Our findings, together with previous work showing that Rheb is required for amino acids to activate the mTORC1 pathway (Roccio et al., 2006
;Smith et al., 2005
) and can localize to late endosomes/lysosomes (Saito et al., 2005
; Sancak et al., 2008
), is consistent with a model in which amino acids induce mTORC1 to associate with the endomembrane system of the cell and thus allow it to encounter its activator Rheb. In this model the essential role of the Ragulator-Rag complex is to serve as an amino acid-regulated docking site for mTORC1 on lysosomal membranes (see schematic in ). The proposed link between the Rag and Rheb GTPases in the regulation of the mTORC1 pathway provides an explanation for why activation of mTORC1 occurs only when activators of both Rheb (e.g., growth factors and energy) and the Rags (i.e., amino acids) are available. For technical reasons (Buerger et al., 2006
; Sancak et al., 2008
), it has not been possible to determine the intracellular localization of endogenous Rheb and work using overexpressed GFP-tagged Rheb1 has placed it on various endomembrane compartments, including endosomes and lysosomes (Buerger et al., 2006
; Saito et al., 2005
; Sancak et al., 2008
; Takahashi et al., 2005
). Our results suggests that at some point in its life cycle Rheb must traverse the lysosomal surface in order to encounter mTORC1 and so in our model we have chosen to place Rheb on this compartment (). However, at any given time only a small fraction of cellular Rheb may actually be on the lysosomal surface or, alternatively, some of the mTORC1 within the cell may move to a non-lysosomal endomembrane compartment that also contains Rheb. These issues will only be answered once a definitive location for endogenous Rheb can be determined.
The trimeric p14, p18, and MP1 protein complex, which we call Ragulator, is a Rag-interacting complex that is essential for amino acid signaling to mTORC1 and represents an additional critical component of the TORC1 signaling pathway in mammals and flies. p18 directly interacts with the Rag GTPases (Figure S2A
) as well as with p14 and MP1 (Nada et al., 2009
) and so may serve as a scaffold to bring the Rag GTPases and MP1-p14 next to each other. In vitro we have not detected a direct interaction between the Rag GTPases and either MP1 or p14, but both proteins are, like p18, necessary for localizing the Rag GTPases to the lysosomal surface. p14 is required to maintain normal p18 expression levels (Figure S2B
), suggesting that within cells p14 and MP1 form a crucial part of the Ragulator structure. Given the non-specific nature of the p14 and p18 names, in the future it may be best to rename these proteins, perhaps to names that reflect their essential roles in the mTORC1 pathway.
The location of the Rag GTPases, the Ragulator, and mTORC1 on the lysosomal surface implicates this organelle as the site of a yet to be discovered sensing system that signals amino acid availability to the Ragulator-Rag complex. The lysosomal location of the amino acid sensing branch of the mTORC1 pathway is consistent with increasing evidence that lysosomes, and their yeast counterparts, vacuoles, are at the nexus of amino acid metabolism within cells. Lysosomes are a major site of protein degradation and amino acid recycling and vacuoles store amino acids at high concentrations (reviewed in (Li and Kane, 2009
)). Thus, mTORC1 and its regulators may reside on the lysosomal surface so as to sense a currently unknown aspect of lysosomal function that reflects the intracellular pools of amino acids.
It is interesting to consider the differences and similarities between the still poorly understood amino acid signaling mechanisms employed by the mTORC1 and yeast TORC1 pathways. Consistent with previous work in mammalian cells (Sancak et al., 2008
), the Gtr1p-Gtr2p heterodimer that is orthologous to RagA/B-RagC/D, interacts with yeast TORC1 when Gtr1p is GTP-loaded (Binda et al., 2009
). TORC1 and the Gtr proteins are located on the surface of the vacuole (Berchtold and Walther, 2009
; Binda et al., 2009
), the yeast equivalent of lysosomes, but, unlike in mammals, yeast TORC1 does not leave the vacuolar surface upon amino acid deprivation although amino acids do control the interaction of TORC1 with Gtr1p-Gtr2p (Binda et al., 2009
). This finding suggests that there must exist a distinct mechanism for retaining TORC1 at the vacuolar surface and that in yeast the interaction between TORC1 and Gtr1p-Gtr2p serves other purposes besides controlling the intracellular location of TORC1. In contrast, our current work argues that in mammals the main role of the Rag GTPase and the associated Ragulator complex is to control the association of mTORC1 with the cellular endomembrane system, in particular, lysosomes. Rheb, which is essential for the activation of mTORC1 by all upstream signals, does not appear to be part of the TORC1 pathway in yeast (reviewed in (Berchtold and Walther, 2009
)). As we suggest that the Rag-dependent and amino acid-regulated translocation of mTORC1 to the lysosomal surface may ultimately be a mechanism for controlling the access of mTORC1 to Rheb, the absence of Rheb in the yeast TORC1 pathway may make regulation of TORC1 localization unnecessary. That known Rag- and Gtr-interacting proteins share no sequence homology also suggests that the mechanisms through which the Rag and Gtr GTPases regulate mTORC1 and yeast TORC1, respectively, have diverged. Although it is clear that the Ragulator and EGO complexes both control the intracellular localization of the Rag (this paper) and Gtr (Gao and Kaiser, 2006
) GTPases, respectively, whether these complexes have additional functions remains to be determined.
Previous studies suggest that MP1-p14-p18 complex plays an adaptor role in the MAP Kinase (MAPK) pathway (reviewed in (Dard and Matthias, 2006
)) and our current findings do not contradict these results. However, considering the very strong inhibition of the mTORC1 pathway that occurs in cells lacking p14 or p18, it seems possible that some of the impairment in MAPK signaling observed in those cells reflects an altered feedback signaling from Akt to the MAPK pathway. For example, in Ragulator-null cells, Akt is slightly activated, almost certainly because the well-known inhibitory signal from mTORC1 to PI3K is absent. As Akt suppresses MAPK signaling by phosphorylating and inhibiting Raf (Zimmermann and Moelling, 1999
), it is conceivable that the activation of Akt that occurs in Ragulator-null cells could account, at least in part, for the inhibition of MAPK signaling that has been observed in these cells.
Mice lacking either p14 or p18 die around embryonic day 7.5–8 and have obvious growth defects (Nada et al., 2009
; Teis et al., 2006
). We would not be surprised if, when generated, mice lacking the Rag proteins die at around the same age and present similar defects. On the other hand, mice lacking the core mTORC1 component raptor die earlier (before embryonic day 6.5) than p14- and p18-null mice (Guertin et al., 2006
). This may be expected because although loss of p14 or p18 completely blocks mTORC1 activation by amino acids, cells lacking the Ragulator proteins are likely to retain a low residual level of mTORC1 activity that may be sufficient to support development further than in embryos completely lacking mTORC1 function. Lastly, our results suggest that the strong growth retardation observed in humans with a mutation that reduces p14 expression (Bohn et al., 2006
), is a result of partial suppression of the mTORC1 pathway. If this turns out to be the case, it would represent the first human example of a loss of function mutation in a positive component of the mTORC1 pathway.