DOI: 10.1126/science.1207056, 678 (2011);334 Science

, et al.Roberto Zoncu-ATPase+Mechanism That Requires the Vacuolar H

mTORC1 Senses Lysosomal Amino Acids Through an Inside-Out

This copy is for your personal, non-commercial use only.

clicking here.colleagues, clients, or customers by , you can order high-quality copies for yourIf you wish to distribute this article to others

  here.following the guidelines

can be obtained byPermission to republish or repurpose articles or portions of articles

  ): November 8, 2011 www.sciencemag.org (this infomation is current as of

The following resources related to this article are available online at

http://www.sciencemag.org/content/334/6056/678.full.htmlversion of this article at:

including high-resolution figures, can be found in the onlineUpdated information and services,

http://www.sciencemag.org/content/suppl/2011/11/03/334.6056.678.DC1.html can be found at: Supporting Online Material

http://www.sciencemag.org/content/334/6056/678.full.html#relatedfound at:

can berelated to this article A list of selected additional articles on the Science Web sites

http://www.sciencemag.org/content/334/6056/678.full.html#ref-list-1, 15 of which can be accessed free:cites 25 articlesThis article

http://www.sciencemag.org/content/334/6056/678.full.html#related-urls2 articles hosted by HighWire Press; see:cited by This article has been

http://www.sciencemag.org/cgi/collection/cell_biolCell Biology

subject collections:This article appears in the following

registered trademark of AAAS. is aScience2011 by the American Association for the Advancement of Science; all rights reserved. The title

CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theScience

on

Nov

embe

r 8,

201

1w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

low-level E3 activity toward yUbc12Met. The struc-ture indicated that Asp substitutions for yDcn1P

Leu110 or Leu173 would approach yUbc12Met’s Nterminus to balance the positive charge. Also,an Ala replacement for the Tyr190 “clamp”wouldnot force a charged yUbc12Met’s N terminusdirectly into the hydrophobic pocket. Indeed,the three Dcn1P mutants showed enhanced E3activity specifically toward unacetylated yUbc12(Fig. 4A).

We asked whether a structure-based mutationcould compensate for in vivo defects in cullin ned-dylation resulting from loss of NatC-mediatedyUbc12 acetylation. Thus, we expressed hemag-glutinin (HA)–taggedDcn1 or the Tyr190→Alamutant(we could not express comparable levels of theother mutants in yeast), in strains deleted for Dcn1alone, or bothDcn1 and the NatC subunitMak10.As with the in vitro enzymology and improvedbinding, the Tyr190→Ala mutant rescued thedefect in yCul1~yNedd8 conjugate formationthat resulted from lack of NatC activity (Fig. 4).

We showed N-terminal acetylation of Ubc12to be an avidity enhancer, contributing a criticalinteraction within a highly interconnected ned-dylation complex. As only part of molecular rec-ognitionwithin largemulticomponent complexes,many interactions depending on N-terminal acety-lation likely remain unknown and may be auxil-iary (2, 20, 25). Our study raises the question ofwhether rules dictating N-terminal acetylationdetermined evolution of interactions controllingfunctions ofN-terminally acetylated proteins. Spec-ificity may also involve proximal elements, suchas Ubc12’s N-terminal helix. Because N-acetyl-

methionine can be completely enwrapped in ahydrophobic environment where it would be un-favorable to bury the positive charge masked byacetylation, we propose that N-acetyl-methioninecan serve as a distinctive residue type allowingburial of proteinN-termini into hydrophobic pock-ets of interactingproteins. SuchN-acetyl-methioninebinding sites may serve as targets for small mol-ecules disrupting these critical interactions.

References and Notes1. T. Arnesen, PLoS Biol. 9, e1001074 (2011).2. T. Arnesen et al., Proc. Natl. Acad. Sci. U.S.A. 106, 8157

(2009).3. P. Van Damme et al., PLoS Genet. 7, e1002169

(2011).4. C. H. Yi et al., Cell 146, 607 (2011).5. S. H. Askree et al., Proc. Natl. Acad. Sci. U.S.A. 101,

8658 (2004).6. T. Kanki et al., Mol. Biol. Cell 20, 4730 (2009).7. S. J. Dixon et al., Proc. Natl. Acad. Sci. U.S.A. 105,

16653 (2008).8. R. Behnia, B. Panic, J. R. Whyte, S. Munro, Nat. Cell Biol.

6, 405 (2004).9. S. R. Setty, T. I. Strochlic, A. H. Tong, C. Boone, C. G. Burd,

Nat. Cell Biol. 6, 414 (2004).10. C. S. Hwang, A. Shemorry, A. Varshavsky, Science 327,

973 (2010).11. Y. Ye, M. Rape, Nat. Rev. Mol. Cell Biol. 10, 755

(2009).12. D. T. Huang et al., Nat. Struct. Mol. Biol. 11, 927

(2004).13. D. T. Huang et al., Nature 445, 394 (2007).14. T. Kurz et al., Mol. Cell 29, 23 (2008).15. A. Y. Kim et al., J. Biol. Chem. 283, 33211

(2008).16. D. C. Scott et al., Mol. Cell 39, 784 (2010).17. T. Kurz et al., Nature 435, 1257 (2005).18. G. Huang, A. J. Kaufman, Y. Ramanathan, B. Singh,

J. Biol. Chem. 286, 10297 (2011).

19. I. Sarkaria et al., Cancer Res. 66, 9437 (2006).20. B. Polevoda, F. Sherman, J. Mol. Biol. 325, 595

(2003).21. B. Polevoda, F. Sherman, J. Biol. Chem. 276, 20154

(2001).22. D. Lammer et al., Genes Dev. 12, 914 (1998).23. X. Yang et al., J. Biol. Chem. 282, 24490

(2007).24. G. H. Bird, F. Bernal, K. Pitter, L. D. Walensky, Methods

Enzymol. 446, 369 (2008).25. B. Polevoda, F. Sherman, Biochem. Biophys. Res.

Commun. 308, 1 (2003).Acknowledgments: This was supported by American

Lebanese Syrian Associated Charities–St. Jude, NIH,and Howard Hughes Medical Institute (B.A.S.), grantsfrom NIH and Millennium Pharmaceuticals ( J.W.H.),and Damon Runyon Cancer Research Foundation (E.J.B.).We thank S. Gygi, I. Kurinov, C. Ralston, R. Cassell,P. Rodrigues, K. Kodali, V. Pagala, R. Schekman,D. W. Miller, S. Bozeman, D. J. Miller, J. Bollinger, andC. Rock for assistance, reagents, and/or discussions.D.C.S., J.K.M., B.A.S., and St. Jude Children’s ResearchHospital have applied for a patent on uses of Ubc12N-terminal acetylation for inhibiting neddylation.Research Collaboratory for Structural Bioinformaticsstructural accession codes: 3TDI, 3TDU, 3TDZ. Authorcontributions: D.C.S., J.K.M., and E.J.B. designed,performed, and analyzed experiments; D.C.S. andB.A.S. wrote the manuscript, with all authorscontributing; J.W.H. and B.A.S. advised and assistedon all aspects.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/science.1209307/DC1Materials and MethodsFigs. S1 to S10Table S1References (26–44)

3 June 2011; accepted 9 September 2011Published online 22 September 2011;10.1126/science.1209307

mTORC1 Senses Lysosomal Amino AcidsThrough an Inside-Out MechanismThat Requires the Vacuolar H+-ATPaseRoberto Zoncu,1,2,3,4 Liron Bar-Peled,1,2,3 Alejo Efeyan,1,2,3 Shuyu Wang,1,2,3

Yasemin Sancak,1,2,3 David M. Sabatini1,2,3,4,5*

The mTOR complex 1 (mTORC1) protein kinase is a master growth regulator that is stimulatedby amino acids. Amino acids activate the Rag guanosine triphosphatases (GTPases), whichpromote the translocation of mTORC1 to the lysosomal surface, the site of mTORC1 activation.We found that the vacuolar H+–adenosine triphosphatase ATPase (v-ATPase) is necessary foramino acids to activate mTORC1. The v-ATPase engages in extensive amino acid–sensitiveinteractions with the Ragulator, a scaffolding complex that anchors the Rag GTPases to thelysosome. In a cell-free system, ATP hydrolysis by the v-ATPase was necessary for amino acidsto regulate the v-ATPase-Ragulator interaction and promote mTORC1 translocation. Results obtainedin vitro and in human cells suggest that amino acid signaling begins within the lysosomallumen. These results identify the v-ATPase as a component of the mTOR pathway and delineatea lysosome-associated machinery for amino acid sensing.

Amino acids are the building blocks ofproteins and intermediates in lipid andadenosine triphosphate (ATP) synthesis.

They also initiate a signaling cascade that leads toactivation of the master growth regulator mTORcomplex 1 (mTORC1). This multicomponent pro-

tein kinase integrates inputs from growth fac-tors as well as nutrient and energy supplies tocontrol many biosynthetic and catabolic pro-cesses (1). Most signals upstream of mTORC1converge on TSC1-TSC2, a heterodimeric tumorsuppressor that negatively regulates the Rhebguanosine triphosphatase (GTPase), which is anessential activator of mTORC1 protein kinaseactivity (2, 3). In contrast, amino acids signal tomTORC1 by promoting its binding to a distinctfamily of GTPases, the Rag GTPases (4, 5). TheRags form heterodimers consisting of RagA orRagB, which are highly similar to each other,bound to RagC or RagD, which are also highlyrelated. In an amino acid–sensitive fashion, theRag GTPases recruit mTORC1 to the surface oflysosomes, which also contain Rheb (5). The

1Whitehead Institute for Biomedical Research, Nine CambridgeCenter, Cambridge, MA 02142, USA. 2Department of Biology,Massachusetts Institute of Technology (MIT), Cambridge, MA02139, USA. 3David H. Koch Institute for Integrative CancerResearch at MIT, 77 Massachusetts Avenue, Cambridge, MA02139, USA. 4Broad Institute, Seven Cambridge Center, Cam-bridge, MA 02142, USA. 5Howard Hughes Medical Institute,MIT, Cambridge, MA 02139, USA.

*To whom correspondence should be addressed. E-mail:sabatini@wi.mit.edu

4 NOVEMBER 2011 VOL 334 SCIENCE www.sciencemag.org678

REPORTS

on

Nov

embe

r 8,

201

1w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

trimeric Ragulator complex, which comprisesthe p18, p14, and MP1 proteins, anchors the RagGTPases to the lysosome and, like the Rags, isnecessary for mTORC1 activation by amino acids(6). Together, the Ragulator and Rag heterodimerform an amino acid–regulated docking site formTORC1 on the lysosomal surface.

Amino acid signaling has been proposed tobegin, alternatively, at the plasma membrane orinside the cell, but this key issue remains unsettled(7–10). The localization of the Rag GTPases onlysosomes, but not on other Rheb-containing endo-membranes, suggests that this organelle has animportant role in amino acid signaling tomTORC1.To determine whether lysosome-associated pro-cesses and proteins participate in the activationof mTORC1 by amino acids, we used RNA in-terference (RNAi) inDrosophila S2 cells to reducethe expression of a number of genes with rolesin lysosomal biogenesis and function (table S1).Double-strandedRNAs (dsRNAs) targetingmostof the genes did not affect the amino acid–inducedphosphorylation of the ribosomal protein S6 ki-nase (dS6K) on T398, a readout of dTORC1 ac-tivity (table S1). In contrast, dsRNAs targetingvhaAC39, vha16, vha100-1, and vha100-2, all en-coding components of the vacuolar H+-ATPase(v-ATPase), suppressed dS6K phosphorylationto degrees similar to that of a dsRNA targeting

dRagC (table S1, Fig. 1, A and B, and fig. S1A).The dsRNAs to vhaAC39 also decreased the sizeof S2 cells (Fig. 1C). Consistent with the resultsinDrosophila cells, lentiviral short-hairpin RNAs(shRNAs) targeting human ATP6V0c, the ortho-log ofDrosophila vha16, suppressed amino acid–induced phosphorylation of S6K1 in humanembryonic kidney (HEK) 293Tcells (Fig. 1D andfig. S1B). These results implicate the v-ATPasein the activation of mTORC1 by amino acids.

The v-ATPase consists of multicomponentV0 and V1 domains and operates through an in-completely understoodmechanism inwhich eachcycle of ATP hydrolysis by the V1 sector gener-ates torque that rotates the membrane domain ofV0, known as the rotor. In turn, this movement en-ables the transfer of protons into the lysosomal lu-men, causing its acidification (11). The macrolidesconcanamycin A (ConA) and salicylihalamide A(SalA) are structurally diverse inhibitors of thev-ATPase that do not have other known targets(11–14). In 293T cells, both ConA and SalA in-hibited amino acid–induced phosphorylationof S6K1 in a concentration-dependent manner(Fig. 1, E and F). The inhibition of S6K1 phos-phorylation occurred after short (15- to 60-min)treatment times (fig. S2A) and without con-comitant alterations in lysosomal morphology(fig. S2, B and C) or inhibition of Akt phos-

phorylation, a readout of growth factor signaling(Fig. 1, E and F).

The finding that the v-ATPase and its activityare necessary for mTORC1 activation by aminoacids led us to consider potential roles for it inthe pathway. One possibility is that the v-ATPasefunctions downstream of amino acids and is partof the amino acid–induced signaling pathwaythat culminates in mTORC1 activation. Anotherconceivable function is that the proton gradientgenerated by the v-ATPase is required for aminoacids to be transported into the cellular compart-ment where an amino acid sensor is located. Tobypass the transport function, we tested whetherthe v-ATPase is required for alcohol ester deriv-atives of amino acids to activate mTORC1. Theseesters freely diffuse across membranes and, with-in the cytoplasm and lysosomes, are hydrolyzedby esterases into native amino acids (15). A mix-ture of amino acid esters or leucine methyl esteractivated mTORC1 with efficiencies comparableto those of their respective native amino acids(fig. S1, C andD).Moreover, ConA also inhibitedthe S6K1 phosphorylation induced by amino acidesters (Fig. 1G and fig. S1E). Consistent withthese findings, SalA also suppressed mTORC1activation induced by cycloheximide, which, byinhibiting translation, boosts concentrations ofintracellular amino acids (7, 16) (Fig. 1H). Thus,

Fig. 1. Requirement of the v-ATPase formTORC1 activation by amino acids. (A)dsRNA-mediated depletion of vhaAC39 in Drosophila S2 cells. Cells weredeprived for amino acids for 1.5 hours and then stimulated with completemedium for 30 min. Proteins from cell lysates were analyzed for phosphoryl-ation of dS6K at threonine 398 (T398). Depletion of vhaAC39 by two distinctdsRNAs is compared to that of dRagC. (B) dsRNA-mediated depletion of bothvha100-1 and vha100-2 in S2 cells suppresses amino acid–induced T398 phos-phorylation of dS6K. (C) Cell size measurement after depletion of vhaAC39 inS2 cells with two dsRNAs (red and blue) compared to a control dsRNA (black). (D)S6K1 phosphorylation at T389 in HEK-293T cells treated with shRNA targetingGFP, RagC and RagD, and V0c. Cells were deprived of amino acids for 50 minand, where indicated, stimulated for 10 min. Immunoblotting was used to

detect the indicated proteins. (E) S6K1 phosphorylation in HEK-293T cellsdeprived of amino acids for 50 min in the presence of the indicated concen-trations of ConA and then stimulated for 10 min with amino acids. (F) S6K1phosphorylation in HEK-293T cells deprived of amino acids for 50 min in thepresence of the indicated concentrations of SalA and restimulated for 10 minwith amino acids. (G) ConA blocks mTORC1 activation by alcohol esters of aminoacids. HEK-293T cells were deprived of amino acids for 50 min and thenstimulated for 10 min with amino acids or alcohol esters of amino acids in thepresence of 2 mM ConA where indicated. (H) Activation of mTORC1 by intra-cellular amino acids. HEK-293T cells were deprived of amino acids for 50 minand stimulated with amino acids or cycloheximide in dimethyl sulfoxide (DMSO)or 2 mM SalA. Immunoblotting was used to detect the indicated proteins.

www.sciencemag.org SCIENCE VOL 334 4 NOVEMBER 2011 679

REPORTS

on

Nov

embe

r 8,

201

1w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

these results are consistent with the v-ATPasehaving a role downstream of intracellular aminoacids in the initiation or propagation of the aminoacid–induced signal to mTORC1.

A key event in amino acid signaling is theRagGTPase–mediated translocation ofmTORC1to the surface of lysosomes (5, 6). In cells treatedwith ConA or SalA or depleted of ATP6V0c,mTOR failed to cluster onto lysosomes in re-sponse to amino acids and instead was foundin a diffuse staining pattern (Fig. 2, A and B,and fig. S3, A to C). Unlike the Ragulator (6), thev-ATPase appears not to be necessary for an-choring the Rag GTPases to the lysosomal sur-face, because pharmacological or RNAi-mediatedinhibition of the v-ATPase did not affect lysosomallocalization of RagC (Fig. 2C and fig. S3, C andD). To test whether the v-ATPase might functionupstream of the Rag GTPases, we used a RagBmutant that is constitutively active (RagBGTP)and renders mTORC1 signaling insensitive toamino acid starvation (4, 5). If the v-ATPase isrequired for the activation of the Rag GTPases,

expression of RagBGTP should rescue the de-fects in mTOR lysosomal recruitment and S6K1phosphorylation caused by ConA and SalA.Indeed, in cells stably expressing RagBGTP, thelysosomal localization of mTOR and the phos-phorylation of S6K1 were insensitive not onlyto amino acid starvation but also to ConA andSalA treatment (Fig. 2, D and E, and fig. S3F).Consistent with these results, in knockin mouseembryonic fibroblasts (MEFs) that express ac-tive RagAGTP from the endogenous RagA locus,SalA (Fig. 2F) and ConA (fig. S3G) did notblock the constitutive S6K1 phosphorylationcaused byRagAGTP.Collectively, these results placethe v-ATPase downstream of amino acids but up-stream of the activation of the Rag GTPases; theyalso exclude its involvement in other regulatory inputsto mTORC1, such as controlling Rheb activity (6).

We tested whether a physical interaction ex-ists between the v-ATPase and the Rags or Ragu-lator or both. Semi-quantitativemass spectrometricanalyses of anti-FLAG immunoprecipitates pre-pared from 293T cells expressing FLAG-tagged

Ragulator components (p18 or p14) or RagBrevealed the presence of many subunits of thev-ATPase (Fig. 3A). Immunoblot assays with an-tibodies to endogenous V0 (c and d1) and V1 (A,B2, and D) subunits confirmed that Ragulatorcoimmunoprecipitateswith theV0 andV1domains(Fig. 3, B and C), whereas the Rags coimmuno-precipitate withV1 subunits only (fig S4, A andB).Although easily detected in immunoblot assays ascoimmunoprecipitating with Ragulator, the c sub-unit of V0 was not detected by mass spectrom-etry, probably because of its highly hydrophobicnature. The v-ATPase did not coimmunopre-cipitate with lysosomal (LAMP1) or cytoplasmic(Metap2) control proteins (Fig. 3, B andC). In vitroassays with purified recombinant proteins verifieda direct interaction between the V0 component d1and p18, but not p14, and between the V1 com-ponent Dwith p18 and, to a lesser degree, with p14(Fig. 3D). No direct interactions were detectedbetween the Rag GTPases and purified v-ATPasesubunits (fig. S4C), which is consistent with therelatively low abundance of v-ATPase subunits

Fig. 2. Requirement of the v-ATPase for lysosomal recruit-ment ofmTORC1 by the RagGTPases. (A) Immunofluorescenceimages of mTOR and LAMP2 in HEK-293T cells deprived ofamino acids (a.a.) (top) or deprived and then stimulated(bottom) in the presence of DMSO (left) or 2.5 mMSalA (right).(B) HEK-293T cells expressing a lentivirally encoded shRNAtargeting GFP (left) or V0c (right) were deprived of aminoacids (top) or deprived and then stimulated (bottom). (C)Staining for RagC and LAMP2 in HEK-293T cells deprivedof amino acids (top) or deprived and then stimulated(bottom) in the presence of DMSO (left) or 2.5 mM SalA(right). (D) HEK-293T cells stably expressing the constitu-tively active RagBQ99L mutant (293T RagBGTP) were deprived of amino acids(top) or deprived and stimulated (bottom) in the presence of DMSO (left) or 2.5mM ConA (right). (E) S6K1 phosphorylation in HEK-293T cells and HEK-293TRagBGTP cells deprived of amino acids for 50min in the presence of DMSO or2 mM SalA and stimulated for 10 min with amino acids. (F) S6K1 phos-

phorylation in wild-type MEFs (RagA+/+) or in MEFs homozygous for the con-stitutive active RagA Q66L mutant (RagAGTP/GTP); cells were deprived of aminoacids for 50 min in the presence of DMSO or 2.5 mM SalA and stimulated for10 min with amino acids. In all images, insets show selected fields that weremagnified five times and their overlays. Scale bars represent 10 mm.

4 NOVEMBER 2011 VOL 334 SCIENCE www.sciencemag.org680

REPORTS

on

Nov

embe

r 8,

201

1w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

in the RagB immunoprecipitates analyzed bymass spectrometry (Fig. 3A). Thus, in addition toscaffolding the Rag GTPases to the lysosomal sur-face, Ragulator provides a physical and functionallink between the v-ATPase and the Rag GTPases.

Consistent with amino acids acting upstreamof the v-ATPase, amino acids regulated the inter-action between the V1 domain of v-ATPase andRagulator and Rag GTPases. Amino acid starva-tion and stimulation strengthened and weakened,

respectively, the interaction (Fig. 3E and fig. S4D).In contrast, amino acids did not affect the bind-ing of Ragulator with the V0 domain of thev-ATPase (Fig. 3E) or of the V1 and V0 subunitswith each other [as does glucose starvation (17)]

Fig. 3. Interaction of the v-ATPase with theRagulator-Rag GTPases. (A) Cartoon summarizingmass spectrometry analyses of immunoprecipi-tates from HEK-293T cells expressing FLAG-p18(left), FLAG-p14 (center), and FLAG-RagB (right).v-ATPase subunits are color-coded according totheir peptide representation (scale at the farright). (B) Binding of Ragulator to the V0 do-main. HEK-293T cells stably expressing FLAG-tagged p18 and p14 were lysed and subjectedto FLAG immunoprecipitation (IP) followed by

immunoblotting for V0c and V0d1. FLAG-LAMP1 and FLAG-Metap2 served as negative controls. (C) Binding of Ragulator to the V1 domain. HEK-293T cells stablyexpressing FLAG-tagged p18, p14, LAMP1, and Metap2 were lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for V1A,V1B2, and V1D. (D) (Top) In vitro binding assays in which purified FLAG-p18 and FLAG-p14 were incubated with recombinant V0d1 fused to glutathioneS-transferase (HA-GST-V0d1), immobilized on glutathione agarose beads. Samples were subjected to immunoblotting for FLAG to detect bound Ragulatorcomponents. HA-GST-Rap2A served as a negative control. (Bottom) In vitro binding assays in which purified FLAG-p18 and FLAG-p14 were incubated withrecombinant V1D fused to glutathione S-transferase (HA-GST-V1D). HA-GST-metap2 served as a negative control. (E) The Ragulator-V1 interaction, but notthe Ragulator-V0 interaction, is regulated by amino acids. HEK-293T cells stably expressing FLAG-tagged p18, p14, and Metap2 were deprived of amino acidsfor 90 min or deprived and then stimulated with amino acids for 15 min. After lysis, samples were subjected to FLAG immunoprecipitation and immunoblottingfor the indicated v-ATPase subunits. (F) SalA blocks regulation of the Ragulator-V1 interaction by amino acids. HEK-293T cells stably expressing FLAG-p14 weredeprived of amino acids for 90 min, or deprived and then stimulated with amino acids for 15 min, in the presence of DMSO or 2 mM SalA. Samples were lysed,FLAG-immunoprecipitated, and immunoblotted for the indicated proteins. (G) Cartoon summarizing the Ragulator–v-ATPase interactions identified in (A) to (F).Orange denotes regulation by amino acids; blue indicates lack of regulation.

www.sciencemag.org SCIENCE VOL 334 4 NOVEMBER 2011 681

REPORTS

on

Nov

embe

r 8,

201

1w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

(fig. S4D). Treatment of cells with SalA, likeamino acid deprivation, strengthened the interac-tion between Ragulator and V1 domain subunitsand, moreover, largely prevented amino acidsfrom weakening the interaction (Fig. 3F andfig. S4E). Thus, amino acids induce a struc-tural rearrangement of the v-ATPase–Ragulator–Rag GTPase complex that is blocked by SalA(Fig. 3G).

The v-ATPase is large and complex and hasmany functions that cannot easily be teased apartin live cells (11, 18–22). Thus, to better understandits role in amino acid signaling to mTORC1, wedeveloped a cell-free system that recapitulates theamino acid–induced binding of mTORC1 to theRagGTPases on the lysosomal surface [seemeth-ods in the supporting online material (SOM)].We prepared a light organelle fraction from293T cells expressing FLAG-RagB that hadbeen deprived of amino acids. The organelleswere briefly stimulated with amino acids or ami-

no acid esters and then incubated with cytosolcontaining Myc-tagged Raptor (Fig. 4A andfig. S5A). In this system, amino acids, and es-pecially amino acid esters, increased binding ofMyc-Raptor to FLAG-RagB–containing vesiclesbut not to control vesicles (Fig. 4A and fig. S5B).As expected, in preparations containing the FLAG-RagBGTP mutant, the binding of Myc-Raptor wasconstitutively high and largely insensitive to ami-no acids (fig. S5C and S5D). We also preparedpurified lysosomes by immunoisolating themfrom 293T cells (see methods in SOM) (fig. S6,A to C). Again, amino acid esters induced bind-ing of Myc-tagged Raptor to isolated lysosomes(fig. S6D). Cytosol appeared to be dispensable,because highly purified, FLAG-tagged Raptorshowed amino acid–induced binding to organ-elles containing GST-tagged Rag heterodimers(fig. S6E). Thus, lysosomes contain all the ma-chinery required for sensing amino acids and ac-tivating the Rag GTPases.

In the in vitro system, the alcohol esters ofamino acids were more effective than native ami-no acids in inducing the RagB-Raptor interaction(Fig. 4A). A possible reason for this is that aminoacids must enter and accumulate in lysosomes toinitiate signaling, and that the amino acid esters doso more easily in the in vitro preparation (15). Totest the requirement for an intralysosomal accu-mulation, we used treatments that permeabilizethe lysosomal membrane and thus allow aminoacids to leak out. Treatment of the organelle pre-paration with Streptolysin O, which introducesnanometer-sized holes into the lysosomal mem-brane, or Triton X-100, which dissolves themembranes without disrupting the v-ATPase–Ragulator–Rag interaction, completely suppressedthe effect of amino acids or their esters to pro-mote the binding of Raptor to RagB (Fig. 4B). ThePAT1 (SLC36A1) transporter is a proton-coupledamino acid transporter that localizes specificallyto lysosomes (fig. S7A) and exports amino acids

cytosol input:DMSO:FCCP:

AMP-PNP (1mM):AMP-PNP (10mM):amino acid esters:

Fig. 4. In vitro analysis of mTORC1 activation by amino acids. (A) Cell-freebinding of Myc-Raptor to FLAG-RagB– but not to FLAG-Rap2A–containingvesicles. Organelle preparations were left unstimulated or were stimulatedwith amino acids or amino acid esters and incubated with Myc-Raptor–containingcytosol. After FLAG immunoprecipitation, bound Myc-Raptor was detected byimmunoblotting. (B) Intact FLAG-RagB lysosomes, FLAG-RagB lysosomes per-meabilized with streptolysin O, and FLAG-RagB lysosomes permeabilized withTriton X-100 were left unstimulated, stimulated with amino acids, or stimulatedwith amino acid esters. Myc-Raptor was detected by immunoblotting. (C) S6K1phosphorylation at T389 in HEK-293T cells transiently expressing FLAG-S6K1,FLAG-S6K1 + Myc-PAT1, FLAG S6K1 + HAGST-tagged active Rag mutants, orFLAG-S6K1 + Myc-PAT1 + HAGST-active Rags. Cells were deprived of aminoacids for 50min or starved and then stimulated for 10min (seemethods in SOM).The indicated proteins were detected by immunoblotting. The band pattern ofMyc-PAT1 is probably due to glycosylation. (Right) Immunofluorescence imagesof lysosomes from HEK-293T cells transiently expressing Myc-PAT1 and stainedfor Myc tag (top, red in the merge) and for LAMP2 (center, green in the merge).

(D) Accumulation of 14C-labeled amino acids into lysosomes immunopurifiedfrom HEK-293T cells expressing LAMP1-mRFP-FLAGX2. Lysosomes were eitherleft intact or permeabilized with Triton X-100 or streptolysin O before mea-surement. Overexpression of PAT1 largely abolished amino acid accumulationinside lysosomes. Each value represents the mean T SD of three independentsamples. (E) FLAG-RagB lysosomes were treated with DMSO or 2 mM SalA,activated with amino acid esters, and then incubated with Myc-Raptor. Anorganellar fraction from FLAG-metap2–expressing cells served as a negativecontrol. (F) FLAG-RagB lysosomes were stimulated with amino acid esters inthe presence of the proton ionophore FCCP or the nonhydrolyzable ATP analogAMP-PNP at 1 mM or 10 mM. Organelle samples were then incubated withMyc-Raptor cytosol, followed by FLAG immunoprecipitation and immuno-blotting for Myc-Raptor and endogenous mTOR. (G) Model for inside-outactivation of mTORC1 by lysosomal amino acids. The accumulation of aminoacids inside the lysosomal lumen generates an activating signal that istransmitted to the Rag GTPases via the v-ATPase–Ragulator interaction. Inturn, the Rags physically recruit mTORC1 to the lysosomal surface.

4 NOVEMBER 2011 VOL 334 SCIENCE www.sciencemag.org682

REPORTS

on

Nov

embe

r 8,

201

1w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

out of the lysosomal lumen (23). In intact cells,overexpression of PAT1 completely suppressedmTORC1 activation by amino acids, and thiseffect was fully rescued by coexpression of con-stitutively active RagBGTP (Fig. 4C). In contrast,overexpression of PAT4 (SLC36A4), an aminoacid transporter that does not localize to the ly-sosome, had no effect on mTORC1 activationby amino acids (fig. S7, B and C).

These results strongly suggest that aminoacid signaling begins inside the lysosome. Con-sistent with this possibility, stimulation of aminoacid–starved 293Tcells with 14C-amino acids ledto the rapid appearance of labeled amino acidswithin lysosomes immunoisolated through aFLAG-tagged lysosomal protein (Fig. 4D andfig. S6, A and B). Amino acid accumulation wasreverted by lysosome permeabilization and large-ly prevented by PAT1 overexpression (Fig. 4D).

In the in vitro system, disruption of thev-ATPase by SalA or by shRNA against V0cblocked the amino acid–induced interaction ofRaptor with RagB (Fig. 4E and fig. S5E). SalAcauses structural rearrangements in the v-ATPasethat inhibit both its capacity to hydrolyze ATPand to establish the lysosomal proton gradient(13, 24). Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), an ionophore that dis-sipates the lysosomal proton gradient withoutinterfering with the v-ATPase (25), did not pre-vent the amino acid–induced binding of Raptorto RagB (Fig. 4F). Moreover, amino acids or theiresters did not alter the lysosomal pH of intactcells, nor did they perturb lysosomal acid-ification in vitro (fig. S8, A to C). In contrast,the nonhydrolyzable ATP analog adenosine

5′-(b,g-imido)triphosphate (AMP-PNP), which in-hibits theATPase activity ofV1 and the consequentrotation of the stalk and the V0 proteolipid sub-units, blocked the amino acid–induced interactionbetween Raptor and RagB in a concentration-dependent manner (Fig. 4F and fig. S8D). Thus,ATP hydrolysis and the associated rotation ofthe v-ATPase appear to be essential to relay anamino acid signal from the lysosomal lumen tothe Rag GTPases, whereas the capacity of thev-ATPase to set up the lysosomal proton gradientis dispensable in the in vitro system.

We propose a lysosome-centric inside-outmodel of amino acid sensing by mTORC1 inwhich amino acids must accumulate in the lyso-somal lumen to initiate signaling (Fig. 4G). Thev-ATPase is required for amino acid signalingto mTORC1 and functions between amino acidsand the nucleotide loading of the Rag GTPases.Its placement in the pathway and its amino acid–sensitive interactions with the Rag-Ragulatorcomplex implicate it as an essential componentof the amino acid sensing mechanism.

References and Notes1. R. Zoncu, A. Efeyan, D. M. Sabatini, Nat. Rev. Mol. Cell

Biol. 12, 21 (2011).2. K. Inoki, Y. Li, T. Xu, K. L. Guan, Genes Dev. 17, 1829

(2003).3. A. R. Tee, B. D. Manning, P. P. Roux, L. C. Cantley,

J. Blenis, Curr. Biol. 13, 1259 (2003).4. E. Kim, P. Goraksha-Hicks, L. Li, T. P. Neufeld, K. L. Guan,

Nat. Cell Biol. 10, 935 (2008).5. Y. Sancak et al., Science 320, 1496 (2008).6. Y. Sancak et al., Cell 141, 290 (2010).7. A. Beugnet, A. R. Tee, P. M. Taylor, C. G. Proud, Biochem.

J. 372, 555 (2003).8. J. Bohé, A. Low, R. R. Wolfe, M. J. Rennie, J. Physiol. 552,

315 (2003).

9. G. R. Christie, E. Hajduch, H. S. Hundal, C. G. Proud,P. M. Taylor, J. Biol. Chem. 277, 9952 (2002).

10. B. Wu et al., J. Cell Biol. 173, 327 (2006).11. M. Forgac, Nat. Rev. Mol. Cell Biol. 8, 917 (2007).12. B. J. Bowman, M. E. McCall, R. Baertsch, E. J. Bowman,

J. Biol. Chem. 281, 31885 (2006).13. M. R. Boyd et al., J. Pharmacol. Exp. Ther. 297, 114

(2001).14. M. Huss et al., J. Biol. Chem. 277, 40544 (2002).15. J. P. Reeves, J. Biol. Chem. 254, 8914 (1979).16. D. J. Price, R. A. Nemenoff, J. Avruch, J. Biol. Chem. 264,

13825 (1989).17. S. Bond, M. Forgac, J. Biol. Chem. 283, 36513 (2008).18. C. M. Cruciat et al., Science 327, 459 (2010).19. P. R. Hiesinger et al., Cell 121, 607 (2005).20. A. Hurtado-Lorenzo et al., Nat. Cell Biol. 8, 124 (2006).21. C. Peters et al., Nature 409, 581 (2001).22. Y. Yan, N. Denef, T. Schüpbach, Dev. Cell 17, 387 (2009).23. C. Sagné et al., Proc. Natl. Acad. Sci. U.S.A. 98, 7206 (2001).24. X. S. Xie et al., J. Biol. Chem. 279, 19755 (2004).25. B. E. Steinberg et al., J. Cell Biol. 189, 1171 (2010).Acknowledgments: We thank members of the Sabatini

Lab as well as R. Perera for helpful suggestions,E. Spooner for the mass spectrometric analysis, andJ. De Brabander (University of Texas Southwestern)for salicylihalamide A. Supported by grants from theNational Institutes of Health (CA103866 and AI47389)and Department of Defense (W81XWH-07-0448) toD.M.S., awards from the W.M. Keck Foundation andLAM Foundation to D.M.S., and fellowship support fromthe Jane Coffin Childs Memorial Fund for MedicalResearch and the LAM Foundation to R.Z., from theHuman Frontier Science Program to A.E., and from theMedical Scientist Training Program to S.W. D.M.S. is aninvestigator of the Howard Hughes Medical Institute.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/334/6056/678/DC1Materials and MethodsFigs. S1 to S8Table S1References

15 April 2011; accepted 13 September 201110.1126/science.1207056

RNAP II CTD Phosphorylated onThreonine-4 Is Required for HistonemRNA 3′ End ProcessingJing-Ping Hsin, Amit Sheth, James L. Manley*

The RNA polymerase II (RNAP II) largest subunit contains a C-terminal domain (CTD) with up to52 Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 consensus repeats. Serines 2, 5, and 7 are known to bephosphorylated, and these modifications help to orchestrate the interplay between transcriptionand processing of messenger RNA (mRNA) precursors. Here, we provide evidence that phosphorylationof CTD Thr4 residues is required specifically for histone mRNA 3′ end processing, functioning tofacilitate recruitment of 3′ processing factors to histone genes. Like Ser2, Thr4 phosphorylationrequires the CTD kinase CDK9 and is evolutionarily conserved from yeast to human. Our datathus illustrate how a CTD modification can play a highly specific role in facilitating efficientgene expression.

The carboxyl-terminal domain (CTD) ofthe RNA polymerase II (RNAP II) largestsubunit (Rpb1) consists of Tyr1-Ser2-Pro3-

Thr4-Ser5-Pro6-Ser7 (YSPTSPS) consensus tan-dem repeats, which are conserved from yeast tohuman. The CTD, through phosphorylation onserine residues, links transcription to mRNA pro-

cessing events (1–3). Ser5 is phosphorylated bycyclin-dependent kinase 7, CDK7, a subunit ofthe general transcription factor TFIIH, and thismodification functions to facilitate capping (4, 5).During transcriptional elongation, Ser2 is phos-phorylated by CDK9/P-TEFb, which helps to co-ordinate RNA3′ end processing and transcription

termination (6, 7). Ser7 phosphorylation has beenimplicated in transcription and 3′ end processingof genes encoding certain small noncoding RNAs(8, 9). Tyr1 can also be phosphorylated by thec-Abl kinase (10). Although Thr4 has been re-ported to be phosphorylated in fission yeast (11),there is no evidence that this residue is modifiedin other species or what might the function ofThr4 be.

To investigate CTD function in a genetical-ly tractable vertebrate cell system, we createdan Rpb1 conditional knockout chicken cell line,DT40-Rpb1, by using methods developed previ-ously to study other conserved proteins (12, 13)(fig. S1). These cells express, as the only sourceof Rpb1, a tetracycline (tet)-repressible cDNA en-coding hemagglutinin (HA)–tagged human Rpb1(human and chicken Rpb1 are 97% identical, andthe CTD is very highly conserved among ver-tebrates; fig. S2). After addition of tet, Rpb1 be-came undetectable between 12 and 18 hours(Fig. 1Aand fig. S3), andDT40-Rpb1 cells stopped

Department of Biological Sciences, Columbia University, NewYork, NY 10027, USA.

*To whom correspondence should be addressed. E-mail:jlm2@columbia.edu

www.sciencemag.org SCIENCE VOL 334 4 NOVEMBER 2011 683

REPORTS

on

Nov

embe

r 8,

201

1w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from