Amino Acid Sensing by mTORC1: Intracellular Transporters Mark the Spot
Authors: Deborah C.I. Goberdhan, Clive Wilson, Adrian L. Harris
Journal: Cell Metabolism (2016)

Cell metabolism and growth are matched to nutrient availability via the amino-acid-regulated mechanistic target of rapamycin complex 1 (mTORC1). Transporters have emerged as important amino acid sensors controlling mTOR recruitment and activation at the surface of multiple intracellular compartments. Classically, this has involved late endosomes and lysosomes, but now, in a recent twist, also the Golgi apparatus. Here we propose a model in which specific amino acids in assorted compartments activate different mTORC1 complexes, which may have distinct drug sensitivities and functions. We will discuss the implications of this for mTORC1 function in health and disease. Cell metabolism and growth are matched to nutrient availability via the amino-acid-regulated mechanistic target of rapamycin complex 1 (mTORC1). Transporters have emerged as important amino acid sensors controlling mTOR recruitment and activation at the surface of multiple intracellular compartments. Classically, this has involved late endosomes and lysosomes, but now, in a recent twist, also the Golgi apparatus. Here we propose a model in which specific amino acids in assorted compartments activate different mTORC1 complexes, which may have distinct drug sensitivities and functions. We will discuss the implications of this for mTORC1 function in health and disease. Central to the ability of a cell to adapt to its microenvironment is mechanistic (formerly mammalian) target of rapamycin (mTOR) complex 1 (mTORC1), a critical signaling hub, which is conserved from yeast to humans, regulating both growth and metabolism (Figure 1A). mTORC1 activity is regulated by a wide range of signals (reviewed in Dibble and Manning, 2013Dibble C.C. Manning B.D. Signal integration by mTORC1 coordinates nutrient input with biosynthetic output.Nat. Cell Biol. 2013; 15: 555-564Crossref PubMed Scopus (527) Google Scholar), including growth factor signaling (Gao et al., 2002Gao X. Zhang Y. Arrazola P. 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Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2398) Google Scholar), oxygen levels (Brugarolas et al., 2004Brugarolas J. Lei K. Hurley R.L. Manning B.D. Reiling J.H. Hafen E. Witters L.A. Ellisen L.W. Kaelin Jr., W.G. Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex.Genes Dev. 2004; 18: 2893-2904Crossref PubMed Scopus (1068) Google Scholar), and nutrients, particularly amino acids, as discussed below. Genes encoding the key mTORC1 kinase component, mTOR, were first identified in yeast, as TOR1 and TOR2, through molecular genetic studies, which revealed that they were important targets of the drug rapamycin (Heitman et al., 1991Heitman J. Movva N.R. Hall M.N. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast.Science. 1991; 253: 905-909Crossref PubMed Scopus (1549) Google Scholar, Li et al., 2014Li J. Kim S.G. Blenis J. Rapamycin: one drug, many effects.Cell Metab. 2014; 19: 373-379Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar). The term mTOR was initially used to refer specifically to TOR's mammalian homologs, while nonmammalian TOR was referred to simply as TOR (Hall, 2013Hall M.N. Talks about TORCs: recent advancesin target of rapamycin signalling. On mTOR nomenclature.Biochem. Soc. Trans. 2013; 41: 887-888Crossref PubMed Scopus (18) Google Scholar). However, a second definition, mechanistic TOR, has more recently also been employed in articles where mammalian, vertebrate, and invertebrate TOR are considered together, and for simplicity, we will use the latter term in this review. Following on from the landmark studies in yeast, mTOR and its regulation by amino acids were shown to be conserved in mammals (Sabatini et al., 1994Sabatini D.M. Erdjument-Bromage H. Lui M. Tempst P. Snyder S.H. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs.Cell. 1994; 78: 35-43Abstract Full Text PDF PubMed Scopus (1222) Google Scholar, Hara et al., 1998Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism.J. Biol. Chem. 1998; 273: 14484-14494Crossref PubMed Scopus (1122) Google Scholar). A combination of genetic analysis in flies (Gao et al., 2002Gao X. Zhang Y. Arrazola P. Hino O. Kobayashi T. Yeung R.S. Ru B. Pan D. Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling.Nat. Cell Biol. 2002; 4: 699-704Crossref PubMed Scopus (573) Google Scholar) and biochemical work using human cells (Inoki et al., 2002Inoki K. Li Y. Zhu T. Wu J. Guan K.L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling.Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2398) Google Scholar) led to a step change in thinking: growth factor signaling through PI3-kinase (PI3K) and Akt was shown to lie upstream of mTORC1 (reviewed in Goberdhan and Wilson, 2003Goberdhan D.C. Wilson C. The functions of insulin signaling: size isn't everything, even in Drosophila.Differentiation. 2003; 71: 375-397Crossref PubMed Scopus (109) Google Scholar), partly, but not exclusively, acting through the heterotrimeric tuberous sclerosis complex (TSC) and the monomeric GTPase, Ras homolog enriched in brain (Rheb). The best-characterized downstream function of mTORC1 is the control of mRNA translation. This is achieved via phosphorylation and suppression of the translation initiation inhibitor, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), in conjunction with activation of S6 kinase (S6K), which controls the transcription of a broad range of ribosome biogenesis genes (Chauvin et al., 2014Chauvin C. Koka V. Nouschi A. Mieulet V. Hoareau-Aveilla C. Dreazen A. Cagnard N. Carpentier W. Kiss T. Meyuhas O. Pende M. Ribosomal protein S6 kinase activity controls the ribosome biogenesis transcriptional program.Oncogene. 2014; 33: 474-483Crossref PubMed Scopus (191) Google Scholar). However, mTORC1 has numerous other biochemical targets with important metabolic and cellular functions (Dibble and Manning, 2013Dibble C.C. Manning B.D. Signal integration by mTORC1 coordinates nutrient input with biosynthetic output.Nat. Cell Biol. 2013; 15: 555-564Crossref PubMed Scopus (527) Google Scholar; Figure 1A). This complex metabolic regulatory network, in the classical paradigm, is represented by a model in which microenvironmental inputs funnel through a single mTORC1 hub to give a wide range of outputs (Figures 1 and 2; Chantranupong et al., 2015Chantranupong L. Wolfson R.L. Sabatini D.M. Nutrient-sensing mechanisms across evolution.Cell. 2015; 161: 67-83Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). However, there is increasing awareness that many signaling cascades are controlled at a subcellular level, providing the flexibility for a more refined response within a single cell. Several studies of mTORC1 amino-acid-sensing mechanisms (Thomas et al., 2014Thomas J.D. Zhang Y.J. Wei Y.H. Cho J.H. Morris L.E. Wang H.Y. Zheng X.F. Rab1A is an mTORC1 activator and a colorectal oncogene.Cancer Cell. 2014; 26: 754-769Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, Jewell et al., 2015Jewell J.L. Kim Y.C. Russell R.C. Yu F.X. Park H.W. Plouffe S.W. Tagliabracci V.S. Guan K.L. Metabolism. Differential regulation of mTORC1 by leucine and glutamine.Science. 2015; 347: 194-198Crossref PubMed Scopus (483) Google Scholar, Fan et al., 2015Fan S.J. Snell C. Turley H. Li J.L. McCormick R. Perera S.M. Heublein S. Kazi S. Azad A. Wilson C. et al.PAT4 levels control amino-acid sensitivity of rapamycin-resistant mTORC1 from the Golgi and affect clinical outcome in colorectal cancer.Oncogene. 2015; https://doi.org/10.1038/onc.2015.363Crossref Scopus (38) Google Scholar), discussed below, support the existence of a multi-hub model with at least two forms of mTORC1 (Figure 1B) controlled by amino acids in different parts of the cell. What amino acids regulate mTORC1 activity? Pioneering work in Chinese hamster ovary cells (Hara et al., 1998Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism.J. Biol. Chem. 1998; 273: 14484-14494Crossref PubMed Scopus (1122) Google Scholar, Beugnet et al., 2003Beugnet A. Tee A.R. Taylor P.M. Proud C.G. Regulation of targets of mTOR (mammalian target of rapamycin) signalling by intracellular amino acid availability.Biochem. 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Guan K.L. Metabolism. Differential regulation of mTORC1 by leucine and glutamine.Science. 2015; 347: 194-198Crossref PubMed Scopus (483) Google Scholar, Fan et al., 2015Fan S.J. Snell C. Turley H. Li J.L. McCormick R. Perera S.M. Heublein S. Kazi S. Azad A. Wilson C. et al.PAT4 levels control amino-acid sensitivity of rapamycin-resistant mTORC1 from the Golgi and affect clinical outcome in colorectal cancer.Oncogene. 2015; https://doi.org/10.1038/onc.2015.363Crossref Scopus (38) Google Scholar, Carroll et al., 2016Carroll B. Maetzel D. Maddocks O.D. Otten G. Ratcliff M. Smith G.R. Dunlop E.A. Passos J.F. Davies O.R. Jaenisch R. et al.Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity.eLife. 2016; 5: e11058https://doi.org/10.7554/eLife.11058Crossref PubMed Scopus (14) Google Scholar). Amino acid transporters were initially implicated in mTORC1 regulation as passageways through the plasma membrane, enabling amino acids to enter cells and activate cytoplasmic amino acid sensors (Christie et al., 2002Christie G.R. Hajduch E. Hundal H.S. Proud C.G. Taylor P.M. Intracellular sensing of amino acids in Xenopus laevis oocytes stimulates p70 S6 kinase in a target of rapamycin-dependent manner.J. Biol. Chem. 2002; 277: 9952-9957Crossref PubMed Scopus (109) Google Scholar, Beugnet et al., 2003Beugnet A. Tee A.R. Taylor P.M. Proud C.G. Regulation of targets of mTOR (mammalian target of rapamycin) signalling by intracellular amino acid availability.Biochem. J. 2003; 372: 555-566Crossref PubMed Scopus (250) Google Scholar). Consistent with this, the heterodimeric amino acid transporter CD98, solute-linked carrier (SLC)3A2-SLC7A5 (CD98hc-LAT1), in combination with a glutamine transporter (SLC1A5), activates mTORC1 by exchanging leucine for glutamine to increase intracellular leucine levels (Nicklin et al., 2009Nicklin P. Bergman P. Zhang B. Triantafellow E. Wang H. Nyfeler B. Yang H. Hild M. Kung C. Wilson C. et al.Bidirectional transport of amino acids regulates mTOR and autophagy.Cell. 2009; 136: 521-534Abstract Full Text Full Text PDF PubMed Scopus (1276) Google Scholar). Other cell-surface amino acid transporters, e.g., LAT1 and LAT3 (Wang et al., 2013Wang Q. Tiffen J. Bailey C.G. Lehman M.L. Ritchie W. Fazli L. Metierre C. Feng Y.J. Li E. Gleave M. et al.Targeting amino acid transport in metastatic castration-resistant prostate cancer: effects on cell cycle, cell growth, and tumor development.J. Natl. 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Genetic screening of a broad range of amino acid transporters in flies highlighted members of the proton-assisted amino acid transporter (PAT) or SLC36 family as having a particularly potent effect in promoting growth in vivo and activating mTORC1 in a cell-autonomous manner: for example, overexpressing fly PAT family members in the developing eye or wing increases organ growth (Figure 3A; Goberdhan et al., 2005Goberdhan D.C. Meredith D. Boyd C.A. Wilson C. PAT-related amino acid transporters regulate growth via a novel mechanism that does not require bulk transport of amino acids.Development. 2005; 132: 2365-2375Crossref PubMed Scopus (118) Google Scholar). The ability of PATs to promote growth increases significantly when growth rates and mTORC1 signaling in the eye are stimulated by activated PI3K (Figure 3B; Ögmundsdóttir et al., 2012Ögmundsdóttir M.H. Heublein S. Kazi S. Reynolds B. Visvalingam S.M. Shaw M.K. Goberdhan D.C. Proton-assisted amino acid transporter PAT1 complexes with Rag GTPases and activates TORC1 on late endosomal and lysosomal membranes.PLoS ONE. 2012; 7: e36616Crossref PubMed Scopus (105) Google Scholar), a signaling defect frequently associated with human cancer. Consistent with this context-dependent effect on growth, the fly PAT, Pathetic (Path), is more critical for the growth of neurons with large dendrites than those with small dendrites during normal development (Lin et al., 2015Lin W.Y. Williams C. Yan C. Koledachkina T. Luedke K. Dalton J. Bloomsburg S. Morrison N. Duncan K.E. Kim C.C. Parrish J.Z. The SLC36 transporter Pathetic is required for extreme dendrite growth in Drosophila sensory neurons.Genes Dev. 2015; 29: 1120-1135Crossref PubMed Scopus (20) Google Scholar). Subsequent analysis in HEK293 and MCF-7 breast cancer cells has shown that the two broadly expressed human PATs, PAT1 and PAT4, are required for amino-acid-dependent mTORC1 activation and cell proliferation. These two human PATs can also promote growth in transgenic flies in vivo (Heublein et al., 2010Heublein S. Kazi S. Ogmundsdóttir M.H. Attwood E.V. Kala S. Boyd C.A. Wilson C. Goberdhan D.C. Proton-assisted amino-acid transporters are conserved regulators of proliferation and amino-acid-dependent mTORC1 activation.Oncogene. 2010; 29: 4068-4079Crossref PubMed Scopus (121) Google Scholar). Before these studies, human PAT1 and PAT2 had already been shown to transport alanine, glycine, and proline by proton-coupled secondary active transport (Figure 4B; Boll et al., 2002Boll M. Foltz M. Rubio-Aliaga I. Kottra G. Daniel H. Functional characterization of two novel mammalian electrogenic proton-dependent amino acid cotransporters.J. Biol. Chem. 2002; 277: 22966-22973Crossref PubMed Scopus (139) Google Scholar, Chen et al., 2003Chen Z. Kennedy D.J. Wake K.A. Zhuang L. Ganapathy V. Thwaites D.T. Structure, tissue expression pattern, and function of the amino acid transporter rat PAT2.Biochem. 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A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1.Science. 2013; 340: 1100-1106Crossref PubMed Scopus (706) Google Scholar), and RagC mutations have recently been linked to follicular lymphoma (Okosun et al., 2016Okosun J. Wolfson R.L. Wang J. Araf S. Wilkins L. Castellano B.M. Escudero-Ibarz L. Al Seraihi A.F. Richter J. Bernhart S.H. et al.Recurrent mTORC1-activating RRAGC mutations in follicular lymphoma.Nat. Genet. 2016; 48: 183-188Crossref PubMed Scopus (124) Google Scholar). Furthermore, amino acid starvation and inactivation of the Rag heterodimer have been linked to recruitment of the tumor suppressor TSC to LELs (Demetriades et al., 2014Demetriades C. Doumpas N. Teleman A.A. Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2.Cell. 2014; 156: 786-799Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, Demetriades et al., 2016Demetriades C. Plescher M. Teleman A.A. Lysosomal recruitment of TSC2 is a universal response to cellular stress.Nat. Commun. 2016; 7: 10662https://doi.org/10.1038/ncomms10662Crossref PubMed Scopus (96) Google Scholar, Carroll et al., 2016Carroll B. Maetzel D. Maddocks O.D. Otten G. Ratcliff M. Smith G.R. Dunlop E.A. Passos J.F. Davies O.R. Jaenisch R. et al.Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity.eLife. 2016; 5: e11058https://doi.org/10.7554/eLife.11058Crossref PubMed Scopus (14) Google Scholar), while another group has reported that TSC localization is primarily regulated by growth factor signaling (Menon et al., 2014Menon S. Dibble C.C. Talbott G. Hoxhaj G. Valvezan A.J. Takahashi H. Cantley L.C. Manning B.D. Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome.Cell. 2014; 156: 771-785Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). But how are amino acids sensed at the LEL surface? Since the V-ATPase proton pump is part of the LEL-located mTORC1 supercomplex and its interactions are modified by amino acids, it was proposed as the sensor (Zoncu et al., 2011Zoncu R. Bar-Peled L. Efeyan A. Wang S. Sancak Y. Sabatini D.M. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase.Science. 2011; 334: 678-683Crossref PubMed Scopus (1157) Google Scholar). The LEL-localized human PAT1, however, coimmunoprecipitates with RagC (Ögmundsdóttir et al., 2012Ögmundsdóttir M.H. Heublein S. Kazi S. Reynolds B. Visvalingam S.M. Shaw M.K. Goberdhan D.C. Proton-assisted amino acid transporter PAT1 complexes with Rag GTPases and activates TORC1 on late endosomal and lysosomal membranes.PLoS ONE. 2012; 7: e36616Crossref PubMed Scopus (105) Google Scholar); this led to an alternative model in which the amino acid sensing is carried out by amino acid transporters based on their ability to bind amino acids. More recently, SLC38A9, a member of an amino acid transporter family with structural similarities to PATs, was highlighted as an amino-acid-sensitive regulator of the LEL-located mTORC1 supercomplex (Figure 2; Wang et al., 2015Wang S. Tsun Z.Y. Wolfson R.L. Shen K. Wyant G.A. Plovanich M.E. Yuan E.D. Jones T.D. Chantranupong L. Comb W. et al.Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1.Science. 2015; 347: 188-194Crossref PubMed Scopus (542) Google Scholar, Rebsamen et al., 2015Rebsamen M. Pochini L. Stasyk T. de Araújo M.E. Galluccio M. Kandasamy R.K. Snijder B. Fauster A. Rudashevskaya E.L. Bruckner M. et al.SLC38A9 is a component of the lysosomal amino acid sensing machinery that controls mTORC1.Nature. 2015; 519: 477-481Crossref PubMed Scopus (450) Google Scholar, Jung et al., 2015Jung J. Genau H.M. Behrends C. Amino acid-dependent mTORC1 regulation by the lysosomal membrane protein SLC38A9.Mol. Cell. Biol. 2015; 35: 2479-2494Crossref PubMed Scopus (173) Google Scholar). Coimmunoprecipitation experiments with different components of this complex consistently pull down SLC38A9, but not PAT1 or other amino acid transporters. This strong interaction is dependent on SLC38A9's cytosolic N-terminal tail, which has a high affinity for the Rag/Ragulator complex. As discussed later, the stability of this interaction may explain why other transporters were not pulled down under the conditions employed for these studies. SLC38A9 expressed in proteoliposomes (Figure 4B; Wang et al., 2015Wang S. Tsun Z.Y. Wolfson R.L. Shen K. Wyant G.A. Plovanich M.E. Yuan E.D. Jones T.D. Chantranupong L. Comb W. et al.Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1.Science. 2015; 347: 188-194Crossref PubMed Scopus (542) Google Scholar, Rebsamen et al., 2015Rebsamen M. Pochini L. Stasyk T. de Araújo M.E. Galluccio M. Kandasamy R.K. Snijder B. Fauster A. Rudashevskaya E.L. Bruckner M. et al.SLC38A9 is a component of the lysosomal amino acid sensing machinery that controls mTORC1.Nature. 2015; 519: 477-481Crossref PubMed Scopus (450) Google Scholar) binds to several amino acids with different affinities. It potentially is involved in arginine sensing (Wang et al., 2015Wang S. Tsun Z.Y. Wolfson R.L. Shen K. Wyant G.A. Plovanich M.E. Yuan E.D. Jones T.D. Chantranupong L. Comb W. et al.Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1.Science. 2015; 347: 188-194Crossref PubMed Scopus (542) Google Scholar), though it has higher affinity for other amino acids, such as glutamine (Rebsamen et al., 2015Rebsamen M. Pochini L. Stasyk T. de Araújo M.E. Galluccio M. Kandasamy R.K. Snijder B. Fauster A. Rudashevskaya E.L. Bruckner M. et al.SLC38A9 is a component of the lysosomal amino acid sensing machinery that controls mTORC1.Nature. 2015; 519: 477-481Crossref PubMed Scopus (450) Google Scholar). Like some PATs (Goberdhan et al., 2005Goberdhan D.C. Meredith D. Boyd C.A. Wilson C. PAT-related amino acid transporters regulate growth via a novel mechanism that does not require bulk transport of amino acids.Development. 2005; 132: 2365-2375Crossref PubMed Scopus (118) Google Scholar, Pillai and Meredith, 2011Pillai S.M. Meredith D. SLC36A4 (hPAT4) is a high affinity amino acid transporter when expressed in Xenopus laevis oocytes.J. Biol. Chem. 2011; 286: 2455-2460Crossref PubMed Scopus (29) Google Scholar) and SLC38A2 (Pinilla et al., 2011Pinilla J. Aledo J.C. Cwiklinski E. Hyde R. Taylor P.M. Hundal H.S. SNAT2 transceptor signalling via mTOR: a role in cell growth and proliferation?.Front. Biosci. (Elite Ed.). 2011; 3: 1289-1299PubMed Google Scholar), SLC38A9 may act as a transceptor because of its close association with the mTORC1 supercomplex. The high degree of evolutionary conservation in other aspects of mTORC1 regulation, however, suggests that in the absence of a fly SLC38A9 homolog, this transporter cannot by itself resolve the amino-acid-sensing puzzle. Where do transporters like PAT1 and SLC38A9 sense amino acids? The primary focus has been on the LEL lumen, although it is also possible that sensing at the cytosolic side could be involved. Labeled extracellular amino acids rapidly enter the LELs (Zoncu et al., 2011Zoncu R. Bar-Peled L. Efeyan A. Wang S. Sancak Y. Sabatini D.M. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase.Science. 2011; 334: 678-683Crossref PubMed Scopus (1157) Google Scholar), probably by a combination of endocytosis and uptake across the LEL membra