Role of the Rag GTPases in Amino Acid sensing and mTORC1 signaling

To sustain metabolism and homeostatic functions, cells need to acquire energy-rich nutrients from their environment. These are broken down in catabolic pathways to provide energy for ATP generation. Nutrients are also the building blocks of complex biomolecules and effectively define the composition of biomass. To balance energy metabolism and biomass production cells need to be able to switch from catabolic to anabolic metabolism. This switch is regulated by the kinase mTOR, which was identified as the target of the immunosuppressant Rapamycin. mTOR was shown to react cell-autonomously to amino acid availability by upregulating translation and downregulating autophagy. Beyond this, mTOR was demonstrated to upregulate various other anabolic pathways, with major implications for human disease and the ageing process. The proteins that facilitate mTOR activation in response to amino acids are the dimeric Rag GTPases, which are localized at the lysosomal surface. An active dimer is composed of a smaller and larger Rag monomer, however there are two paralog genes for the smaller (Rag A and RagB) and the larger (RagC and RagD) monomer. Although many regulators of the Rag GTPases have been identified, the role of paralog Rag GTPase genes has not been thoroughly investigated. Our hypothesis was that the paralog Rag GTPase proteins are non-redundant and facilitate different signaling events in the mTORC1 pathway. We tested this hypothesis by using gene editing tools to knock-out endogenous Rag GTPase genes, obtaining a quadruple knock-out cell line. We used this cell line for a reconstitution approach, in which we re-expressed all four possible Rag dimer combinations. We performed functional mTOR assays and were able to report novel, non-redundant functions of the paralogs. All dimer combinations rescued phosphorylation of the substrate S6K, which controls translation. However, only the Rag GTPase dimers containing the RagD paralog are able to rescue the phosphorylation of lysosomal transcription factors TFEB and TFE3. We investigated TFE3-dependent transcription and were able to confirm a downregulation by RagD-, but not RagC-containing dimers. We studied the regulatory mechanism of substrate specificity and found stronger localization of RagD-containing dimers at the lysosomal surface. We identified the regions of the RagD protein that enable it to regulate the subset of lysosomal mTOR substrates. Moreover, we discovered that cancer-associated gain-of-function mutations enabled the paralog RagC protein to also facilitate lysosomal substrate phosphorylation. Finally, we demonstrated, that the RagB, but not the RagA paralog rendered mTOR activity resistant to amino acid starvation. We identified a novel mode of regulating mTOR substrate selectivity and amino acid response. Thus, we were able to uncover a whole new level of mTOR regulation by the paralogs of Rag GTPases with major implication for the pathways’ function