Journal: Cell

Article Title: mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient.

doi: 10.1016/j.cell.2017.09.046

Figure Lengend Snippet: Figure 1. A Mutant of SLC38A9 that Does Not Interact with Arginine Cannot Signal Arginine Sufficiency to mTORC1 (A) Schematic depicting domains of SLC38A9 and the location of the I68A and T133W point mutations. Transmembrane segment 1 of SLC38A9 shares sequence similarity with members of the APC superfamily of transporters. F13H10.3 is likely the C. elegans homolog of SLC38A9. (B) The T133W, but not the I68A, mutant of SLC38A9 is deficient in arginine transport in vitro. SDS-PAGE and Coomassie blue staining was used to analyze recombinant proteins purified from HEK293T cells. (C) Interaction of wild-type SLC38A9 and the T133W mutant, but not the Ragulator-Rag-binding mutant I68A or the control protein metap2, with endogenous Ragulator (p18 and p14) and Rag GTPases (RagA and RagC). HEK293T cells were transfected with the indicated cDNAs, and lysates were prepared and subjected to anti-FLAG immunoprecipitation and analyzed by immunoblotting. (D) Loss of SLC38A9 inhibits activation of mTORC1 by arginine, but not leucine. Cells starved of the indicated amino acid for 50 min were stimulated for 10 min with leucine or arginine, and cell lysates analyzed for the specified proteins and phosphorylation states. (E) For arginine to activate mTORC1 signaling, SLC38A9 must be able to interact with both arginine and Rag-Ragulator. Wild-type and SLC38A9-null cells stably expressing the indicated proteins were analyzed as in (D). See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Glutathione Agarose Pierce 16100 Cholorquine Sigma C6628-25G Experimental Models: Cell Lines HEK293T ATCC CRL-3216 Mia-PaCa Gift from Rushika Perera N/A 8988T Gift from Rushika Perera N/A KP4 Gift from Rushika Perera N/A KRAS G12D/+ P53 / mouse PaCa cells Gift from Matthew G. Vander Heiden N/A HeLa ATCC ATCC CCL-2 Experimental Models: Organisms/Strains Male C57BL/6J mice 6-8 weeks Charles River 027 Oligonucleotides Primer sgSLC38A9 mouse Fwd: ATGCTATGTGTAT AGTCCAT This paper N/A Primer sgSLC38A9 mouse Rev: ATGGACTATACAC ATAGCAT This paper N/A Recombinant DNA pLJM1-FLAG-metap2 This Paper N/A pLJM60-FLAG-SLC38A9 Wang et al., 2015 Addgene 71858 pLJM60-FLAG-SLC38A9 I68A Wang et al., 2015 Addgene 71864 pLJM60-FLAG-SLC38A9 T133W This Paper #101833 pLJM60-FLAG-SLC38A9 delta110 Wang et al., 2015 Addgene 71861 pLJC5-FLAG-SLC38A9 This Paper N/A pLJC5-3XHA-TMEM192 This Paper #102930 pLJC5-2XFLAG-TMEM192 This Paper #102929 pLJC6-3XHA-TMEM192 This Paper N/A pLJC6-2XFLAG-TMEM192 This Paper N/A Software and Algorithms Prism version 6.0.1 GraphPad https://www.graphpad.com Other PD10 gel filtration Column General Electric 17085101 Extruder Set with Holder/Heating Block Avanti 610000

Techniques: Mutagenesis, Sequencing, In Vitro, SDS Page, Staining, Recombinant, Binding Assay, Control, Transfection, Immunoprecipitation, Western Blot, Activation Assay, Phospho-proteomics, Stable Transfection, Expressing

Journal: Cell

Article Title: mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient.

doi: 10.1016/j.cell.2017.09.046

Figure Lengend Snippet: Figure 2. Arginine, at Concentrations Found in Lysosomes, Promotes the Interaction of SLC38A9 with Rag-Ragulator (A) Whole-cell and lysosomal arginine and leucine concentrations. HEK293T cells were starved of the indicated amino acid for 50 min and re-stimulated with it for 10 min. The RPMI condition represents the non-starved state. Whole-cell and lysosomal arginine and leucine concentrations (mM) were measured using the LysoIP method described in the STAR Methods. Bar graphs show mean ± SEM (n = 3). (B) In vitro, arginine promotes the interaction of SLC38A9 with the Rag-Ragulator complex in a dose-dependent manner. Purified HA-GST-RagC/HA-RagB and HA-Ragulator were immobilized on glutathione affinity resin and incubated with FLAG-SLC38A9 in the presence of the indicated concentrations of arginine. HA- GST-Rap2A was used as a control. Proteins captured in the glutathione resin pull-down were analyzed by immunoblotting for the indicated proteins using anti- epitope tag antibodies. (C) Arginine and lysine, but not other amino acids, promote the interaction of SLC38A9 with Rag-Ragulator in vitro. Experiment was performed as in (B), except that all amino acids were at 1 mM. (D) Arginine does not promote the interaction of SLC38A9 T133W with Rag-Ragulator. The experiment was performed as in (B), except that arginine was used at 500 mM. See also Figure S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Glutathione Agarose Pierce 16100 Cholorquine Sigma C6628-25G Experimental Models: Cell Lines HEK293T ATCC CRL-3216 Mia-PaCa Gift from Rushika Perera N/A 8988T Gift from Rushika Perera N/A KP4 Gift from Rushika Perera N/A KRAS G12D/+ P53 / mouse PaCa cells Gift from Matthew G. Vander Heiden N/A HeLa ATCC ATCC CCL-2 Experimental Models: Organisms/Strains Male C57BL/6J mice 6-8 weeks Charles River 027 Oligonucleotides Primer sgSLC38A9 mouse Fwd: ATGCTATGTGTAT AGTCCAT This paper N/A Primer sgSLC38A9 mouse Rev: ATGGACTATACAC ATAGCAT This paper N/A Recombinant DNA pLJM1-FLAG-metap2 This Paper N/A pLJM60-FLAG-SLC38A9 Wang et al., 2015 Addgene 71858 pLJM60-FLAG-SLC38A9 I68A Wang et al., 2015 Addgene 71864 pLJM60-FLAG-SLC38A9 T133W This Paper #101833 pLJM60-FLAG-SLC38A9 delta110 Wang et al., 2015 Addgene 71861 pLJC5-FLAG-SLC38A9 This Paper N/A pLJC5-3XHA-TMEM192 This Paper #102930 pLJC5-2XFLAG-TMEM192 This Paper #102929 pLJC6-3XHA-TMEM192 This Paper N/A pLJC6-2XFLAG-TMEM192 This Paper N/A Software and Algorithms Prism version 6.0.1 GraphPad https://www.graphpad.com Other PD10 gel filtration Column General Electric 17085101 Extruder Set with Holder/Heating Block Avanti 610000

Techniques: In Vitro, Incubation, Control, Western Blot

Journal: Cell

Article Title: mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient.

doi: 10.1016/j.cell.2017.09.046

Figure Lengend Snippet: Figure 6. SLC38A9 Is Required for Amino Acids Produced via Autophagy to Activate mTORC1 and to Support Cell Proliferation (A) Loss of ATG7 prevents the autophagy-mediated reactivation of mTORC1 that occurs after long-term leucine deprivation. Wild-type and ATG7-null HEK293T cells were deprived of leucine for either 50 min or the indicated time points and, where specified, re-stimulated for 10 min with leucine. Cell lysates were analyzed by immunoblotting for the total levels and phosphorylation states of the indicated proteins. (B) Loss of SLC38A9 prevents the autophagy-mediated reactivation of mTORC1 that occurs after long-term leucine deprivation. Wild-type or SLC38A9-null HEK293T cells were deprived of leucine for 50 min or the indicated time points and, where indicated, re-stimulated with leucine for 10 min. Cell lysates were analyzed by immunoblotting for the levels and phosphorylation states of the indicated proteins. (C) mTORC1 signaling does not reactivate after long-term leucine deprivation in cells expressing the T133W SLC38A9 mutant. Wild-type or SLC38A9-null HEK293T cells stably expressing the indicated proteins were starved for leucine for 50 min or 8 hr and, where indicated, re-stimulated with leucine for 10 min. Lysates were analyzed as in (A). (D) In cells lacking SLC38A9, lysosomal leucine concentrations do not drop upon starvation for leucine despite its depletion at the whole-cell level. Metabolite profiling of lysosomes from wild-type and SLC38A9-null cells deprived of leucine for the indicated times is shown. Fold changes are relative to concentrations of cells cultured in RPMI. Bar graphs show mean ± SEM (n = 3). See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Glutathione Agarose Pierce 16100 Cholorquine Sigma C6628-25G Experimental Models: Cell Lines HEK293T ATCC CRL-3216 Mia-PaCa Gift from Rushika Perera N/A 8988T Gift from Rushika Perera N/A KP4 Gift from Rushika Perera N/A KRAS G12D/+ P53 / mouse PaCa cells Gift from Matthew G. Vander Heiden N/A HeLa ATCC ATCC CCL-2 Experimental Models: Organisms/Strains Male C57BL/6J mice 6-8 weeks Charles River 027 Oligonucleotides Primer sgSLC38A9 mouse Fwd: ATGCTATGTGTAT AGTCCAT This paper N/A Primer sgSLC38A9 mouse Rev: ATGGACTATACAC ATAGCAT This paper N/A Recombinant DNA pLJM1-FLAG-metap2 This Paper N/A pLJM60-FLAG-SLC38A9 Wang et al., 2015 Addgene 71858 pLJM60-FLAG-SLC38A9 I68A Wang et al., 2015 Addgene 71864 pLJM60-FLAG-SLC38A9 T133W This Paper #101833 pLJM60-FLAG-SLC38A9 delta110 Wang et al., 2015 Addgene 71861 pLJC5-FLAG-SLC38A9 This Paper N/A pLJC5-3XHA-TMEM192 This Paper #102930 pLJC5-2XFLAG-TMEM192 This Paper #102929 pLJC6-3XHA-TMEM192 This Paper N/A pLJC6-2XFLAG-TMEM192 This Paper N/A Software and Algorithms Prism version 6.0.1 GraphPad https://www.graphpad.com Other PD10 gel filtration Column General Electric 17085101 Extruder Set with Holder/Heating Block Avanti 610000

Techniques: Produced, Western Blot, Phospho-proteomics, Expressing, Mutagenesis, Stable Transfection, Cell Culture