Mechanisms of nutrient sensing by the lysosome

In response to amino acids and glucose, a specialized molecular scaffold that includes the heterodimeric Rag GTPases, the pentameric Ragulator complex and the vacuolar H+ ATPase (V-ATPase) recruits mTORC1 to the surface of the lysosome, a key step for mTORC1 activation.  Intriguingly, the internal amino acid pool of the lysosome may play an especially important role in regulating the V-ATPase/Ragulator/Rag GTPase complex and triggering mTORC1 translocation. This lysosome-centric model of mTORC1 regulation is consistent with the observation that the lysosome is the first place where amino acids are generated and accumulate during a starvation-response process known as autophagy. Moreover, it reflects the evolutionarily conserved role of the lysosome as the cellular site for nutrient storage, which is especially prominent in the vacuole, the yeast and plant equivalent of the lysosome.

Studying lysosomal mTORC1 translocation in cells and in vitro. (A) Time-lapse of HEK-293T cell expressing a GFP-tagged mTORC1 subunit. Following amino acid stimulation, mTORC1 clusters to intracellular spots corresponding to lysosomes.  (B) Schematic of an vitro binding assay combining purified lysosomes to epitope-tagged mTORC1. (C) A single FLAG affinity bead following the mTORC1 binding assay. Yellow indicates co-localization of LAMP1 and mTORC1 on the bead surface.

Studying lysosomal mTORC1 translocation in cells and in vitro. (A) Time-lapse of HEK-293T cell expressing a GFP-tagged mTORC1 subunit. Following amino acid stimulation, mTORC1 clusters to intracellular spots corresponding to lysosomes.  (B) Schematic of an vitro binding assay combining purified lysosomes to epitope-tagged mTORC1. (C) A single FLAG affinity bead following the mTORC1 binding assay. Yellow indicates co-localization of LAMP1 and mTORC1 on the bead surface.

The lysosome-centric model of amino acid sensing poses intriguing questions. How do amino acids gain access to the lysosomal lumen, and what mechanisms establish and maintain the lysosomal amino acid pool?  How does the V-ATPase regulate the function of Ragulator and Rag GTPases? Do other nutrient signals cross-talk with the core amino acid-sensing machinery? We are addressing these questions using in vitro biochemical assays of mTORC1 binding and V-ATPase activity, in combination with loss- and gain-of function and advanced live cell imaging. These studies will shed light on how the lysosome senses its internal status and relays this information to downstream signaling effectors to regulate growth and metabolism.