| Summary: | Multicellular eukaryotes readily adjust their growth in response to environmental cues. A central nutrient sensing pathway crucial for this process requires the mechanistic target of rapamycin complex 1 (mTORC1), which integrates various inputs such as amino acids, growth factor signaling and energy levels. Amino acids promote the localization of mTORC1 to the lysosomal surface, where it can then be activated in response to growth factor availability. While the Rag GTPases mediate the recruitment of mTORC1 to the lysosomal surface, they also have a mutually exclusive and nutrient regulated interaction with SLC38A9, a lysosomal amino acid transporter. How the mutually exclusive mTORC1-Rag and the SLC38A9-Rag interaction is achieved and how it alters the function of each component is not completely understood.
We found the Rag GTPases are necessary to promote SLC38A9 transport independent of mTORC1 kinase activity. Moreover, we attained the structures of Raptor-Rag-Ragulator and SLC38A9-Rag-Ragulator at 3.2 Å and 3.6 Å resolutions, respectively, and generated separation of function mutants of RagA. Using these constructs, we show that perturbation of Rag GTPase binding to SLC38A9, but not mTORC1, causes the accumulation of a distinct set of non-polar, mostly essential amino acids (Tyrosine, Leucine, Phenylalanine, and Isoleucine). We also show that “inactive” Rags, competent to bind SLC38A9, promote its transport activity in proteoliposomes reconstituted with wild-type SLC38A9. We believe the purpose of this regulation is to efflux amino acids from the lysosome during starvation.
Overall, we identified the mechanism by which mTORC1 regulates the efflux of essential amino acids from lysosomes to be through the Rag-Ragulator complex. This work ascribes an alternative function to the Rag-Ragulator complex independent of its ability to convey the availability of nutrients to mTORC1. Furthermore, we show this direct gating mechanism plays a role in regulating the efflux of essential amino acids from lysosomes of mouse hepatocytes in-vivo during periods of starvation and refeeding. Thus, the studies described in this thesis provide new insights to the role of the Rag GTPases by genetic, biochemical, and structural techniques. Further mechanistic insights into Rag-Ragulator binding to SLC38A9 will reveal how the Rag GTPases regulate its transport function, along with that of other interactors, in various physiological contexts.
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