Cell Biology

I Love Leucine

Science Signaling  01 May 2012:
Vol. 5, Issue 222, pp. ec121
DOI: 10.1126/scisignal.2003167

The mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which contains the kinase mTOR and the regulatory component Raptor, couples the availability of nutrients, such as amino acids, with signaling pathways that mediate cellular growth and proliferation. Amino acids trigger the translocation of mTORC1 to the lysosome, where it interacts with the Rag family of guanosine triphosphatases (GTPases) through an association between RagC and Raptor. Han et al. and Bonfils et al. now show that leucyl-transfer RNA (tRNA) synthetase (LRS), which catalyzes the “charging” or aminoacylation of leucine to tRNA and also edits or proofreads amino acids, acts as a leucine sensor for mTORC1. However, these groups proposed different underlying mechanisms (see commentary by Segev and Hay). Han et al. found that LRS immunoprecipitated with mTOR and Raptor in a leucine-dependent manner. An siRNA directed against LRS reduced the activation of mTORC1 in response to amino acids, leucine, and isoleucine (to a lesser extent) and reduced the lysosomal translocation of mTOR and Raptor in response to amino acid stimulation. In transfected cells, LRS interacted with RagD, but not with RagA, RagB, or RagC, and the association of endogenous LRS with ectopically expressed RagD increased after leucine treatment. In addition, the amino acid–induced interaction of RagD with Raptor was increased by overexpression of LRS and decreased by knockdown of LRS. Phe50 and Tyr52 in LRS form the hydrophobic pocket that accommodates the side chain of leucine, and a form of LRS containing alanine substitutions at these sites (Phe50→Ala/Tyr52→Ala; F50A/Y52A) did not interact with RagD. More LRS interacted with wild-type RagD or a mutant that mimicked the GTP-bound form (Gln121→Leu; Q121L) than with a RagD mutant that mimicked the GDP-bound form (Ser77→Leu; S77L), and LRS colocalized with RagD Q121L but not with RagD S77L. In vitro assays demonstrated that a C-terminal fragment of LRS (which interacts with RagD but not with leucine or ATP) increased GTP hydrolysis by RagD, indicating that LRS acts as a GTPase-activating protein (GAP) for RagD. Leucine-stimulated GTP hydrolysis by RagD was increased in transfected cells expressing wild-type LRS but not the F50A/Y25A mutant. Furthermore, leucine-stimulated activation of mTORC1 was increased in cells expressing wild-type LRS but not a form of LRS with mutations in a putative GAP motif. Using mass spectrometry and biochemical assays, Bonfils et al. independently showed that Cdc60, the yeast homolog of LRS, interacted with Gtr1, the yeast homolog of RagA and RagB, but not Gtr2, the yeast homolog of RagC and RagD, a result that differs from the findings of Han et al. Bonfils et al. found that the ability of Cdc60 to increase TORC1 activity did not require its aminoacylation activity (a finding also reported by Han et al.) but rather its amino acid–editing activity, because treatment with DHBB, a compound that traps uncharged tRNALeu in the editing active site, suppressed TORC1 activity in wild-type cells. DHBB also reduced the amount of GTP-loaded Gtr1. Norvaline inhibits the editing activity of LRS, and treating cells with norvaline inhibited TORC1. The CP1 domain, which mediates the editing activity of LRS, interacted with Gtr1 but not with Gtr2, the yeast homolog of RagC and RagD. Bonfils et al. propose that leucine deprivation causes LRS to mischarge tRNALeu, triggering conformational changes in the CP1 domain that enable a GAP to access Gtr1 and induce GTP hydrolysis, thereby decreasing TORC1 activity. The reasons for the different LRS binding partners reported by Han et al. and Bonfils et al. and for the distinct mechanisms proposed by the two groups are unclear.

J. M. Han, S. J. Jeong, M. C. Park, G. Kim, N. H. Kwon, H. K. Kim, S. H. Ha, S. H. Ryu, S. Kim, Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway. Cell 149, 410–424 (2012). [PubMed]

G. Bonfils, M. Jaquenoud, S. Bontron, C. Ostrowicz, C. Ungermann, C. De Virgilio, Leucyl-tRNA synthetase controls TORC1 via the EGO complex. Mol. Cell 46, 105–110 (2012). [PubMed]

N. Segev, N. Hay, Hijacking leucyl-tRNA synthetase for amino acid-dependent regulation of TORC1. Mol. Cell 46, 4–6 (2012). [PubMed]