METABOLISM

Lysosomal metabolomics revealsV-ATPase- and mTOR-dependentregulation of amino acid effluxfrom lysosomesMonther Abu-Remaileh,1,2,3,4* Gregory A. Wyant,1,2,3,4* Choah Kim,1,2,3,4

Nouf N. Laqtom,1,2,3,4 Maria Abbasi,1,2,3,4 Sze Ham Chan,1

Elizaveta Freinkman,1† David M. Sabatini1,2,3,4‡

The lysosome degrades and recycles macromolecules, signals to the cytosol and nucleus,and is implicated in many diseases. Here, we describe a method for the rapid isolation ofmammalian lysosomes and use it to quantitatively profile lysosomal metabolites under variouscell states. Under nutrient-replete conditions, many lysosomal amino acids are in rapidexchange with those in the cytosol. Loss of lysosomal acidification through inhibition of thevacuolar H+–adenosine triphosphatase (V-ATPase) increased the luminal concentrations ofmost metabolites but had no effect on those of the majority of essential amino acids. Instead,nutrient starvation regulates the lysosomal concentrations of these amino acids, an effect wetraced to regulation of the mechanistic target of rapamycin (mTOR) pathway. Inhibition ofmTOR strongly reduced the lysosomal efflux of most essential amino acids, converting thelysosome into a cellular depot for them.These results reveal the dynamic nature of lysosomalmetabolites and that V-ATPase- and mTOR-dependent mechanisms exist for controllinglysosomal amino acid efflux.

Lysosomes are membrane-bound organellesbest known for their capacity to degrademacromolecules and recycle their constit-uent metabolites and for their dysfunctionin a groupof raremetabolic disorders known

as lysosomal storage diseases (1, 2). Lysosomes alsoparticipate in signal transduction (3), particularlyin nutrient sensing by the mechanistic target ofrapamycin complex 1 (mTORC1) pathway (4, 5),and are often deregulated in common diseasessuch as cancer (6). Given the critical roles of lyso-somes in producing and sensing many metabo-lites, a better understanding of lysosomal functionrequires uncovering its metabolite content andits regulation in diverse cell states.Traditional techniques for purifying lysosomes,

such as density-based centrifugation, are too slowto preserve what is likely a labile lysosomal me-tabolome (“lysobolome”). To overcome this issue,we used insights from a recently reportedmethodfor the rapid isolation of mitochondria (7) to de-velop an analogous approach for lysosomes. Ourlysosome immunoprecipitation (LysoIP) methoduses antibody tohuman influenza virus hemagglu-

tinin (HA)conjugated tomagneticbeads to immuno-purify lysosomes from human embryonic kidney(HEK) 293T cells expressing transmembraneprotein 192 (TMEM192) fused to three tandemHA epitopes (HA-Lyso cells) (Fig. 1, A and B).TMEM192 is a transmembrane protein (8) thatwe find retains its lysosomal localization uponoverexpression better than other such proteins,such as lysosomal-associated membrane protein1 (LAMP1). Startingwith live cells, it takes ~10minto isolate lysosomes that are highly pure andintact, as judged by the absence of markers forother cellular compartments (Fig. 1C), retentionof cathepsin D activity (Fig. 1D), and capacity totake up radiolabeled arginine in vitro (Fig. 1E).Moreover, tracking of either a lysosomal mem-brane protein (LAMP2), a luminal protein (cathep-sin D), or a small molecule (LysoTracker Red),yielded the same value for the fraction of totalcellular lysosomes purified (Fig. 1F), indicatingthat the lysosomes do not leak soluble contentsduring the purification. Importantly, the LysoIPmethod uses buffers compatible with subsequentanalyses of the lysosomal metabolome by liquidchromatography andmass spectrometry (LC-MS).Because the metabolite content of human lyso-

somes is not established, we used LC-MS to deter-mine the relative abundances of ~150 polar smallmolecules in lysosomes versus control anti-HAimmunoprecipitates from cells stably expressingFlag-taggedTMEM192 (Control-Lyso cells) (fig. S1Aand table S1). Of these, 57 were twice as abundantin the isolated lysosomes and thus deemed lyso-somal metabolites (fig. S1A and table S1). The ly-sosomes did not containmetabolites characteristicof other compartments, suchas the cytosolic glyco-

lytic intermediates fructose 1,6-bisphosphate andlactate or the mitochondrially enriched coenzymeA (7) (fig. S1B).We quantified the concentrations of the 57

metabolites in lysosomal and whole-cell samplesusing standard curves for each and the volumesof lysosomes and intact cells (see the supplemen-tary materials). Lysosomal metabolite concentra-tions correlated highly across biological replicates(r2 = 0.95) (Fig. 1G) and evenwith those obtainedusing the less preferable LAMP1-RFP-3xHA asthe lysosomal antigen tag (r2 = 0.95) (fig. S1C),mitigating concerns that expression of TMEM192,whose function is unknown,might have effects onthe lysosomal metabolome. In the proliferatingcells used in these experiments, the concentra-tions of metabolites tended to be, with a few ex-ceptions, lower in lysosomes than in whole cells(Fig. 1, H and I, and table S2). Two moleculespreviously predicted to be stored in lysosomes,cystine (the oxidized dimeric form of cysteine)and glucuronic acid (9, 10), were indeed enrichedin lysosomes, with concentrations 28- and 5.5-fold greater than those of whole cells, respectively(Fig. 1, H and I, and table S2). All nucleosides(guanosine, adenine, cytidine, uridine, and ino-sine) were lysosomally enriched (9- to 25-fold),consistent with the lysosome also being a depotfor these metabolites, at least in HEK-293T cells(Fig. 1, H and I). The lysosomal concentrationsof proteinogenic amino acids varied widely anddid not correlate well with those in whole cells(Fig. 1I), suggesting that although some lysosomalamino acids are in equilibrium with the rest ofthe cell, others are either sequestered in a dif-ferent compartment or undergo preferred trans-port out of the lysosome and thus show higherconcentrations in the whole-cell samples. Lyso-somes also contained metabolites that are notthought to result from the degradation of macro-molecules and thus are likely transported intolysosomes (Fig. 1I). These include nonproteino-genic amino acids, such as beta-alanine (20 mM),taurine (11 mM), and hypotaurine (12 mM); co-factors and vitamins, such as choline (7 mM) andphosphocholine (94 mM); creatine (274 mM) andphosphocreatine (111 mM); and multiple speciesof carnitines (Fig. 1I). The metabolomic land-scape of the human lysosome is consistent withits role as a recycling center but also indicatesthat the transport of metabolites into lysosomesmay influence lysosomal biology more than iswidely recognized.The multicomponent vacuolar H+–adenosine

triphosphatase (V-ATPase) maintains the lyso-somal lumen at a pHof ~4.5 (11), which is thoughtto be required for the optimal activity of lysosomalhydrolases and to set up a proton gradient withthe cytosol that provides energy for transportersto move metabolites across the lysosomal mem-brane. To directly ask how loss of the acidic pHaffects lysosomal metabolites, we profiled lyso-somes from cells acutely treatedwith the V-ATPaseinhibitors bafilomycin A1 (BafA1) or concanamycinA (ConA) (12, 13) at concentrations that do notinhibitmTORC1 signaling (4) (fig. S2A). Althoughneither had a major impact on the whole-cell

RESEARCH

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1Whitehead Institute for Biomedical Research andMassachusetts Institute of Technology, Department of Biology,9 Cambridge Center, Cambridge, MA 02142, USA. 2HowardHughes Medical Institute, Department of Biology,Massachusetts Institute of Technology, Cambridge, MA 02139,USA. 3Koch Institute for Integrative Cancer Research, 77Massachusetts Avenue, Cambridge, MA 02139, USA. 4BroadInstitute of Harvard and Massachusetts Institute of Technology,7 Cambridge Center, Cambridge, MA 02142, USA.*These authors contributed equally to this work. †Present address:Metabolon, Inc., Research Triangle Park, NC 27709, USA.‡Corresponding author. Email: sabatini@wi.mit.edu

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Fig. 1. LysoIPmethod for rapid immunoisolation of intact lysosomes forabsolute quantification of their metabolite content. (A) Localization ofTmem192-3xHA fusion protein to lysosomes.Tmem192-3xHAand lysosomesweredetected by immunofluorescence with antibodies to the HA epitope tag and thelysosomal marker LAMP2, respectively. Scale bars, 10 mm. Insets representselected fields that were magnified 3.24X. (B) Schematic of the workflow for theLysoIP method. Control-Lyso and HA-Lyso cells refer to cells stably expressing2xFlag-tagged TMEM192 or 3xHA-tagged Tmem192, respectively. (C) The LysoIPmethod isolates pure lysosomes. Immunoblotting for protein markers of varioussubcellular compartments in whole-cell lysates, purified lysosomes, or controlimmunoprecipitates. Lysates were prepared from cells expressing the 2xFlag-tagged TMEM192 (Control-Lyso cells) or 3xHA-tagged Tmem192 (HA-Lyso cells).ER, endoplasmic reticulum. (D to F) Purified lysosomes are intact and retain theircontents. (D) Cathepsin D activity was measured in whole-cell lysates andlysosomes, and immunoprecipitates from Control-Lyso cells served as a negative

control (Control IP) (mean ± SEM; n = 3). (E) Purified lysosomes take upradiolabeled arginine (Arginine [3H]). Lysosomes treated with a detergent wereused as a control (mean ± SEM; n = 3). (F) Calculations of the amounts ofcaptured lysosomes (mean ± SEM; n= 6, P>0.05; N.S., not significant; analysis ofvariance) were similar whether determined by tracking a membrane protein(LAMP2), the activityof the lysosomal protease cathepsinD (CatD),or a lysosome-specific small molecule (LysoTracker). Data are presented as the fraction of thematerial in the initial cell lysate. (G) Absolute quantification of lysosomalmetabolites. Comparison of concentrations of lysosomal metabolites across twobiological replicates, with r2 value shown. (H) Metabolite concentrations inlysosomes and whole cells. Metabolites above the dotted blue line are enrichedin lysosomes. Cys, cystine; Uri, uridine; Gua, guanosine; Ade, adenosine;Cyt, cytidine; Ino, inosine; GA, glucuronic acid. (I) Whole-cell and lysosomalconcentrations of 57 metabolites in HEK-293Tcells (mean ± SEM; n = 5).n indicates the number of independent biological replicates.

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Fig. 2. The efflux from lysosomes of most non-essential, but not essential, amino acidsrequires the proton gradient. (A) Changes inmetabolite concentrations in whole cells and lyso-somes upon V-ATPase inhibition. Principal compo-nents analyses of changes in metaboliteconcentrations in whole cells (circle) or lysosomes(square) after treatment for 1 hour with 200 nMbafilomycin A1 (BafA1, blue) or concanamycin A(ConA, purple). Dimethyl sulfoxide (DMSO) vehicle–treated cells were used as control (vehicle, green).(B) Most metabolites accumulate in lysosomes uponV-ATPase inhibition, whereas their levels in wholecells are not affected. P values are for comparisonsbetween metabolite concentrations in whole-cell(triangle) or lysosome (circle) samples shown in (A)(n = 3 for each treatment; dotted line representsP = 0.05). (Lower panel) Heat map of fold changes(log2) in metabolite concentrations after V-ATPaseinhibition relative to vehicle treatment. Gray boxes indicate undetectedmetabolites. (C and D) Accumulation of most nonessential, but not essential,amino acids in lysosomes upon V-ATPase inhibition. Fold changes in whole-celland lysosomal concentrations of amino acids in BafA1- or ConA-treated cellsrelative to vehicle-treated cells (mean ± SEM; n = 3, *P < 0.05). (E) Tracing ofexogenously added alanine and isoleucine in live cells. Cells were incubated inmedium containing 15N-labeled alanine and isoleucine for the indicated timepoints and then subjected to LysoIP. Data are presented as the fraction of thetotal pool of the amino acid that is 15N-labeled inwhole cells (black) or lysosomes(red) (mean±SEM;n=3 in each timepoint). (F) Dependenceof lysosomal efflux

of alanine, but not that of isoleucine, on the proton gradient. Cells treated with orwithout ConA were incubated in medium containing 15N-labeled alanine andisoleucine for 1 hour (pulse period), which was then replaced with mediumcontaining the natural 14N-containing isotope for the indicated timepoints (chaseperiod).The fold change in the fraction of 15N-labeled amino acid remaining inthe whole cells (circle) or lysosomes (square) was measured [mean ± SEM;n= 3; k (inmin−1) is the rate constant for the decay of the 15N-labeled amino acidfrom the lysosome, and the asterisk indicates nonoverlapping 95% confidenceintervals of the calculated k values between the treatments].Two-tailed t testswere used for comparisons between groups.

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metabolome, both caused large changes in themetabolome of the lysosome (Fig. 2A and fig.S2B). This emphasizes the value of LysoIP forstudying an organelle that in HEK-293T cellsoccupies only 2 to 3% of the total cell volume.V-ATPase inhibition caused the accumulationof many metabolites in lysosomes (Fig. 2B andtable S3), and only the concentration of cystinedropped significantly (P ≤ 0.01 in either treat-ment; two-tailed t test) (fig. S2C), consistent within vitro work showing that the lysosomal entry ofcysteine requires the pH gradient (14). Althoughall the nonessential amino acids accumulated inlysosomes upon V-ATPase inhibition (Fig. 2C)—with proline, alanine, and glycine being the mostaffected (Fig. 2C)—seven of the nine essentialamino acids did not, with histidine and threoninebeing the exceptions (Fig. 2D). Given that lyso-somes harbor several well-characterized proton-dependent amino acid transporters, such aslysosomal amino acid transporter 1 (LYAAT-1)(15), lysosomal accumulation of the nonessentialamino acids caused by V-ATPase inhibitionmayresult from their decreased efflux. We thereforeundertook pulse-chase experiments using 15N-labeled alanine, a representative pH-dependentamino acid, and isoleucine, a non–pH-dependentone (Fig. 2, C to F). In live cells, both enteredlysosomes, with isoleucine doing somore rapidlythan alanine (Fig. 2E). ConA treatment slowedthe efflux of alanine, but not that of isoleucine,from lysosomes (Fig. 2F), consistent with proton-dependent transporters mediating the efflux ofthis and other nonessential amino acids fromlysosomes. Furthermore, the failure of V-ATPaseinhibition to affect the lysosomal levels of mostessential amino acids, particularly the nonpolarones, raises the question of what, if anything,regulates their abundance.To investigate this, we examined other con-

ditions that might affect lysosomal metabolites,including nutrient starvation. We starved cellsof all amino acids for 60 min and measuredthe concentrations of amino acids in lysosomesand in whole cells. Concentrations of most non-essential amino acids did not drop in eithersample, consistent with the capacity of cells tosynthesize them. In contrast, the concentrationsof most essential amino acids, including thosethat were insensitive to V-ATPase inhibition,diminished in the whole-cell samples, but mostshowed little, if any, change in lysosomes (Fig. 3Aand table S4). Thus, amino acid starvation appearsto inhibit the lysosomal egress of many essentialamino acids.Given that a major consequence of amino

acid starvation is inhibition of mTORC1 (Fig. 3B)(16, 17), we asked whether mTORC1 regulatesthe abundance of amino acids in lysosomes.Consistent with this possibility, in cells thatlack functional GATOR1 [DEPDC5 knockout(KO) cells] and thus have amino acid–insensitivemTORC1 signaling (18), amino acid starvationdid decrease the concentrations of lysosomalamino acids (Fig. 3A and 3B). Moreover, in-hibition of the kinase activity of mTOR withTorin1 (19) increased the lysosomal concentra-

tions of six of the seven V-ATPase–insensitiveamino acids (leucine, phenylalanine, isoleucine,tryptophan, methionine, and valine) and oftyrosine, while having small effects on mostother amino acids, including histidine andserine, as well as many additional metabolites(Fig. 3, C and D; fig. S3A; and table S5). Torin1also increased the lysosomal concentrations ofnucleosides, although in this case the effect wasalso seen in whole cells (Fig. 3E). Of the sevenamino acidsmost strongly affected by Torin1, allare nonpolar and essential, with the exception oftyrosine, which is generated from the essentialamino acid phenylalanine (20). Importantly,other chemically distinct ATP-competitive in-hibitors of mTOR, including AZD8055 andWYE-132 (21, 22), also increased the concentration ofthese seven amino acids (fig. S3B), and mTORinhibition had similar effects across multiple celllines (fig. S3C). Although Torin1, AZD8055, andWYE-132 inhibit both mTORC1 and mTOR com-plex 2 (mTORC2), inhibition of only mTORC1with the allosteric inhibitor rapamycin or withlower concentrations of Torin1 also increasedthe concentration of these amino acids, albeitto smaller extents (fig. S3, B and D). mTORC1 isessential for cell survival, but it is possible togenerate cells lacking rictor, a critical mTORC2-specific component needed for phosphorylationof the protein kinase Akt (23). Loss of rictor didnot increase lysosomal amino acid concentra-tions, and, importantly, Torin1 increased the abun-dance of the seven amino acids in lysosomes evenmore in cells lacking rictor than inwild-type cells(fig. S3E). Thus, mTORC1 appears to mediatethe effects of mTOR inhibition on lysosomalamino acids.Because mTORC1 inhibits autophagy (24–28),

a potential explanation for the effects of Torin1is that it activates autophagic flux to such adegree that the production of metabolites bylysosomal macromolecular degradation exceedsthe capacity of lysosomes to export them. Wetested this possibility in cells lacking ATG7 (fig.S3F), which encodes a key component of theautophagy machinery (29). For most metabo-lites, loss of autophagy almost completely elim-inated the Torin1-induced increases in theirlysosomal concentrations, but it had only minoreffects on those of the seven strongly affectedamino acids (leucine, tyrosine, phenylalanine,isoleucine, tryptophan, methionine, and valine)(Fig. 3, D and E; fig. S3G; and table S5). mTORinhibition also activates the proteasome (30), butbortezomib, a proteasomal inhibitor (31), hadno effect on the capacity of Torin1 to increaseabundance of lysosomal amino acids (Fig. 3Fand fig. S3H). Lastly, mTORC1 inhibition sup-presses mRNA translation (32), but the proteinsynthesis inhibitor cycloheximide did not mimicthe effects of the mTOR inhibitors on lysosomalamino acid levels, although it didmildly increasewhole-cell and lysosomal pools of the mTOR-regulated amino acids (fig. S3L). Thus, mTORC1regulates the lysosomal concentrations of a largelydistinct set of amino acids from those affectedby the V-ATPase (Fig. 3G) through a mechanism

that does not involve autophagy, the proteasome,or protein synthesis (Fig. 3G).Given that mTORC1 does not affect the seven

amino acids through established downstreamprocesses, we considered the possibility that itcontrols their flux across the lysosomal mem-brane.We used 15N-labeled amino acids tomon-itor the transport of four of themTOR-regulatedamino acids (leucine, tyrosine, phenylalanine,and isoleucine) and a control amino acid (serine)into lysosomes in live cells. When added to theculture media, the labeled amino acids rapidlyexchanged with the 14N-containing amino acidsalready in lysosomes (Fig. 2E and Fig. 4A). Incells treatedwith orwithout cycloheximide, Torin1caused lysosomal accumulation of 15N-labeledleucine, tyrosine, phenylalanine, and isoleucine,but not serine (fig. S4, A and B, and Fig. 4B),demonstrating that mTOR regulates the move-ment of free amino acids across the lysosomalmembrane independently of their incorporationinto protein. Pulse-chase experiments revealedthat mTOR inhibition slows the efflux of leucine,tyrosine, phenylalanine, and isoleucine, but notthat of serine, from lysosomes but not fromwholecells (Fig. 4C and fig. S4C).We recently identified the multipass protein

SLC38A9 as a lysosomal effluxer of many es-sential nonpolar amino acids (33). Its loss ledto the accumulation in lysosomes of the sevenamino acids most affected by mTOR inhibitionand greatly reduced their efflux from lysosomes(Fig. 4D and fig. S4D). In cells lacking SLC38A9,Torin1 did not boost the already high lysosomalconcentrations of the seven amino acids (Fig. 4D).Thus, mTOR inhibition and loss of SLC38A9 donot have additive effects on lysosomal aminoacids, suggesting that mTORC1 regulates the ly-sosomal abundance of amino acids through amechanism that involves SLC38A9. Loss ofSLC38A9 greatly impaired the capacity of cellsto survive amino acid starvation and of theGCN2pathway, which senses uncharged tRNAs, to re-turn to baseline activity levels upon prolongedstarvation (Fig. 4, E and F). Thus, the efflux ofthemTORC1-regulated essential amino acids fromlysosomes is important for the cellular responseto starvation.Our data show that mTORC1 has a previously

unknown role in promoting the efflux of essen-tial amino acids from the lysosome into thecytosol (Fig. 4G). mTORC1 inhibition leads to thesequestration of these amino acids in the lyso-some by slowing their movement across the lyso-somal membrane, in effect converting it into astorage compartment for them. We speculatethat this function of mTORC1 is important forpreventing the inappropriate use of essentialamino acids during amino acid starvation, a statein which lysosomal and proteasomal protein deg-radation are thought to be a major source ofamino acids (30, 34, 35). One can imagine thefollowing scenario: Early in a starvation period,mTORC1 becomes profoundly inhibited, which,by suppressing SLC38A9 and perhaps other trans-porters, prevents the exit from lysosomes of es-sential amino acids. Over time, as proteolysis

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partially restores amino acid levels, mTORC1 be-comes sufficiently reactivated so that essentialamino acids are released into the cytosol at afaster rate to be used to execute the ongoing geneexpression program that cells induce to adapt tostarvation (3, 34). In this regard, it is interestingthat Torin1, which completely inhibits mTORC1,

causes a greater accumulation of amino acidsin lysosomes than rapamycin, which only par-tially inhibits it (19, 36–38). This pattern is alsotrue for several other processes downstream ofmTORC1, such as autophagy and protein syn-thesis (19, 36–38), and may indicate that themechanisms through whichmTORC1 regulates

lysosomal amino acid efflux, such as throughSLC38A9, are also sensitive to the exact amount ofmTORC1 activity, allowing for distinct outcomesat different levels. HowmTORC1 affects SLC38A9function is unknown, and it may do so indirectlyor directly. Activated mTORC1 resides on thelysosomal surface (5, 39), so it has the correct

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Fig. 3. mTOR regulates the lysosomal levels of essential nonpolar aminoacids in an autophagy-independent manner. (A) mTORC1 regulates theabundance of amino acids in lysosomes upon amino acid starvation. A heat mapshows fold changes (log2) in amino acid (AA) concentrations in whole-cellsamples or lysosomes of wild-type and DEPDC KO cells after amino acidstarvation for 60 min relative to cells cultured in medium with all amino acids(n = 2 for each time point). (B) Amino acid starvation inhibits mTORC1 signaling.Immunoblotting was used to monitor the levels and phosphorylation state ofS6 kinase (S6K1) in the same samples as in (A). Raptor served as a loadingcontrol. (C) Pharmacological inhibition of mTOR leads to the accumulation ofmanymetabolites in lysosomes. Cells were treated with 250 nM Torin1 or DMSO(vehicle) for 1 hour, and the lysosomal metabolite concentrations weredetermined and compared (n = 3). Red lines indicate threefold change inlysosomal concentration in Torin1- relative to vehicle-treated cells. (D) Lysosomalaccumulation of nonpolar essential amino acids and tyrosine in an autophagy-

independent manner after mTOR inhibition. Fold changes in the whole-cell andlysosomal concentrations of amino acids in wild-type and Atg7-null cells treatedwith Torin1 relative to vehicle-treated cells (mean ± SEM, n = 3, *P < 0.05;N.S., not significant). Histidine and serine served as examples of autophagy-dependent amino acids. (E) Upon mTOR inhibition, nucleosides accumulate inlysosomes in amostly autophagy-dependentmanner. Fold changes in whole-celland lysosomal concentrations of nucleosides in the same cells as in (D)(mean ± SEM; n = 3, *P < 0.05). (F) Proteasome activity is dispensable for thelysosomal accumulation of amino acids upon mTOR inhibition. Fold changes inthe whole-cell and lysosomal concentrations of amino acids in cells treated with250 nM Torin1 or Torin1 together with 5 mM Bortezomib relative to DMSO- orBortezomib-treated cells, respectively (mean ± SEM; n = 3, *P < 0.05; N.S, notsignificant). (G) Proteogenic amino acids can be divided into those whoselysosomal levels are regulated by V-ATPase- or mTOR-dependent mechanisms.Two-tailed t tests were used for comparisons between groups.

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Fig. 4. mTOR controls the efflux of nonpolar essential amino acids andtyrosine from lysosomes. (A) Tracing of exogenously added leucine, tyrosine,phenylalanine, andserine in livecells.Cellswere incubated inmediumcontaining theindicated 15N-labeled amino acids for various times and subjected to the LysoIPmethod. Data are presented as the fraction of the total pool of the amino acid inwhole cells (black) or lysosomes (red) that is 15N-labeled (mean ± SEM; n = 3 ineach time point). (B) Independently of protein synthesis, mTOR inhibition leads tothe accumulation in lysosomes of leucine, tyrosine, phenylalanine, and isoleucine,but not serine. Data are presented as the fold change in the whole-cell andlysosomal abundance of the indicated 15N-labeled amino acid after Torin1treatment, relative to the DMSO vehicle treatment.The experiment was performedas indicated in the figure in the presence of 50 mg/mLcycloheximide (CHX) (mean ±SEM; n = 3, P < 0.005).This experiment was performed using Atg7-null cells.(C) mTOR controls the lysosomal efflux of nonpolar essential amino acids as well astyrosine. Cells treated with 250 nM Torin1 or DMSO (vehicle) were incubated inmedium containing the indicated 15N-labeled amino acids for 1 hour (pulse period).This mediumwas then replaced with media containing the natural 14N-isotope ofthe amino acid for the indicated time points (chase period). Data are presented asthe fold change in the fraction of 15N-labeled amino acid remaining in whole cells

(triangle) or lysosomes (circle) at each time point [mean ±SEM, n≥ 3; k (inmin−1) isthe rate constant for the decay of the 15N-labeled amino acid from lysosomes,and the asterisk indicates nonoverlapping 95% confidence intervals of thecalculated k between the treatments].This experiment was performed usingAtg7-null cells. (D) mTORC1 regulates the lysosomal abundance of essentialnonpolar amino acids and tyrosine in an SLC38A9-dependent manner. Foldchanges in the lysosomal concentrations of indicated amino acids inwild-type andSLC38A9-null cells treated with Torin1 relative to vehicle-treated wild-type cells(mean ± SEM; n = 3). (E) Loss of SLC38A9 impairs the fitness of cells understarvation conditions.Wild-type, SLC38A9-null, and SLC38A9-null addback(SLC38A9-null + SLC38A9) cells were seeded in medium containing all aminoacids (full media) or lacking leucine, isoleucine, tyrosine, and phenylalanine (-LIYF)for 3 days, at which point cell numbers were measured (mean ± SD; n = 3,*P < 0.001). (F) SLC38A9 is required for inhibiting the GCN2 pathway after aprolonged amino acid starvation period. Immunoblotting was used tomonitor thelevels and phosphorylation state of eIF2a. Raptor served as a loading control.LIYF indicates leucine, isoleucine, tyrosine, and phenylalanine. (G) A modelproposing a role for mTORC1 in regulating the efflux of amino acids fromlysosomes.Two-tailed t tests were used for comparisons between two groups.

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localization to control SLC38A9 or its regulators.The fact that SLC38A9 also signals arginine levelsto mTORC1 (40–42) suggests that SLC38A9 ispart of a sophisticated system for coordinatingmTORC1 activity and lysosomal amino acid effluxwith the concentrations of cytosolic and lysosomalamino acids. Our findings provide an example ofthe utility of LysoIP for uncovering a new func-tion for lysosomes—the sequestering of essentialamino acids uponmTORC1 inhibition. Themethodthat we described may be useful for studyingthe emerging roles of lysosomes and for probingthemetabolic state of the lysosome in the variousdiseases in which it is implicated.

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ACKNOWLEDGMENTS

We thank all members of the Sabatini laboratory for helpfulinsights, particularly J. R. Cantor, W. W. Chen, and J. M. Orozco;C.A. Lewis and T. Kunchok from the Whitehead InstituteMetabolite Profiling Core Facility; and N. S. Gray (Dana-FarberCancer Institute) for Torin1. This work was supported bygrants from NIH (R01 CA103866, R01 CA129105, and R37AI47389) and Department of Defense (W81XWH-15-1-0230) toD.M.S., from Department of Defense (W81XWH-15-1-0337)to E.F., and from the European Molecular Biology OrganizationLong-Term Fellowship to M.A.-R.; a Saudi Aramco Ibn KhaldunFellowship for Saudi Women to N.N.L.; and fellowship support fromthe National Defense Science and Engineering Graduate Fellowship(NDSEG) Program to G.A.W. D.M.S. is an investigator of theHoward Hughes Medical Institute.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/358/6364/807/suppl/DC1Materials and MethodsFigs. S1 to S5Tables S1 to S5References (43–46)

11 May 2017; accepted 3 October 2017Published online 26 October 201710.1126/science.aan6298

Abu-Remaileh et al., Science 358, 807–813 (2017) 10 November 2017 7 of 7

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efflux from lysosomesLysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid

and David M. SabatiniMonther Abu-Remaileh, Gregory A. Wyant, Choah Kim, Nouf N. Laqtom, Maria Abbasi, Sze Ham Chan, Elizaveta Freinkman

originally published online October 26, 2017DOI: 10.1126/science.aan6298 (6364), 807-813.358Science 

, this issue p. 807Scienceof inhibition of the mTOR (mechanistic target of rapamycin) protein kinase complex.amino acids) is a regulated process. Amino acid transport was inhibited under conditions of nutrient depletion as a result under various conditions showed that efflux from the lysosome of most essential amino acids (but not that of most othermembranes that could be used to rapidly precipitate purified lysosomes on magnetic beads. Analysis of their contents

engineered cultured human cells to produce a protein tag on lysosomalet al.mass spectrometry. Abu-Remaileh A new technique allows rapid purification of lysosomes and metabolic profiling by liquid chromatography and

Regulated lysosomal efflux of amino acids

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REFERENCES

http://science.sciencemag.org/content/358/6364/807#BIBLThis article cites 46 articles, 28 of which you can access for free

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