracellular amino acid pool within lysosome is a signal that stimulates the nutrient-sensing mTORC1 signalling pathway. The signal transduction cascade has garnered much attention, but little is known about the sequestration of the signalling molecules within the lysosome. Using human HEK293 cells as a model, we found that suppression of the BHD syndrome gene FLCN reduced the leucine level in lysosome, which correlated with decreased mTORC1 activity. Both consequences could be reversed by supplementation with high levels of leucine, but not other tested amino acids. Conversely, overexpressed FLCN could sequester lysosomal leucine and stimulate mTORC1 in an amino acid limitation environment. These results identify a novel function of FLCN: it controls mTORC1 by modulating the leucine signal in lysosome. Furthermore, we provided evidence that FLCN exerted this role by inhibiting the accumulation of the amino acid transporter PAT1 on the lysosome surface, thereby maintaining the signal level within the organelle.

Glycosylation affects the stability and subcellular distribution of human PAT1 protein

“…C, lower panel). This result has been attributed to the reduced amino acids pool in the lysosome, which was caused by the excessive lysosome‐anchored PAT1 . Surprisingly, we found that mTORC1 activities were either unchanged or modestly increased in three different cell lines stably expressing the EGFP‐PAT1 3NQ (Fig.…”

“…In a previous study , we generated stable 293 cell lines expressing EGFP‐PAT1, which encodes a human PAT1 fusion protein with an N‐terminal EGFP tag. Using these cells as a model, we found that some EGFP‐PAT1 proteins were present on the lysosome and that overexpression of EGFP‐PAT1 decreased mTORC1 activity . Similar results have been reported by other studies through checking either the endogenous PAT1 (for the localization assay) or chimeric PAT1 fused with different tags .…”

“…Recent studies have revealed an important role of PAT1 during the regulation of mTORC1. Suppression of PAT1 has been shown to inhibit mTORC1 in different human cell types and the Drosophila animal model . However, the precise mechanism underlying the control of mTORC1 by PAT1 remains elusive.…”

“…In one mechanism, PAT1 was found to activate mTORC1 through direct interactions with the Rag proteins (components of the amino acids signal‐sensing machinery) and was suspected to participate in the signal transduction process . In the second mechanism, PAT1 plays a more upstream role by reducing the amino acid levels within the lysosome and hence inhibits mTORC1 . Currently, how PAT1 can either activate or inhibit mTORC1 is not fully understood yet .…”

Lysosome: The metabolic signaling hub

“…FLCN is recruited to the lysosome by interaction with RagA/B GDP and requires GATOR1 GAP activity; FLCN recruitment to the lysosome is thus likely regulated by the same set of amino acid sensors upstream of GATOR1/2 that act to regulate mTORC1 68 . One possible explanation for the localization of FLCN to the lysosome following amino acid depletion was recently discovered: FLCN sequesters lysosomal leucine when amino acids are limited by blocking PAT1, a lysosomal amino acid transporter, thus helping to preserve mTORC1 activity during nutrient limitation 69 .…”