The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates 1 – 3 . However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy 4 , 5 , is phosphorylated by mTORC1 via a substrate-specific mechanism mediated by RagGTPases. Thus, TFEB phosphorylation is strictly dependent on amino acid-mediated activation of RagC/D GTPase but, unlike other mTORC1 substrates such as S6K and 4E-BP1, insensitive to growth factor-induced Rheb activity. This mechanism plays a crucial role in Birt-Hogg-Dubé (BHD) syndrome, a disorder caused by mutations of the RagC/D activator folliculin (FLCN) and characterized by benign skin tumors, lung and kidney cysts and renal cell carcinoma 6 , 7 . We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and paradoxical mTORC1 hyperactivity observed in BHD syndrome. Remarkably, depletion of TFEB in a kidney-specific mouse model of BHD syndrome fully rescued the disease phenotype and associated lethality and normalized mTORC1 activity. Together, these findings identify a substrate-specific control mechanism of mTORC1, whose dysregulation leads to kidney cysts and cancer.

During starvation the transcriptional activation of catabolic processes is induced by the nuclear translocation and consequent activation of transcription factor EB (TFEB), a master modulator of autophagy and lysosomal biogenesis. However, how TFEB is inactivated upon nutrient refeeding is currently unknown. Here we show that TFEB subcellular localization is dynamically controlled by its continuous shuttling between the cytosol and the nucleus, with the nuclear export representing a limiting step. TFEB nuclear export is mediated by CRM1 and is modulated by nutrient availability via mTOR-dependent hierarchical multisite phosphorylation of serines S142 and S138, which are localized in proximity of a nuclear export signal (NES). Our data on TFEB nucleo-cytoplasmic shuttling suggest an unpredicted role of mTOR in nuclear export.

The mechanistic Target Of Rapamycin Complex 1 (mTORC1) is recruited to the lysosome by Rag GTPases and regulates anabolic pathways in response to nutrients. Here we find that MiT/TFE transcription factors, master regulators of lysosomal and melanosomal biogenesis and autophagy, control mTORC1 lysosomal recruitment and activity by directly regulating the expression of RagD. In mice this mechanism mediated adaptation to food availability after starvation and physical exercise and played an important role in cancer growth. Up-regulation of MiT/TFE genes in cells and tissues from patients and murine models of renal cell carcinoma, pancreatic ductal adenocarcinoma, and melanoma triggered RagD-mediated mTORC1 induction, resulting in cell hyper-proliferation and cancer growth. Thus, this transcriptional regulatory mechanism enables cellular adaptation to nutrient availability and supports the energy-demanding metabolism of cancer cells.

Metabolite accumulation in lysosomal storage disorders (LSDs) results in impaired cell function and multi-systemic disease. Although substrate reduction and lysosomal overload-decreasing therapies can ameliorate disease progression, the significance of lysosomal overload-independent mechanisms in the development of cellular dysfunction is unknown for most LSDs. Here, we identify a mechanism of impaired chaperone-mediated autophagy (CMA) in cystinosis, a LSD caused by defects in the cystine transporter cystinosin (CTNS) and characterized by cystine lysosomal accumulation. We show that, different from other LSDs, autophagosome number is increased, but macroautophagic flux is not impaired in cystinosis while mTOR activity is not affected. Conversely, the expression and localization of the CMA receptor LAMP2A are abnormal in CTNS-deficient cells and degradation of the CMA substrate GAPDH is defective in Ctns−/− mice. Importantly, cysteamine treatment, despite decreasing lysosomal overload, did not correct defective CMA in Ctns−/− mice or LAMP2A mislocalization in cystinotic cells, which was rescued by CTNS expression instead, suggesting that cystinosin is important for CMA activity. In conclusion, CMA impairment contributes to cell malfunction in cystinosis, highlighting the need for treatments complementary to current therapies that are based on decreasing lysosomal overload.

The mechanism of cytoskeleton remodeling during exocytosis is not well defined. A combination of vesicular dynamics and functional studies shows that the Rab27a effector JFC1 and the RhoA-GTPase–activating protein Gem-interacting protein are necessary for RhoA regulation, actin depolymerization, and vesicular transport through the actin cortex during exocytosis.