SIGNAL TRANSDUCTION

Crystal structure of the humanlysosomal mTORC1 scaffold complexand its impact on signalingMariana E. G. de Araujo,1* Andreas Naschberger,2*† Barbara G. Fürnrohr,2*Taras Stasyk,1 Theresia Dunzendorfer-Matt,2 Stefan Lechner,2 Stefan Welti,2

Leopold Kremser,3 Giridhar Shivalingaiah,2 Martin Offterdinger,4 Herbert H. Lindner,3

Lukas A. Huber,1,5‡ Klaus Scheffzek2‡

The LAMTOR [late endosomal and lysosomal adaptor and MAPK (mitogen-activated proteinkinase) and mTOR (mechanistic target of rapamycin) activator] complex, also known as“Ragulator,” controls the activity of mTOR complex 1 (mTORC1) on the lysosome.The crystalstructure of LAMTOR consists of two roadblock/LC7 domain–folded heterodimers wrappedand apparently held together by LAMTOR1, which assembles the complex on lysosomes. Inaddition, the Rag guanosine triphosphatases (GTPases) associated with the pentamer throughtheir carboxyl-terminal domains, predefining the orientation for interaction with mTORC1.In vitro reconstitution and experiments with site-directed mutagenesis defined thephysiological importance of LAMTOR1 in assembling the remaining components to ensurefidelity of mTORC1 signaling. Functional data validated the effect of two short LAMTOR1 aminoacid regions in recruitment and stabilization of the Rag GTPases.

The LAMTOR [late endosomal and lysosomaladaptor andMAPK (mitogen-activated pro-tein kinase) andmTOR (mechanistic targetof rapamycin) activator] complex is a pen-tameric complex [LAMTOR1 to LAMTOR5;

also known as p18, p14, MP1 (MEK binding part-ner 1), C7orf59, and HBXIP (hepatitis B virusX-interacting protein)] on late endosomes andlysosomes (1). LAMTOR2 and LAMTOR3 scaf-fold MEK1 (MAPK kinase 1) and ERK1 or ERK2(Ras-dependent extracellular signal–regulated ki-nase) to lysosomes, providing spatial and temporalspecificity in theMAPKpathway (2). Furthermore,LAMTOR anchors the Rag guanosine triphospha-tases (GTPases) to the lysosomal surface (3) andmay serve as a guanine nucleotide exchange fac-tor (GEF) toward theRagproteins (i.e., the “Rags”),contributing to mTOR complex 1 (mTORC1) acti-vation. Hence, it was also named “Ragulator” (1).Lysosomal mTORC1 activation depends on conco-mitant inputs controlled byRheb andRagGTPases(4). Nutrient availability leads to GTP loading ofRagA or RagB, which then recruit Raptor to targetmTORC1 to lysosomes (5, 6). LAMTOR controlsmany cellular processes: embryonic development;tissue homeostasis; cell cycle progression; recep-tor trafficking; focal adhesion turnover; migra-tion; maturation and biogenesis of lysosomes;

and growth factor and amino acid signaling (7–11).To explore how these functions are executed andcoordinated,we solved the crystal structures of thepentameric LAMTOR and of its complex with theC-terminal domains (CTDs) of the Rags at resolu-tions of 2.3 and 2.9 Å, respectively.We crystallized the pentameric LAMTOR com-

plex (Fig. 1, figs. S1 to S3, and supplementarytext). The crystal structure (Fig. 1A and table S1)comprises two heterodimers (the first containingLAMTOR2 and LAMTOR3, the second encom-passing LAMTOR4 and LAMTOR5) surroundedby LAMTOR1. Despite lacking the C-terminalhelix, LAMTOR4 adopts a roadblock fold and

interacts with LAMTOR5 through b-sheet aug-mentation, displaying the same dimerizationmode as LAMTOR2 and LAMTOR3 (12–14). Thetilted arrangement between two neighboring pro-teins of each heterodimer is similar to that of therelated trimeric TORC1-recruiting Ego complexin yeast (fig. S4), indicating structural conser-vation of the roadblock fold within eukaryotes(15, 16). Although interactions within the hetero-dimers are prominent and numerous, few con-tacts could be detected between heterodimers.Thus, the heterotetramer appears to be held to-gether largely by its surrounding interactionswith LAMTOR1.Although the version of LAMTOR1 that was

crystallized included residues 21 to 161, electrondensity was only resolved for residues 80 to 149.We used electrophoresis and mass spectrometryanalysis to exclude major proteolytic degrada-tion during the preparation and crystallizationprocess (fig. S5). Thus, we could not see residues21 to 80, probably because of flexibility in thecrystal. LAMTOR1 discontinued at the N and Ctermini, leaving an apparently accessible spaceon the solvent-exposed side of the LAMTOR2and LAMTOR3 heterodimer. The a2 helices ofboth LAMTOR2 and LAMTOR3 are thought tomediate interaction with other proteins (12, 13).The pentameric structure showed that the absentC-terminal helices of LAMTOR4 and LAMTOR5were spatially complemented by helices a4 anda5 of LAMTOR1 (Fig. 1B), closely resembling theinteraction mode observed for Ego2 and Ego1(15) (fig. S4). The structurally resolved region ofLAMTOR1 is largely helical and stabilizes thecomplex by forming a U-shaped belt around thetwo heterodimers, providing additional contactsthat may contribute to increased affinity be-tween the different subunits (Fig. 2). We iden-tified three areas of contact between LAMTOR1and LAMTOR3, LAMTOR4, or LAMTOR5 (Fig. 2,figs. S6 and S7, and supplementary text).

RESEARCH

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1Division of Cell Biology, Biocenter, Medical University ofInnsbruck, 6020 Innsbruck, Austria. 2Division of BiologicalChemistry, Biocenter, Medical University of Innsbruck, 6020Innsbruck, Austria. 3Division of Clinical Biochemistry,Biocenter, Medical University of Innsbruck, 6020 Innsbruck,Austria. 4Division of Neurobiochemistry-Biooptics, Biocenter,Medical University of Innsbruck, 6020 Innsbruck, Austria.5Austrian Drug Screening Institute, 6020 Innsbruck, Austria.*These authors contributed equally to this work. †Present address:Division of Genetic Epidemiology, Medical University of Innsbruck,6020 Innsbruck, Austria. ‡Corresponding author. Email: lukas.a.huber@i-med.ac.at (L.A.H.); klaus.scheffzek@i-med.ac.at (K.S.)

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LAMTOR3LAMTOR1

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(view in fig. S4)LAMTOR1

Fig. 1. The crystal structure of the pentameric LAMTOR complex. (A) The overall structureconsists of two roadblock heterodimers formed by LAMTOR2 (purple) and LAMTOR3 (blue) orLAMTOR4 (orange) and LAMTOR5 (yellow), respectively, surrounded by LAMTOR1 (red). N and Ctermini are labeled. (B) The side view of the complex shows the complementation of helices absentin the roadblock domains of LAMTOR4 and LAMTOR5 by helices of LAMTOR1.

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To investigate the physiological relevance ofthe extended LAMTOR1 conformation in the pen-tameric complex, we generated truncations andalanine mutants to specifically abolish the inter-action to each of the remaining LAMTOR sub-units (Fig. 3A). The choice ofmutated residueswasbased on structurally identified contacts (Fig. 2and fig. S1). For instance, in LAMTOR1_LT2*.SH,we mutated V148 and D149 of LAMTOR1 to ala-

nine. LAMTOR1 variants were tagged with astreptavidin-binding peptide and a hemagglutinin(HA) epitope, hereafter designated as an SH tag.LAMTOR1WT.SH andmutants, as well as SH.GFP(GFP, green fluorescent protein) as a control, wereexpressed in modified human embryonic kidney(HEK) 293 Flp-In T-REx cells (hereafter HEK293)(Fig. 3B). LAMTOR1WT.SHcoimmunoprecipitatedall other LAMTOR proteins, Rags, SLC38A9 (the

lysosomal amino acid transporter), and compo-nents of the BORC (for BLOC1–related complex):namely, Snapin, C10orf32, and C17orf59 (17–21).The majority of mutants neither formed penta-meric complexes nor interactedwith their knownpartners. LAMTOR1_1-147.SH associatedweaklywith the remainingLAMTORsubunits. LAMTOR1_LT2*.SHmutations interfered with complex sta-bility but permitted association with the Rags. Incontrast, LAMTOR1_1-150.SH associatedwith theremaining LAMTORandBORC components butdid not recruit either Rags or SLC38A9. Thus,the belt-like function of LAMTOR1 appears to befundamental for LAMTOR stability, and the Cterminus (residues 150 to 161) of LAMTOR1 isessential for association with the Rags.We then generated a LAMTOR1 hypomorph cell

line (hereafter LAMTOR1HM) (fig. S8 and supple-mentary text) and transiently transfected it withLAMTOR1WT.SH and the same LAMTOR1 mu-tants previously tested in HEK293 cells. They allcolocalized with endogenous LAMP1 (lysosomal-associatedmembraneprotein 1) (fig. S8C), indicatingthat differences observed in the interactome analy-sis were not due tomislocalization. To functionallyaddress the role of the C terminus of LAMTOR1 inanchoring the Rags to the lysosomal surface (3),we established LAMTOR1HM cell lines stably ex-pressing LAMTOR1WT.HA, LAMTOR1_1-106.HA,or LAMTOR1_1-150.HA (fig. S9A). LAMTOR1WT.HArestored the interaction with the Rags and SLC38A9(fig. S9B), whereas LAMTOR1_1-150.HA did not. Inwild-type cells, RagC was recruited to LAMP1 struc-tures (Fig. 3C). LAMTOR1 deletion resulted in im-paired recruitment of RagC that could be restoredby expression of LAMTOR1WT.HA but not byLAMTOR1_1-106.HAorLAMTOR1_1-150.HA(Fig.3C).Complementarily, we observed colocalization ofendogenous RagC with LAMTOR1WT.HA but notwith LAMTOR1_1-106.HA or LAMTOR1_1-150.HA(fig. S9, C and D). Next, we tested the cell lines fortheir signaling properties (Fig. 3D). Control cellsresponded to withdrawal of amino acids with lowphosphorylation of both p70S6K (ribosomal pro-tein S6 kinase beta-1) and downstream S6. Reex-posure of control cells to all essential amino acidsand glutamine readily increased phosphorylationof p70S6K and S6, indicating mTORC1 activation.LAMTOR1HM cells showed impaired p70S6K andS6 phosphorylation when reexposed to aminoacids, and this defect was rescued by expression ofLAMTOR1WT.HAbut not by LAMTOR1_1-150.HA.Although defective in mTORC1 signaling,LAMTOR1_1-150.HA cells contained stable penta-meric LAMTOR complexes (Fig. 3D). Thus, the Cterminus of LAMTOR1 appears to be requiredto recruit the Rags, thereby controlling aminoacid–dependent activation of mTORC1. The 12 C-terminal residues of LAMTOR1 are highly con-served and contain two prominent charged (KEE)and hydrophobic (LVV) clusters (Fig. 3 and fig.S1), which we next mutated to alanine triplets.LAMTOR1_KEE.SH and LAMTOR1_LVV.SH in-teracted with LAMTOR3 and LAMTOR2, butmutation of KEE impaired the association withtheRags, andLVVmutation completely abolishedit (Fig. 3E).

de Araujo et al., Science 358, 377–381 (2017) 20 October 2017 2 of 4

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Fig. 2. Interactions mediated by LAMTOR1 within the LAMTOR complex. Chains are coloredas in Fig. 1 [LAMTOR1 (red), LAMTOR2 (purple), LAMTOR3 (blue), LAMTOR4 (orange), andLAMTOR5 (yellow)]; N and C termini are labeled. Amino acid residues are labeled usingsingle-letter codes [A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N,Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr]. (A) Overview of the pentamericLAMTOR complex showing Ca of LAMTOR1 residues mutated in this study as spheres. Regionsencircled in gray are shown in the close-up views in (C) to (F). (B) Cartoon representation of theLAMTOR complex. Interacting residues (distance cutoff: 3.6 Å) are connected by black lines. Theflexible N- and C-terminal regions of LAMTOR1 are symbolized by red dashed lines. (C to F) Close-upviews of LAMTOR1 interacting with LAMTOR5 (C), LAMTOR4 (D and F), and LAMTOR3 (E). Polarcontacts are indicated by dashed lines.

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The CTD of Gtr1 associates with Ego1 andEgo2 (22), and dimerized CTDs of the Rags arenecessary for LAMTOR1 binding (23). The terminiof the resolved LAMTOR1 portions are in prox-imity to the LAMTOR2 and LAMTOR3 regionsthat were previously thought to be potential ef-fector interaction sites (Fig. 1A) (12). Therefore, wetested whether the Rags might directly bindLAMTOR through their CTDs.Weperformedmassspectrometry after cross-linking to analyze theheptameric LAMTOR-RagA T21N-RagC Q120Lcomplex (supplementary text and figs. S3 and S10).We detected intra- and intermolecular contacts

among the LAMTOR subunits coherent with thecrystal structure. Consistent with a previousmodel(23), we detected cross-linked peptides betweenthe RagA CTD and LAMTOR2 and between theRagC CTD and LAMTOR3 but not between the Gdomains of the Rags and LAMTOR components.As deduced from the crystal structure of het-

erodimeric Gtr1 and Gtr2, the CTDs of the Ragsare predicted to form a stable roadblock hetero-dimer (23, 24) and coimmunoprecipitate LAMTOR1invivo (23). PurifiedRagCTDsandLAMTORelutedas a stable heptameric complex, as confirmed bysize exclusionchromatographyandmass spectrom-

etry (fig. S11). Thus, in vitro, the Rags CTDs weresufficient to interact with the LAMTOR complex.The crystal structure of LAMTOR with Rag

CTDs revealed CTDs binding to the predictedregion of LAMTOR2 and LAMTOR3 with ad-ditional contact surfaces provided by LAMTOR1(Fig. 4, fig. S12, and table S1) (23). In contrast tothe pentameric LAMTOR structure, LAMTOR1residues 47 to 64 form a helix in the heptamericcomplex (Fig. 4 and figs. S13 and S14). We havereconstituted the LAMTOR1HM cell line with aversion of LAMTOR1 in which N64, I66, and V68(hereafter NIV) were mutated to alanines (fig. S1).

de Araujo et al., Science 358, 377–381 (2017) 20 October 2017 3 of 4

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Fig. 3. Requirement of the C terminus of LAMTOR1 for recruitment of Rags to endomembranes and aminoacid–dependent activation of mTORC1. (A) Schematic representation of LAMTOR1 mutants with annotatedpositions at which LAMTOR1 was truncated. Asterisks indicate mutants in which the structurally identified contactsites between LAMTOR1 and each of the remaining LAMTOR proteins were exchanged to alanine. LT, LAMTOR.(B) Expression of SH.GFP, the LAMTOR1.SH wild type, and LAMTOR1.SH mutants was induced by incubating thecorresponding HEK293 cell lines with tetracycline. Streptavidin (STREP) immunoprecipitates were analyzed byimmunoblotting. SH, StrepII-HA-tag; GFP, green fluorescent protein; HA, hemagglutinin. n = 2 independentbiological experiments. (C) Indirect immunofluorescence images of HeLa wild type (WT), LAMTOR1HM, and rescuecell lines kept under normal growth conditions. Merged and single-channel images of endogenous lysosomal markerLAMP1 (red) and endogenous RagC (green) are indicated. Representative cells are shown. Scale bars, 10 mm. (D) HeLawild type, LAMTOR1HM, and rescue cell lines were starved for amino acids for 5 hours and then stimulated withamino acids for 10 min. Obtained lysates were analyzed by immunoblotting. n = 2. (E) Expression of SH.GFP,LAMTOR1.SH wild type, LAMTOR1 truncation mutants, and mutants in which the KEE (residues 151 to 153) or LVV(residues 154 to 156) of LAMTOR1 were mutated to alanines was induced by incubating the corresponding HEK293stable cell lines with tetracycline. STREP immunoprecipitates were analyzed by immunoblotting. n = 2.

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Immunoprecipitated LAMTOR1_ NIV.HA failedto recruit the Rags or SLC38A9 despite restoringthe assembly of the LAMTOR complex (fig. S9B),and colocalization of endogenous RagC withLAMTOR1_NIV.HA (fig. S9C) was abolished.The additional density observed at the C ter-

minus of LAMTOR1 (residues 150 to 158) revealedthat the LVV motif contacts the N terminus ofLAMTOR2 (unstructured in the pentamer crystal)that mediates most of the interactions betweenLAMTOR and the RagA CTD. Residues 157 to 160,adjacent to the LVVmotif of LAMTOR1, come closeto the RagA CTD andmay further contribute to thisinteraction (figs. S12 to S14). Together with thefunctional data and the cross-linking analysis (fig.S10C), where three different pairs of cross-linkedpeptides identified the N terminus of LAMTOR2linked to the C terminus of RagA (K295 andK299),

a second essential region for Rags recruitment inthe LAMTOR was identified.We superimposed the structure of the CTDs of

yeast Gtr1 and Gtr2 to the heptameric complex(fig. S15). With some minor deviations, the CTDsof Rag and Gtr proteins adopted a similar fold.This defined the approximate orientation ofthe G domains of the Rags for interaction withmTORC1 (Fig. 4B). Targeting of Gtr1 and Gtr2to the membrane in the absence of the Ego com-plex is sufficient to promote TORC1 activity (15),indicating that in yeast the Ego complex servesas scaffold for the Gtr proteins. In contrast, mam-malian LAMTOR exhibits GEF activity towardRagA and RagB (1). Extrapolating from our hep-tameric complex, the G domains and associatednucleotide binding sites would be far away fromthe LAMTOR components, raising the possibility

of an unusual or allosteric mechanism of nucleo-tide exchange, if no other cellular componentsare required for GEF activity.Altering the Rag-LAMTOR interaction might

represent a previously unknown mechanism forspecific inhibition of mTORC1 (25). The identifi-cation of two small structural motifs (LVV andNIV) in LAMTOR1, necessary for Rag recruitment,may be used to design compounds interferingwith this interaction.

REFERENCES AND NOTES

1. L. Bar-Peled, L. D. Schweitzer, R. Zoncu, D. M. Sabatini, Cell150, 1196–1208 (2012).

2. D. Teis, W. Wunderlich, L. A. Huber, Dev. Cell 3, 803–814 (2002).3. Y. Sancak et al., Cell 141, 290–303 (2010).4. L. Bar-Peled, D. M. Sabatini, Trends Cell Biol. 24, 400–406 (2014).5. Y. Sancak et al., Science 320, 1496–1501 (2008).6. M. Manifava et al., eLife 5, e19960 (2016).7. N. Taub et al., J. Cell Sci. 125, 2698–2708 (2012).8. D. Teis et al., J. Cell Biol. 175, 861–868 (2006).9. J. M. Scheffler et al., Nat. Commun. 5, 5138 (2014).10. N. Schiefermeier et al., J. Cell Biol. 205, 525–540 (2014).11. Y. Takahashi et al., Biochem. Biophys. Res. Commun. 417,

1151–1157 (2012).12. R. Kurzbauer et al., Proc. Natl. Acad. Sci. U.S.A. 101,

10984–10989 (2004).13. V. V. Lunin et al., J. Biol. Chem. 279, 23422–23430 (2004).14. I. Garcia-Saez, F. B. Lacroix, D. Blot, F. Gabel, D. A. Skoufias,

J. Mol. Biol. 405, 331–340 (2011).15. K. Powis et al., Cell Res. 25, 1043–1059 (2015).16. T. Zhang, M. P. Péli-Gulli, H. Yang, C. De Virgilio, J. Ding,

Structure 20, 2151–2160 (2012).17. J. Pu et al., Dev. Cell 33, 176–188 (2015).18. J. Jung,H.M.Genau,C. Behrends,Mol. Cell. Biol.35, 2479–2494 (2015).19. M. Rebsamen et al., Nature 519, 477–481 (2015).20. S. Wang et al., Science 347, 188–194 (2015).21. R. L. Wolfson, D. M. Sabatini, Cell Metab. 26, 301–309 (2017).22. S. Kira et al., Mol. Biol. Cell 27, 382–396 (2016).23. R. Gong et al., Genes Dev. 25, 1668–1673 (2011).24. J. H. Jeong et al., J. Biol. Chem. 287, 29648–29653 (2012).25. L. D. Schweitzer, W. C. Comb, L. Bar-Peled, D. M. Sabatini, Cell

Reports 12, 1445–1455 (2015).

ACKNOWLEDGMENTS

We thank J. Frankel and E. M. Nelsen for depositing 12G10 anti–a-tubulin to the Developmental Studies Hybridoma Bank. We alsothank M. Gstaiger for providing the pTO-HA-STREPIIc-GW-FRT andpCDNA5/FRT/TO/SH/GW constructs, I. Berger (European MolecularBiology Laboratory Grenoble) for support on the design of thesynthetic LAMTOR gene construct and for the acceptor vector,M. Nanao and D. Sanctis at the European Synchrotron RadiationFacility for support with data acquisition, C. Herrmann for technicalsupport, K. Pansi for technical support and maintenance of theinsect cell culture, and W. Kabsch (Max Planck Institute Heidelberg,Germany) and F. McCormick (University of California, San Francisco)for valuable comments and discussion. The obtained data setsfrom LAMTOR pentamer and LAMTOR with RagA and RagC CTDshave been deposited under Protein Data Bank (PDB) IDs 6EHP and6EHR, respectively. The work presented in this manuscript wassupported by grants from the Austrian Science Funds (FWF): P26682to L.A.H. and P28975 to K.S.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/358/6361/377/suppl/DC1Materials and MethodsSupplementary TextFigs. S1 to S16Tables S1 to S3References (26–60)

6 July 2017; accepted 11 September 2017Published online 21 September 201710.1126/science.aao1583

de Araujo et al., Science 358, 377–381 (2017) 20 October 2017 4 of 4

RagCCTD

RagARagACTD LT5

LT4

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C

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Fig. 4. Interaction of Rag GTPases with the LAMTOR complex. (A) Crystal structure of the heptamer.Dark green, RagA CTD; light green, RagC CTD; red, LAMTOR1; purple, LAMTOR2; blue, LAMTOR3; orange,LAMTOR4; yellow, LAMTOR5. N and C termini are labeled. (B) Rotated view of the structure shown in(A) with the G domains, modeled on the basis of the Gtr1 and Gtr2 structures [PDB: 4ARZ (24)] to definetheir positional orientation in the heptameric complex. Experimental cross-links are indicated by blackdotted lines. A cartoon representation is included (bottom right). The antiparallel arrows between tworoadblock domains indicate the central b-sheet augmentation of roadblock heterodimers.

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signalingCrystal structure of the human lysosomal mTORC1 scaffold complex and its impact on

Klaus ScheffzekLechner, Stefan Welti, Leopold Kremser, Giridhar Shivalingaiah, Martin Offterdinger, Herbert H. Lindner, Lukas A. Huber and Mariana E. G. de Araujo, Andreas Naschberger, Barbara G. Fürnrohr, Taras Stasyk, Theresia Dunzendorfer-Matt, Stefan

originally published online September 21, 2017DOI: 10.1126/science.aao1583 (6361), 377-381.358Science 

, this issue p. 377Sciencearound the other subunits to hold them in place and interacts with the Rag guanosine triphosphatases in the complex.mTORC1 at the lysosomal membrane for activation. The structure and functional studies reveal how LAMTOR1 wraps

report the crystal structure of the LAMTOR (or ''Ragulator'') complex that helps assembleet al.proliferation. de Araujo that coordinates input from growth-factor receptors and nutrient availability with metabolism and cell growth and

The mTORC1 (mechanistic target of rapamycin complex 1) complex garners much attention as a signaling hubStructure of human mTORC1 components

ARTICLE TOOLS http://science.sciencemag.org/content/358/6361/377

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2017/09/20/science.aao1583.DC1

CONTENTRELATED http://stke.sciencemag.org/content/sigtrans/11/559/eaat6903.full

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