Subjects

Abstract

Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses1,2,3. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H+-ATPase4,5,6,7,8. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator–RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.

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Accessions

Primary accessions

ArrayExpress

Data deposits

The protein-protein interactions have been submitted to the IMEx (http://www.imexconsortium.org) consortium through IntAct (http://www.ebi.ac.uk/intact/) and assigned the identifier IM-23283. The SLC network has the IntAct accession number EBI-9975668 and the RAGA-RAGC-LAMTOR network is EBI-9975664. RNA-Seq data is available in ArrayExpress (http://www.ebi.ac.uk/arrayexpress) under the accession number E-MTAB-3102.

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Acknowledgements

We thank D. M. Sabatini, S. Wang and Z. Tsun for discussing results before publication and generously providing Flag–SLC38A9 and Flag–METAP2 stably expressing cells, all members of the Superti-Furga laboratory for discussions, the Bennett laboratory for the proteomic analyses, F. Pauler and the Barlow laboratory for the RNA-seq analysis and M. Gstaiger for providing expression vectors. This work was supported by the Austrian Academy of Sciences, ERC grant to G.S.-F. (i-FIVE 250179), EMBO long-term and Marie Curie fellowships to M.R. (ALTF 1346-2011, IEF 301663), EMBO long-term fellowship to R.K.K. (ALTF 314-2012), Swiss NSF fellowship (P300P3_147897) to B.S., Vienna Science and Technology Fund (WWTF VRG10-001) and the Austrian Science Fund (FWF P 25522-B20) to C.K., the Italian Ministry of Instruction University and Research, PON-ricerca e competitività 2007-2013 (no. PON01_00937) to C.I., the Austrian Federal Ministry for Science and Research (GenAu projects, APP-III and BIN-III) to L.A.H., K.L.B. and G.S.-F., the Austrian Science Fund MCBO/SFB021 to L.A.H.

Author information

Author notes

    • Elena L. Rudashevskaya

    Present address: Institute of Medical Chemistry, Medical University of Vienna, 1090 Vienna, Austria.

Affiliations

  1. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria

    • Manuele Rebsamen
    • , Richard K. Kandasamy
    • , Berend Snijder
    • , Astrid Fauster
    • , Elena L. Rudashevskaya
    • , Manuela Bruckner
    • , Stefania Scorzoni
    • , Kilian V. M. Huber
    • , Johannes W. Bigenzahn
    • , Leonhard X. Heinz
    • , Keiryn L. Bennett
    •  & Giulio Superti-Furga
  2. Department DiBEST (Biology, Ecology and Earth Sciences), University of Calabria, 87036 Arcavacata di Rende, Italy

    • Lorena Pochini
    • , Michele Galluccio
    •  & Cesare Indiveri
  3. Biocenter, Division of Cell Biology, Innsbruck Medical University, 6020 Innsbruck, Austria

    • Taras Stasyk
    • , Mariana E. G. de Araújo
    • , Przemyslaw A. Filipek
    •  & Lukas A. Huber
  4. Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria

    • Claudine Kraft

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Contributions

M.R. and G.S.-F. conceived the study. L.P., M.G. and C.I. designed and performed transport assays. M.R., T.S., M.E.G.d.A., E.L.R., M.B., K.L.B., L.A.H. and G.S.-F. designed and performed TAP-mass spectrometry experiments. M.R., M.E.G.d.A., B.S., A.F., M.B., S.S. and P.A.F. performed the other experiments. M.R., L.A.H. and G.S.-F. designed the other experiments. R.K.K. and B.S. performed bioinformatic data and image analysis. K.V.M.H., J.W.B., L.X.H., C.K. generated reagents and provided scientific insight. M.R. and G.S.-F. wrote the manuscript. All authors contributed to the discussion of results and participated in manuscript preparation.

Competing interests

A patent has been filed with data generated in this manuscript where M.R. and G.S.-F. are listed as inventors.

Corresponding author

Correspondence to Giulio Superti-Furga.

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DOI

https://doi.org/10.1038/nature14107

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