Biochemical and physiological exploration of the nutrient sensing pathway upstream of mTORC1

Abstract

The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase controls growth in response to environmental cues. Aberrant mTORC1 activity is linked to numerous diseases including cancer. Understanding the regulation of mTORC1 will facilitate therapeutic developments. Amino acids promote the translocation of mTORC1 to the lysosomal surface, a process dependent on the Rag GTPases nucleotide state, which is regulated by several multi-component complexes. Leucine and arginine are known activators of mTORC1 with reported corresponding sensors.

Despite years of research, two essential questions still remain: 1. What and how other inputs impact mTORC1 activity? 2. What are the physiological functions of the nutrient sensing pathway?

We identified SAMTOR as a previously uncharacterized protein that inhibits mTORC1 signaling by interacting with GATOR1, the GTPase activating protein (GAP) for RagA/B. The methyl donor Sadenosylmethionine (SAM) disrupts the SAMTOR-GATOR1 complex by binding directly to SAMTOR with a dissociation constant of approximately 7 μM. In cells, methionine starvation reduces SAM levels below this dissociation constant and promotes the association of SAMTOR with GATOR1, thereby inhibiting mTORC1 signaling. Methionine-induced activation of mTORC1 requires the SAM binding capacity of SAMTOR. Thus, SAMTOR is a SAM sensor that links methionine and one carbon metabolism to mTORC1.

In parallel, I explored the physiological roles of the nutrient sensing pathway in Drosophila melanogaster. Recent work in cultured cells established Sestrin as a conserved cytosolic leucine sensor, but its role in the organismal response to dietary leucine remains elusive. I found that Sestrin null flies (Sesn-/- ) fail to inhibit mTORC1 or activate autophagy upon leucine deprivation and survive worse on a low leucine diet. Knock-in flies expressing a leucine-binding deficient Sestrin mutant (SesnL431E) show decreased and leucine-insensitive mTORC1 activity. Interestingly, we found that flies can discriminate between food with or without leucine in a Sestrin-dependent manner. Leucine regulates mTORC1 activity in glial cells and a knockdown of Sesn in these cells reduces the ability of flies to detect leucinefree food. Thus, nutrient sensing by mTORC1 is not only necessary for flies to adapt to, but also to detect, a diet deficient in an essential nutrient.