SLC38A9 is a component of the lysosomal amino acid-sensing machinery that controls mTORC1

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 responses1-3. 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+ -ATPase 4-8. 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, while 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.

acid withdrawal, while 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.
Keywords mTOR; solute carrier proteins; amino acid transport; metabolism; cancer Amino acids are essential for mTORC1 activity, as growth factors cannot efficiently activate mTOR in their absence 5,9 . Notwithstanding the growing number of proteins involved in the activation of mTOR at the lysosomal surface, the molecular nature of the amino acid sensing mechanisms have remained elusive 1,2,4,[9][10][11][12][13] . Several members of the solute carrier (SLC) group belonging to families capable of transporting amino acids at the plasma membrane have been shown to regulate mTOR activity 14 , raising the possibility that SLCs may also be involved in the lysosomal sensing. We hypothesized the existence of an ubiquitously expressed SLC belonging to the a family competent for amino acid transport 15 with a subcellular localisation compatible with lysosomal amino acid sensing. Among the list of SLCs robustly expressed in two different cell lines, we focused on member 9 of the SLC38 family as it was completely uncharacterized, showed vesicular staining 16 and had been associated to lysosomes by proteomic analysis 17 (Extended Data Fig. 1a). The SLC38 family contains eleven members, and is part of a phylogenetic cluster of amino acid transporters comprising the SLC32 and SLC36 families 18 (Extended Data Fig. 1b). SLC38A9 is predicted to encompass eleven transmembrane helices and a 120-residue cytoplasmic N-terminal region. Treatment with peptide-N-glycosidase (PNGase) F showed that SLC38A9 is highly glycosylated and enabled detection of the endogenous protein (Extended Data Fig. 2a-b). Supporting a possible role in growth regulatory pathways, silencing of SLC38A9 by short hairpin RNA (shRNA) in HEK293T cells resulted in a reduction of cell size and cell proliferation, (Extended Data Fig. 2c-d).
To test whether SLC38A9 would associate with the complex regulating mTORC1, we engineered HEK293 cells to express tagged SLC38A9 in an inducible fashion and verified the localisation of the protein to lysosomes (Extended Data Fig. 3a-c). We purified endogenously assembled protein complexes using tandem affinity purification (TAP) coupled to one-dimensional gel-free liquid chromatography tandem mass spectrometry (LC-MS/MS). The gel-free approach was critical as upon boiling SLC38A9 formed insoluble aggregates that failed to enter SDS-polyacrylamide gels (Extended Data Fig. 2e-f). The analysis identified all the five members of the Ragulator/LAMTOR complex and the four RAG GTPases as specific interactors of SLC38A9 (Fig. 1a, Extended Data 3d). Such collective high sequence coverage of all components of the Ragulator/RAG GTPases complex strongly indicated that SLC38A9 was an additional uncharacterized member. When co-expressed in HEK293T cells, SLC38A9 co-immunoprecipitated with LAMTOR1 and overexpressed LAMTOR1 bound endogenous SLC38A9 (Fig. 1b-c). We validated complex membership entirely at the endogenous level in different cell lines. Immunoprecipitation of SLC38A9 resulted in the specific recruitment of endogenous RAGA and LAMTOR1 and, conversely, immunoprecipitated RAGA bound SLC38A9 (Fig. 1d). This association was not observed when SLC38A9 was silenced, confirming specificity. Association of endogenous SLC38A9 and RAGA was demonstrated in HeLa and K562 cells (Fig. 1e-f) and in murine NIH/3T3 fibroblasts and RAW 264.7 macrophages (Extended Data Fig. 2g-h). To further challenge specificity, we applied the identical proteomic strategy to the two highest expressed members of the SLC38 family, SLC38A1 and SLC38A2, and SLC36A1/PAT1, which has been previously associated with the Ragulator/RAG GTPase complex 19 . Despite very high bait recovery, none of the Ragulator/RAG GTPase complex members was identified among the interactors, highlighting that the association of SLC38A9 with this complex is a unique property of this family member (Extended Data 3d). Moreover, when we immunoprecipitated SLC38A9, SLC38A1, SLC38A2, SLC36A1/PAT1 as well as a lysosomal member of the SLC38 family, SLC38A7 20 , and a second member of the SLC36 family SLC36A4/PAT4, only SLC38A9 co-immunoprecipitated endogenous LAMTOR1, LAMTOR3, RAGA and RAGC, with both low and high expression levels (Fig. 1g).
Immunostaining of tagged SLC38A9 in HeLa cells revealed extensive colocalization with the late endosome/lysosome markers LAMP1, CD63 and the late endosome/multivesicular bodies lipid LBPA, but not with early endosome (EEA1) or Golgi (Giantin) markers ( Full membership to this multiprotein complex would entail physical association with any of the several detected members in reciprocal purifications. We performed affinity purification coupled to mass spectrometry with LAMTOR1, 3, 4 and 5, as well as RAGA and RAGC GTPases. At the core of the interacting network obtained by combining the six independent purifications we found all the expected members of the Ragulator/RAG GTPases complex, RAPTOR as well as SLC38A9 (Fig. 2a, Extended Data Fig. 5a). The overall low sequence coverage of SLC38A9 could be ascribed to inefficient proteolytic cleavage of the inaccessible transmembrane portions of the protein as it mirrored the coverage obtained when SLC38A9 was used as bait (Extended Data Fig. 5b-c). The interaction of endogenous SLC38A9 with all baits was confirmed by immunoprecipitation ( Fig. 2b-c). The quality of the proteomic survey was also indicated by detection of the subunit VA0D1 of the v-ATPase complex 7 and the FLCN-FNIP2 complex 11 . Interestingly, we did not detect any other SLC member of the amino acid transporter families in any of the purifications with the members of Ragulator/GTPases complex, indicating that SLC38A9 is, at least in this cellular system, the only prominently interacting SLC.
Deletion studies indicated that the N-terminal cytoplasmic tail of SLC38A9 (amino acids 1-112), devoid of any transmembrane region, was sufficient and required to bind the Ragulator/RAG GTPases complex, whereas this interaction was completely lost when the remaining eleven transmembrane-containing region (113-561), that retains lysosomal localisation, was used (Fig. 2d, Extended Data 4c-d). We further mapped the minimal interacting region to amino acids 31-112 and identified four conserved motifs in this portion (Extended Data 6a-b). Mutation of any of the first three motifs completely abolished binding, while disruption of the fourth had no effect (Fig. 2e, Extended Data 6c). Importantly, none of the described mutations affected lysosomal targeting of SLC38A9 (Extended Data 4e-h). Whereas the N-terminal cytoplasmic region is evolutionary conserved across SLC38A9 proteins, we could not detect any significant homology with the N-terminal region of any other SLC38 family member. These results defined the unique cytoplasmic portion of SLC38A9 as responsible for the interaction with the lysosomal mTOR-activating machinery.
SLC38 family are commonly competent for the transport of glutamine 18,20 which, together with leucine and arginine, are considered the main amino acids involved in the regulation of mTORC1 9,14,21 . We monitored the transporter competence of SLC38A9 towards these amino acids in liposomes reconstituted with purified recombinant SLC38A9 (Extended Data 7a). In proteoliposomes, the cytoplasmic tail located at the outside face of the vesicles corresponding to the orientation observed in lysosomes (Extended Data 7b). Addition of [ 3 H]-glutamine resulted in a time-dependent transport (Fig 3a) that required intraliposomal sodium, but not addition of external sodium, and was most active at acidic pH (pH 5.5-6.5) (Extended Data 7c and 7e, not shown), consistent with the lysosomal localisation of the natural protein. Moreover, point mutation of the putative sodium-binding site (N128A) 22 moderately affected transport (Extended Data 7d). Membrane potential artificially created by potassium gradients in the presence of valinomycin 23 both positive outside or inside did not influence the transport activity of SLC38A9 (not shown). Competition experiments showed that some polar amino acids were capable of competing efficiently for glutamine transport whereas MeAIB, an inhibitor of system A SLC38 family members, had no effect (Fig 3b, (Fig 3c). The low ability of arginine to compete with glutamine transport, as previously reported also for SLC38A7 20 , may reflect differences in binding and/or transport properties for the two amino acids. The initial uptake rate calculated for 10 μM glutamine was 0.42 ± 0.10 nmol/mg of protein/minute, which is moderate when compared to other reconstituted transporters 23 . If a physiological role of SLC38A9 is to assess the intralysosomal availability of amino acids, then it is also relevant to measure its efflux-enabling activity. We monitored efflux of [ 3 H]-glutamine from proteoliposomes and measured a rate of 1.7 ± 0.30 nmol/mg/min (Fig 3d), which is higher than the uptake but overall still lower than several other amino acid transporters measured with the same approach 23 . This suggests that SLC38A9 may be a low capacity transporter similar to SLC38A7 20 and resembling the properties of amino acid sensors described in yeast 24 and Drosophila 25 .
The ability of RAG GTPase heterodimers to recruit mTOR by binding Raptor is critically dependent on the nucleotide loading status and the resulting conformation of the two GTPase partners 5 . By immunoprecipitating different combinations of RAGA/B-RAGC nucleotide-binding mutant heterodimers we could recapitulate the regulated interactions with RAPTOR and LAMTOR proteins 8,11 and observed that SLC38A9 binding to RAG GTPases was dramatically influenced by their mutational state, even more than what was observed for the Ragulator complex (Fig 3e, Extended Data 8). The low affinity nucleotide binding mutants RAGA T21N and RAGB T54N showed a strong increase in SLC38A9 recruitment, contrasting with the behaviour of RAGC S75N that abolished the binding of SLC38A9 to the heterodimer. GTP-bound RAGA Q66L /B Q99L mutants showed also reduced SLC38A9 binding (Fig 3e, Extended Data 8). These results indicate that the interaction of SLC38A9 with the critical GTPases moieties of the complex is highly conformation specific. In cells stably expressing tagged SLC38A9, amino acid starvation strengthened the interaction between SLC38A9 and endogenous RAGC and, to a minor extent, RAGA, without significantly affecting LAMTOR1 and LAMTOR3 recruitment (Fig 3f). Similarly, amino acid stimulation reduced the amount of recruited RAGC and RAGA. Altogether, the amino acid-sensitive character of these binding properties are evocative of the ones exerted by Ragulator 8 and Folliculin 11 and point to a possible function of SLC38A9 in modulating the nucleotide status of the RAG GTPases. Amino acid sensitivity required the transmembrane region, as the recruitment of RAGC by the N-terminal region alone was not affected by amino acid availability (Fig 3g). This is consistent with the notion that the eleven transmembrane helices-encompassing region is the moiety physically engaging amino acids and required to convey sensitivity.
Withdrawal of amino acids results in rapid inactivation of mTORC1. Cells stably expressing SLC38A9 showed sustained mTORC1 activation upon amino acid starvation, as monitored by the phosphorylation of the substrates S6 kinase and ULK-1 (Fig 4a, Extended Data 9a). This resulted in a delayed and reduced induction of autophagy upon amino acid starvation, as shown by quantification of LC3B relocalisation to autophagosomes (Fig 4b, Extended Data 9b), as well as sustained phosphorylation and delayed nuclear translocation of the transcription factor TFEB 26 (Extended Data 9c). Sustained mTOR activity triggered by SLC38A9 expression during starvation was inhibited by Torin 1 (Extended Data 9e). In contrast, the v-ATPase inhibitor Concanamycin A had no effect in this setting, whereas it efficiently blocked mTORC1 activation induced by amino acid stimulation (Extended Data 9e-f). This suggests that the v-ATPase complex and SLC38A9 concur in the control of mTORC1 activity by amino acids. Most likely, the high expression levels of SLC38A9 resulted in an active signalling state that bypasses the v-ATPase input. Indeed, expression of the N-terminal region appears to be sufficient to confer prolonged mTORC1 activation, suggesting that this moiety assumes an active conformation independently of the transmembrane region (Fig 4c, Extended Data 9d). Altogether, the data indicate that SLC38A9 is an upstream positive regulator of mTORC1 function.
Accordingly, silencing of SLC38A9 in HEK293T by shRNA resulted in a reduction of amino acid-induced mTORC1 activation (Fig 4d). Cells left in culture for longer times manifested a weaker phenotype possibly due to compensatory adaptive mechanisms (not shown). We therefore silenced SLC38A9 by small interfering RNA (siRNA) and observed suppression of amino acid-induced mTORC1 activation in both HEK293T and HeLa cells with an efficiency that was comparable with knockdown of LAMTOR1 (Fig 4f, Extended Data 10a). Depletion of SLC38A9 also impaired mTORC1 activation induced by cycloheximide, which mimics amino acid stimulation by blocking protein synthesis and thus inducing accumulation of intracellular amino acids 5 (Fig 4e). This further suggests that SLC38A9 participates in mTORC1 activation at the lysosome rather than contributing to the import of extracellular amino acids at the plasma membrane. Moreover SLC38A9 levels did not appear to be induced upon amino acid starvation, in contrast to several SLCs responsible for importing amino acids at the plasma membrane (Extended Data 10b-c).
Altogether, the work presented here identifies SLC38A9 as a novel integral component of the lysosomal machinery that controls mTORC1 activity in response to amino acids ( Fig  4g). SLC38A9 is the first member of the entire machinery shown to be competent for binding and transporting amino acids. As other solute carrier proteins, it should be eminently druggable 27 . We failed to observe a strong dependence on SLC38A9 in amino acid stimulation-induced mTOR lysosomal recruitment, which could be due to technical reasons or, more intriguingly, to separate, partly independent, mechanisms controlling localisation and activation of mTOR. Together with the adaptation observed upon prolonged SLC38A9 silencing, this suggests that additional sensing components are likely to operate in this pathway. Considering the low transport capacity and the physical association with the Ragulator/RAG GTPase complex, it is reasonable to consider SLC38A9 a transceptor-type of SLC 24,28-30 , reminiscent of yeast amino acid sensors GAP1 and ssy1p, in which amino acid engagement is used for allosteric signal transduction rather than mere transport.

Cells
HEK293T, Raw264.7, NIH/3T3 and K562 cells were obtained from ATCC and DMSZ. HeLa were provided by M. Hentze. HEK293 Flp-In TREx cells that allow doxycyclinedependent transgene expression were from Invitrogen. Cells were kept in DMEM (Sigma) or RPMI medium (PAA Laboratories) supplemented with 10% (v/v) FBS (Invitrogen) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) and checked for mycoplasma by PCR or ELISA.

Transfections, cell lysis, deglycosylation, immunoprecipitations and fractionation
Cells were transfected with Polyfect (Qiagen) and used for experiments after 24 hours. For lysis, cells were resuspended in Nonidet-40 lysis buffer (1% NP-40, 50mM Hepes pH7.4, 250 mM NaCl, 5 mM EDTA, Halt phosphatase inhibitor cocktail (ThermoScientific), one tablet of EDTA-free protease inhibitor (Roche) per 50 ml) on ice for 5 min. Lysates were cleared by centrifugation in a microcentrifuge (13000r.p.m., 10min, 4C). Proteins were quantified with BCA (Pierce). For immunoprecipitations, lysates were precleared on Sepharose6 beads (Sigma) (40 min with rotation, 4 °C) and then incubated either with HA-, V5-or FLAG-coupled beads (3h with rotation, 4 °C) or with primary antibody and protein G-sepharose (GE healthcare) (14h with rotation, 4 °C). Beads were recovered and washed four times with lysis buffer before analysis by SDS-PAGE and immunoblotting. When required, a mouse anti-rabbit IgG (conformation specific) antibody was used for immunoblot and revealed with an anti-mouse HRP-conjugated antibody to avoid detection of immunoglobulin heavy chains. In case of detection of endogenous SLC38A9, samples were treated with PNGase (NEB, 250U for 30 ul, 1h, 37 °C) before SDS-PAGE. Nuclearcytoplasm cell fractionation was performed as previously described 32 .

Generation of stably expressing cells
HEK293T cells expressing codon optimized FLAG-tagged SLC38A9 isoform 1 (GenScript) were generated using a modified pLKO.1 lentiviral vector having a CMV promoter (pLJM60). Lentiviruses were produced by co-transfection of the lentiviral transfer vector with the ΔVPR envelope and CMV VSV-G packaging plasmids into HEK293T cells using the XTremeGene 9 transfection reagent (Roche). The media was changed 24 hours posttransfection to DMEM supplemented with 30% IFS. Virus-containing supernatants were collected 48 and 72 hours after washing, filtered and used for spin infection (2,200 rpm for 1 hour) of target HEK293T cells in presence of 8 μg/mL polybrene. 24 hours after infection, the virus was removed and the cells selected with puromycin. HEK293T expressing codon optimized untagged or SII-HA tagged SLC38A9 (GenScript) full length, C-terminal (113-561) or N-terminal region (aa 1-112) were generated using a modified pMSCV retroviral vector. Cells expressing EGFP-LC3B or TFEB-STHA were generated by infecting FLAG-SLC38A9 or FLAG-METAP2 stable cells using a modified pMSCV retroviral vector and blasticidin selection.

Proteomics
Flp-in HEK293 T-Rex cell lines inducibly expressing SII-HA-tagged SLC38A9, SLC38A1, SLC38A2, SLC36A1, RAGA, RAGC, GFP or LAMTOR complex subunits were generated as described 33 . Tandem affinity STREP-HA purifications were performed as previously described 31 . In brief, cells were stimulated with doxycycline/tetracycline for 24 h to induce expression of SII-HA-tagged bait proteins. LAMTOR3 pulldowns were done using murine protein version and performed after 9h starvation in serum free medium. Protein complexes were isolated by TAP using streptavidin agarose followed by elution with biotin, and a second purification step using HA-agarose beads. Proteins were eluted with 100 mM formic acid, neutralized with triethylammonium bicarbonate (TEAB) and digested with trypsin, and the peptides were analysed by LC-MS/MS.

MS data analysis and interaction data filtering
Peak list data were extracted from RAW files using ProteoWizard (release 3.0.3201 -http:// proteowizard.sourceforge.net/) and searched against human SwissProt database version v2013.01_20130110 (37,261 sequences and common contaminants). The search engines MASCOT (v2.3.02, MatrixScience, London, UK) and Phenyx (v2.5.14, GeneBio, Geneva, Switzerland) 34 were used. The searches were submitted to MASCOT using in-house perl scripts at precursor and fragment ions mass tolerances ±10 ppm and ± 0.6 Da, respectively. Using the high-confidence identifications from this search, precursor and fragment ion masses were recalibrated for a second-pass search on MASCOT and Phenyx with precursor and fragment ions mass tolerances ±4 ppm and ± 0.3 Da, respectively. One tryptic missedcleavage was permitted. Carbamidomethyl cysteine and oxidized methionine were set as fixed and variable modifications, respectively. A false discovery rate of < 0.25% and < 0.1% were used for proteins and peptides, respectively, as described 35 . SAINT AP-MS filtering software 36 was used to filter the interactions using GFP TAP as negative control. All prey proteins with a SAINT AvgP of > 0.95 were identified as high-confidence interactors. In addition, proteins with a spectral count of 1 or a CRAPome 37 frequency of >0.1 were excluded. For the LAMTOR-RAG network, we retained only those proteins that interacted with all the bait proteins (RAGD was not detected in RAGC pulldown).

Immunofluorescence
HEK293T cells were plated on fibronectin-coated glass coverslips and, after 16 hours, induced with doxycycline. After 24h, cells were washed with PBS, fixed (PBS, 4% formaldehyde) and permeabilized (PBS, 0.3% Saposin, 10% FBS). Slides were incubated with anti-HA (sc-805 Santa Cruz), anti-LAMP1 (ab25630 Abcam) or anti-LAMP2 (sc-18822 Santa Cruz) antibodies (1 hour, 25 °C, PBS, 0.3% Saposin, 10% FBS). After three washes slides were incubated with goat anti-mouse AlexaFluor568 or anti-rabbit AlexaFlour488 antibodies (Invitrogen, 1 hour, 25 °C, PBS, 0.3% Saposin, 10% FBS). After DAPI staining, slides were washed three times and mounted on coverslips with ProLong Gold (Invitrogen). Images were taken with a Zeiss Laser Scanning Microscope (LSM) 700. Images were exported from lsm files to tiff files, and analysed using custom Matlab code. Nuclei and cell outlines were detected based on the DAPI and combined immunofluorescence stains respectively, and colocalization measurements were restricted to cytoplasmic regions. Colocalization was measured as the percentage of SCL38A9 (green) pixel values above background that are also above background in the LAMP1 or LAMP2 (red) channel. The SLC38A9 and LAMP1 or LAMP2 colocalization was verified to be robust to variations in the background threshold, and also shows up as significant pixel value correlations between the red and green channels.
HeLa cells were seeded to 80% confluency for transfection (Lipofectamine LTX, Invitrogen). 24hours post transfection, cells were split into glass coverslips and incubated for another 24hours. Cells were then washed with PBS and fixed in 4% PFA in cytoskeleton buffer (20mM Pipes pH6.8, 150mM NaCl, 5mM EGTA, 5mM glucose and 10mM MgCl2). Permeabilization and blocking were preformed simultaneously by incubating the cells in cytoskeleton buffer supplemented with 0,025% saponin and 50mM NH4Cl. Cells were then incubated with primary antibodies diluted in blocking buffer for 2hours at RT. Cells were then washed 6 times in cytoskeleton buffer supplemented with 50mM NH4Cl and incubated with the secondary antibodies diluted in blocking buffer for 45 minutes at RT. Upon washing 6 times in cytoskeleton buffer supplemented with 50mM NH4Cl, the coverslips were mounted using Vectashield hardening medium (vectorlabs). Z-stack images were taken with an SP5 Laser Scanning confocal Microscope (Leica) and the 63x oil immersion objective (na 1.4). Original images were deconvoluted using Huygens professional Deconvolution and Analysis Software (Scientific Volume imaging). The Z-stack ids files were then visualized in ImageJ (open source version), converted into a color stack image and a representative Z plane was selected. The single plane images were finally converted to Adobe Photoshop CS6 format. Representative cells are shown in all figures at the same exposure and magnification.

Cell size and autophagosome measurements
HEK293T cells transduced with shRNA against SLC38A9 or GFP cells were seeded 24 h before fixation (PBS, 4% formaldehyde), permeabilized (PBS, 0.3% Saposin, 10% FBS) and stained with DAPI. Images were taken by automated microscopy using the PerkinElmer Operetta with 20x magnification in confocal mode. Images were analysed using CellProfiler (www.cellprofiler.org), CellClassifier (http://www.pelkmanslab.org/?page_id=63), Population Context measurement code (https://www.pelkmanslab.org/?page_id=1150) and custom Matlab code written specifically for this study. CellProfiler was used to detect individual nuclei on each image, and iterative machine learning using CellClassifier was applied to detect properly segmented interphase nuclei. Population context measurement code was used to measure the local cell density of each individual cell, and cell size measurements were restricted to sparse cells to avoid local crowding from confounding the measurements. We used the typical nucleus diameter (i.e. the diameter of a circle with the same area as that measured for each nucleus) as a robust proxy for cell size 38 . We confirmed that the cell size reduction induced by SLC38A9 shRNA treatment were present for a broad range of different local cell densities. EGFP-LC3B and SLC38A9 or METAP2 expressing cells were seeded in 96 well plates for imaging. After 24 hour cells were washed with PBS and starved for amino acid and serum for the indicated time. Three by three images were acquired per well with the Operetta at 20x magnification on living cells to minimize disruption of EGFP-LC3B positive autophagosomes. After imaging cells were fixed, DAPI stained and reimaged. Autophagosomes were quantified from the GFP channel using custom Matlab analysis, based on a thresholding of the integrated Laplacian of Gaussian transformation for diameters between 8 and 30 pixels. Candidate spots with a local GFPsignal enrichment of less than 42% were discarded, and remaining spots were considered autophagosomes, and normalized to the cell number and area for each condition. Each condition was measured in three replicate wells accounting for over 85000 cells. Adjusted hill curves were fit and data were normalized to the maximum fitted value in the METAP2 control cell line.

Cell proliferation measurements
HEK293T cells transduced with shRNA against SLC38A9 or GFP were seeded and counted every 24 h using Casy (Roche).

Amino acids starvation and stimulation
HEK293T cells grown in RPMI were washed with PBS and starvation was performed by incubating the cells for 50 min in amino acid-free RPMI without serum. Cell were then stimulated for 10 or 20 min by the addition of RPMI containing a two time concentrated solution of amino acids. After stimulation, the final concentration of amino acids in the media was the same as in RPMI. In case of cycloheximide treatment, amino acid-starved cells were stimulated by addition of cycloheximide diluted in amino acid-free RPMI at a final concentration of 25 μg/ml. HeLa cells grown in RPMI were stimulated for 10 or 20 min by the addition of RPMI containing a two time concentrated solution of amino acids and insulin (1 uM final concentration, Sigma, I9278). Concanamycin A (sc202111 Santa Cruz) was used at 5μM and Torin 1 (4247, Tocris Bioscience,) at 250 nM. Amino acid-free RPMI medium powder (R8999-04A, US biological) was complemented with sodium bicarbonate and sodium phosphate, dissolved in water, adjusted to pH7.4 and filtered. RPMI containing a two time concentrated solution of amino acids was obtained by complementing amino acid-free RPMI medium with RPMI 1640 amino acids solution (R7131, Sigma), adjusted to pH7.4 and filtered. L-glutamine (59202C, Sigma) was added shortly before usage.

RNA sequencing
RNA was extracted using TRI reagent (SIGMA), treated with DNAse I (DNA free kit, Ambion) and RiboZero kit (Epicentre) to remove ribosomal RNA. The library was prepared using ScriptSeq kit version 1 (Epicentre -strand specific library). Sequencing was performed on Illumina HiSeq 2000. Sequence reads in fastq format was aligned against RefSeq hg19 build (as downloaded on 2.9.2011) using TopHat and FPKM values were calculated using Cufflinks.

Cloning, expression and purification of recombinant human SLC38A9
The human SLC38A9 cDNA was optimized according to E. coli codon usage by GenScript. In this optimized gene, the Codon Adaptation Index (CAI) was upgraded from 0.63 (wild type) to 0.87, the GC content and unfavourable peaks were optimized to prolong the half-life of the mRNA and a ribosome binding site was removed. The optimized cDNA was then sub-cloned cloned into expression vector (pH6EX3-His 6 -hSLC38A9) 39 . The plasmid was used to transform E. coli Lemo21(DE3)pLysS (NEB). Selection on LB-agar was performed as previously described 39 . 0.1 mM rhamnose was added to modulate RNA polymerase expression. After addition of 0.4 mM IPTG cells were grown at 39°C for 2 h. Cells were treated as previously described 39 . The protein patterns of the cell lysate fractions were analyzed by SDS-PAGE. The insoluble cell fraction (about 1.5 mg proteins) from cells expressing SLC38A9 or empty vector transfected cells, was washed with 100 mM Tris/HCl and resuspended in 100 mM β-ME, 3.5 M urea, 0.5 % sarkosyl, 200 mM NaCl, 10% glycerol, 20 mM Tris/HCl pH 8.0 and centrifuged at 12,000 g for 10 min at 4 °C. The resulting supernatant (about 1 mL) was applied onto a column (0.5 cm × 2.5) filled with His select nickel affinity gel (Sigma) pre-conditioned with 8 mL of 0.1 % sarkosyl, 200 mM NaCl, 10% glycerol, 10 mM Tris/HCl pH 8.0. The elution was performed with 10 mL of 0.1 % C 12 E 8 , 150 mM NaCl, 10% glycerol, 5 mM DTE, 10 mM Tris/HCl pH 8.0 (washing buffer), 1.4 mL of the same buffer plus10 mM imidazole; then the purified protein fraction (4-7 μg protein) was eluted by 1.4 mL of the same buffer plus 50 mM imidazole.

Reconstitution of SLC38A9 in proteoliposomes and transport measurements
The purified fractions from SLC38A9 or empty vector preparation were reconstituted by removing the detergent as previously described 40 with a batch-wise procedure from a mixture of 400 μL of protein (about 2 μg protein in 0.1 % C 12 E 8 , β-ME 6 mM, 10% glycerol, 20 mM Tris/HCl pH 8.0, 150 mM NaCl, 50 mM imidazole), 80 μL of 10 % C 12 E 8 , 100 μL of 10% egg yolk phospholipids (w/v), 20 mM Hepes/Tris pH 6.5. 600 μL of proteoliposomes were passed through a Sephadex G-75 column (0.7 cm diameter × 15 cm height) preequilibrated with 20 mM Hepes/Tris pH 6.5. Transport (uptake) measurement was started adding 10 μM [ 3 H]glutamine or other radioactive substrates as indicated (0.5 μCi/nmol) to 100 μL proteoliposomes aliquots at 25°C. Transport was stopped by applying each sample of proteoliposomes on a Sephadex G-75 column (0.6 × 8 cm) to separate the external from the internal radioactivity. In competition experiments, the indicated amino acids (1 mM) were added together with [ 3 H]-glutamine (10 μM) and transport was measured at 60 min. For efflux measurements, aliquots of the same pool of proteoliposomes passed through a Sephadex G-75 column (0.7 cm diameter × 15 cm height) preequilibrated with 20 mM Hepes/Tris pH 6.5 were incubated with external 10 μM [ 3 H]glutamine. After 120 min of loading, proteoliposomes were passed again through a Sephadex G-75 column (0.7 cm diameter × 15 cm height) preequilibrated with 20 mM Hepes/Tris pH 6.5, for removing the residual external radioactivity. The time course of [ 3 H]glutamine efflux was then measured stopping the efflux reaction at each time interval by applying proteoliposome samples on a Sephadex G-75 column (0.6 × 8 cm) to separate the external from the internal radioactivity. In both uptake and efflux assays, proteoliposomes eluted with 1 mL 50 mM NaCl were collected in scintillation cocktail for counting. The amount of reconstituted recombinant protein was estimated as previously described 39 . Time course data were interpolated by a first order rate equation from which the initial rate of transport was calculated as k × transport at equilibrium. L-Glutamine [3,

Orientation of SLC38A9 in proteoliposomes
After purification, His-SLC38A9 was incubated overnight at 37°C in absence or in presence of 1 U thrombin (GE healthcare) and then assayed by immunoblotting using anti-His or anti-SLC38A9 antibody. To assess the orientation of SLC38A9, reconstituted proteoliposomes were centrifuged at 108.000 × g for 90 minutes, resuspended in 20 mM Hepes/Tris pH 6.5, incubated overnight at 37°C with 1 U thrombin in the same conditions of the purified protein. After incubation proteoliposomes were dissolved by 2.5% SDS and 0.2M Tris/HCl pH 6.8. and immunoblotting analysis was performed as described for the purified protein.

Assay of pH and intraliposomal sodium dependence of the SLC38A9 function
Reconstitution was performed in 20 mM Hepes/Tris buffer at different pH. Transport (uptake) was started by adding to proteoliposomes10 μM [ 3 H]-glutamine in 20 mM Hepes/ Tris buffer at the same pH of the reconstitution mixture and stopped after 30 min. To test sodium dependency, SLC38A9 was purified omitting NaCl from elution buffer. Reconstitution was performed in the absence or in the presence of 20 or 50 mM NaCl and transport (uptake) measurement was performed.

Statistical analysis
A normal distribution of data was assumed and appropriate test were applied.

Europe PMC Funders Author Manuscripts
Europe PMC Funders Author Manuscripts   were starved for the indicated time. LC3B positive autophagosomes were quantified by image analysis. Data were normalized to cell size and plotted relative to the fitted METAP2 maximum. Mean ± s.d of at least three replicate wells. c. HEK293T cells stably expressing the indicated untagged SLC38A9 constructs were treated and analysed as in a. d-e, HEK293T cells transduced with lentivirus-encoded shRNA against SLC38A9 or GFP were starved for 50 min and then stimulated with amino acids (d) or cycloheximide (e, 25μg/ml) for 10 or 20 min. Cell lysates were analysed by immunoblot. f, HEK293T were transfected with siRNA targeting SLC38A9, LAMTOR1 or non targeting control. After 72h, cells were treated as in d.
In (e-f lower) cell lysates were treated with PNGase; *: non-specific band. g, Schematic model of SLC38A9 function in amino acid-induced mTORC1 activation. Results are representative of two (a-e, n=2) or three (f) independent experiments (n=3).