Mitochondrial Threonyl-tRNA Synthetase TARS2 Is Required for Threonine-Sensitive mTORC1 Activation

•Mitochondrial protein TARS2 interacts with lysosomal amino-acid-sensing machinery•Threonine modulates localization and activity of mTORC1 through TARS2•TARS2 binds inactive RagC elevates GTP loading RagA in response to threonine•TARS2 is required for threonine-dependent regulation the signaling pathway Mechanistic target rapamycin complex 1 (mTORC1) controls cell growth proliferation by sensing fluctuations environmental cues such as nutrients, factors, energy levels. The Rag GTPases (Rags) serve a critical module that signals amino acid (AA) availability modulate activity. Recent studies have demonstrated how AAs regulate Rags. Here, we uncover an unconventional activates variations threonine (Thr) levels via mitochondrial threonyl-tRNA synthetase TARS2. 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Rajasekharan Hesketh G.G. Abe K.T. Samavarchi-Tehrani P. Zhu L.Y. map cell.bioRxiv. https://doi.org/10.1101/796391Crossref (0) https://humancellmap.org). Through exploration HumanCellMap database ARSs, noted expected near-exclusive recovery mt-ARSs mt baits. However, also readily recovered interactor sole subunit dataset (LAMTOR1) 1B). Except isoleucyl-tRNA (IARS2), which prey LAMTOR1, albeit comparatively lower level than no were significantly captured LAMTOR1 Consistently, LamTOR1 co-localized IARS2, LARS2 (mt-LARS) 1C). intriguing surprising finding prompted us explore physical association machinery. In contrast reportedly involved lung cancer tumorigenesis AKT/mTOR (Di 2019Di Jin Ma Cong Tian Zhao oncogene IARS2 promotes non-small activating pathway.Front. Oncol. 9: 393Crossref (12) remained unexplored. Our co-immunoprecipitation (coIP) assay revealed interacted only (LamTOR1–LamTOR5) components (mLST8, TEL2, TTI1) (RagA–RagD) S1A). As expected, TARS known binding partner 4EHP (Chapat 2017Chapat Jafarnejad Matta-Camacho Gelbart I.A. Attig Gkogkas C.G. Alain Stern-Ginossar Fabian M.R. al.Cap-binding effects silencing microRNAs.Proc. 114: 5425-5430Crossref (51) 2019Jeong Nguyen L.T. Hwang E.Y. Giong J.S. Yoon J.H. initiation machinery.Nat. Commun. 10: 1357Crossref (23) mainly localizes mitochondria because N-terminal targeting sequence (MTS) (Figures S1B–S1D). detected fraction representing mixture 1D, mito. + lyso.), pure where AA-sensing machinery, LamTOR4, resides. Nonetheless, (10% 15%) present cytoplasm cyto.), implying possibility transient machinery interface lysosomes. LARS functions (Han addition canonical enzymatic charging tRNA. Bearing mind, examined potential Thr-mediated activity, indicated phosphorylation ribosomal (pS6 S240/244) 4E-BP1 (p4E-BP1 T37/46), rapidly decreased depletion within h (75% 60% reduction, respectively) 2A). Whereas less drastic alone same duration (30% reduction), prolonged starvation (12 h) dramatically reduced (85% reduction) low potency likely amount (Novoa 2014Novoa E.M. Camacho Tor Wilkinson Moss Marín-García Azcárate I.G. Bautista Mirando A.C. Francklyn C.S. al.Analogs natural aminoacyl-tRNA inhibitors clear malaria vivo.Proc. 2014; 111: E5508-E5517Crossref (52) affinity compared cellular concentration (Wang 2016Wang Zhou X.L. Ruan Z.R. R.J. Eriani disease-causing point mutation induces both structural functional defects.J. 291: 6507-6520Abstract (22) Abu-Remaileh 2017Abu-Remaileh Laqtom N.N. Abbasi Chan Freinkman metabolomics V-ATPase- mTOR-dependent efflux lysosomes.Science. 807-813Crossref (190) (mTORC2) (pAKT S473) altered S2A). Reduced reversed upon incubation 800 ?M 2C S2B). Moreover, adding (8 ?M) Thr-starved sufficient full 90 min S2C). Notably, stimulating serine valine, two similar Thr, failed rescue recruited mTOR, co-localization LAMP2, enhanced 2D), effectively next whether process. Compared (cells stably expressing short hairpin RNA [shCon]), knockdown [shTARS2]) 12 (64% 85% 2E S2D). fully 15 shCon, shTARS2, even 60 after stimulation 2F S2E), indicating These independent (shRNA) sequences S2F). Furthermore, recruitment abolished shTARS2 2G), presence/absence almost identical shCon S2G). GCN2 deficiency responding uncharged tRNA inhibits eIF2?-ATF4-Sestrin2 (Ye 2015Ye Palm Peng King Lindsten M.O. Koumenis Thompson C.B. sustains suppression deprivation inducing Sestrin2.Genes 2331-2336Crossref (140) neither eIF2? (peIF2? S51), triggered deprivation, nor ATF4 S2H), part regulating pathway. recruiting observed most 3A, S3A, S3B). coIP using truncation mutants S3C, upper panel) showed although TARS21–60 TARS21–124 expressed, probably inherent instability, TARS21–399 panel, lane 10), TARS21–300 (lane 5). result suggested TARS2301–399, intermediate region editing catalytic core domains, RagC. TARS2301–399 contains segment (TARS2382–398) unique higher organisms S3D, panel), deletion did affect panel). Likewise, S3E, RagC1–369 7), RagC1–242 RagC243–369 displayed 9), consistent our endogenous data 3B). corresponding RagD244–370 (only 9 127 different). single substitution S3F, abolish ?2 loss interaction. knockdown, could recover S3G), preferential dependent 3C). Among aminoacylation-defective (C409A R438A) editing-defective (H133A?H137A) (Chen 2018Chen Xue M.Q. synthetase-like aminoacylation activities.Nucleic Acids Res. 3643-3656Crossref (14) C409A mutant lost 3D). C409 coordinates Zn2+ ion, ?-amino group side-chain hydroxyl recognition discrimination site, whereas R438 carboxyl 3?-CCA tRNA, ?-phosphate ATP (Sankaranarayanan 1999Sankaranarayanan Dock-Bregeon Romby Caillet Springer Rees Ehresmann Moras structure synthetase-tRNA(Thr) enlightens repressor zinc ion site.Cell. 1999; 97: 371-381Abstract (247) Torres-Larios 2003Torres-Larios Sankaranarayanan Conformational movements cooperativity acid, synthetase.J. 2003; 331: 201-211Crossref (46) Even though mutations defective, previous bacterial orthologs suggest defective (Nureki 1993Nureki O. Kohno Sakamoto Miyazawa Yokoyama Chemical modification mutagenesis synthetases.J. 1993; 268: 15368-15373Abstract R438A (Minajigi Francklyn, 2008Minajigi RNA-assisted catalysis enzyme: 2?-hydroxyl tRNA(Thr) A76 synthetase.Proc. 105: 17748-17753Crossref (36) Collectively, these results imply must correctly charged Intriguingly, hydrolysis-defective (QL, RagCQ120L) mimicking GTP-RagC 3E). RagC, regardless 3F). GDP-RagA (TL, RagAT21L) (QL) 3G). demonstrate presence Thr. To examine explored parallel (pS6), S4A). similarly elevate 4A). Because GTP-RagA (Kim 2008Kim Goraksha-Hicks Neufeld T.P. nutrient response.Nat. 935-945Crossref (901) replenishing incoming transmitted distinct pathways. Cytoplasmic (Wolfson (Chantranupong detect free AAs, v-ATPase-Ragulator (Rebsamen 2015Rebsamen Pochini Stasyk de Araújo M.E.G. Galluccio Kandasamy R.K. Snijder Fauster Rudashevskaya E.L. Bruckner al.SLC38A9 mTORC1.Nature. 519: 477-481Crossref (384) AAs. determine mechanism levels, pre-treated v-ATPase inhibitor bafilomycin A1 (BafA1) block (Abu-Remaileh Recovery replenishment without BafA1 S4B), detects (GTPases) GEFs, GEFs Rags: (Shen LamTOR4 (shLamTOR4) (shSLC38A9) 4B). 30% shLamTOR4 shSLC38A9 comparable 4C). it lacks GEF, act potent Thr-sensitive presumably modulating yet-unknown GEF. Finally, physiological impact TARS2-mediated