Amino acids are critical activators of TORC1 however the mechanism of amino acid signaling is largely unresolved. Although VPS34 was proposed to mediate nutrient signals to TOR20, 21
, we recently found that Drosophila
null mutations have normal TOR activity45
. Vps34 knockdown had no effect on dS6K phosphorylation (data not shown). In this report, we have identified Rag GTPases as important novel activators of TORC1 pathway in response to amino acids both in Drosophila
and in mammals. Knockdown of dRagA or dRagC dramatically decreased dS6K phosphorylation in response to amino acid stimulation. Overexpression of constitutively active dRagA Q61L increased cell size in fat body and wings, especially in starved Drosophila.
Expression of dominant negative dRagA T16N decreased cell size and this effect was stronger when nutrients were sufficient. Furthermore, dRagA Q61L expression and dRagC
mutation suppressed starvation-induced autophagy and the lethality of Tsc1
mutant animals, respectively. In mammalian cells, overexpression of constitutively active RagA activated TORC1 even in the absence of amino acids, and expression of dominant negative RagA blocked TORC1 activation in response to amino acid stimulation. The relationship between Rag and amino acids is rather specific because constituvely active RagA could not overcome osmotic stress.
Rag physiological role in nutrient response is further supported by the fact that flies expressing dRagA Q61L die earlier than the controls during starvation, presumably due to a failure to attenuate their metabolic activity and growth owing to a false sense of nutrient sufficiency. Additionally, animals expressing dRagA T16N are more resistant to starvation and survive longer in the absence of nutrients.
Our data indicate that dimer formation between RagA/B and RagC/D is important for TORC1 activation. Within the dimer, the function of RagA/B is dominant. In other words, RagA Q66L dominantly activates and RagA T21N dominantly inhibits TORC1 regardless of the nucleotide binding status of the RagC/D in the complex. Nevertheless, RagC/D is likely to be critical for the dimer function, given the effects of dRagC knockdown in S2 cells and the phenotypes of dRagC
mutant animals. Interestingly, the yeast Gtr1 and Gtr2 need to be in GTP-bound and GDP-bound status, respectively, to function26
. Our results suggest that the relationship between RagA and RagC is similar to that between Gtr1 and Gtr2. In addition, RagC may have a GTPase-independent role in stabilizing RagA and may regulate RagA localization or activity, or aid in TORC1 activation.
Studies in yeast have shown that Gtr1/Gtr2 could control intracellular protein trafficking, thereby regulating the localization of the general amino acid permease Gap126
. This observation suggests a possible mechanism whereby Gtr1/2 activate TORC1 by promoting amino acid import and thus regulating nutrient availability. However, our data from mammalian cells are not consistent with this model: complete and extended amino acids depletion fails to inhibit TORC1 activity in cells overexpressing RagA Q66L. Second, we found that the amino acid import was not significantly affected by RagA expression (Fig. S5
). Therefore, it is unlikely that Rag regulates TORC1 by promoting the availability of nutrients. Instead, we favor a model in which Rag acts between amino acids and TORC1 in a pathway parallel to the TSC-Rheb axis (). An interesting possibility is that Rag regulates localization of TORC1 pathway components.
This study identifies Rag GTPases as novel positive regulators of TORC1 in amino acid signaling, a conclusion supported by biochemical studies in mammalian cells and genetic studies in Drosophila. Future study to elucidate the molecular mechanism of Rag in amino acid sensing and TORC1 activation will shed new insight into this important pathway.