The role of the endocytic system in mTORC1 signaling
Flinn, Rory Joseph
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The multisubunit mTORC1 complex integrates signals from growth factors and nutrients to regulate protein translation, cell growth, and autophagy. Growth factor signaling to mTORC1 is well characterized and requires nutrient availability, but the mechanism by which nutrients signal to mTORC1 remains unclear. Strong evidence implicates a role for the endocytic system in regulation of mTORC1. To investigate how the endocytic system might regulate mTORC1 signaling, nutrient and growth factor stimulated phosphorylation of S6K1 was evaluated after perturbation of specific endocytic trafficking processes. Blocking early/late endosomal conversion, by overexpression of a constitutively active Rab5 or knockdown of hVps39, which results in the formation of hybrid early/late endosomes, inhibited nutrient and insulin stimulated mTORC1 signaling. Perturbation of other endocytic trafficking steps did not affect mTORC1 signaling. Formation of hybrid endosomes did not alter nutrient uptake, mTORC1 complex stability/formation, activation of the negative mTORC1 regulator AMPK. Amino acids mediated the localization of mTOR to late endosomes in control cells, and to hybrid endosomes in cells where early/late endosomal conversion had been blocked. The inhibition of mTORC1 by hybrid endosome formation was rescued by overexpression of Rheb, yet not by activation of endogenous Rheb alone. These results suggested that formation of hybrid endosomes altered Rheb-mTORC1 interactions. Late endosomes differ from early endosomes by their intraluminal pH and degradative capacity. We found that alkalinization of late endosomes, but not inhibition of lysosomal degradation, inhibited mTORC1 signaling. Alkalinization of late endosomes did not inhibit the amino acid stimulated localization of mTOR to late endosomes, nor was mTORC1 kinase activity inhibited in vitro by lysosomotropic agents. However, overexpression of Rheb rendered cells insensitive to alkalinization, suggesting that a low intraluminal pH is required for Rheb-mTORC1 interactions and mTORC1 signaling. We conclude that the integrity of late endosomes is essential for mTORC1 signaling.