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

Abstract

The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth.

Keywords: amino acid sensing; autophagy; lysosome; mTOR; micropinocytosis; nutrient sensing.

Figure 1. see also Figure S1: 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 HEK-293T 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). HEK-293T cells were transfected with the indicated cDNAs and lysates 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 minutes were stimulated for 10 minutes 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).

Figure 2. see also Figure S2: Arginine, at concentrations found in lysosomes, promotes the interaction of SLC38A9 with Rag-Ragulator

(A) Whole-cell and lysosomal arginine and leucine concentrations. HEK-293T cells were starved of the indicated amino acid for 50 minutes and re-stimulated with it for 10 minutes. The RPMI condition represents the non-starved state. Whole-cell and lysosomal arginine and leucine concentrations (μM) were measured using the LysoIP method described in 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 μM.

Figure 3. see also Figure S3: Many essential amino acids accumulate in lysosomes lacking SLC38A9

(A) The rapid immuno-isolation method for lysosomes (LysoIP) yields pure lysosomes from wild-type and SLC38A9-null HEK-293T cells as monitored by immunoblotting for protein markers of various subcellular compartments. Lysates and immunoprecipitates were prepared from HEK-293T cells expressing 2xFLAG-TMEM192 (Control-Lyso cells) or 3XHA-TMEM192 (HA-Lyso cells) as described in the methods. (B) Many essential amino acids accumulate in the lysosomes of SLC38A9-null HEK-293T cells. Fold changes are relative to concentrations in wild-type HEK-293T cells and bar graphs show mean ± SEM (n=3; *p<0.05). (C) Overexpression of SLC38A9, but not the control protein metap2, reduces the lysosomal concentrations of most non-polar, essential amino acids (phenylalanine, leucine, isoleucine, tryptophan, and methionine) as well as tyrosine. Fold changes are relative to concentrations in the control metap2-overexpressing HEK-293T cells and bar graphs show mean ± SEM (n= 3; *p<0.05). (D) Expression of wild-type SLC38A9 or its Rag-Ragulator-binding mutant I68A, but not the transport-deficient T133W mutant, reverses the increase in lysosomal amino acid concentrations caused by loss of SLC38A9. Aspartate was used as a control amino acid as it is unaffected by SLC38A9 loss. Lysosomes were analyzed as in (B) and bar graphs are mean ± SEM (n=3; *p<0.05).

Figure 4. see also Figure S4: SLC38A9 is an arginine-regulated high affinity transporter for leucine

(A) In vitro SLC38A9 transports arginine with a K_m of ∼4 mM in the improved transport assay described in the methods. Experiment was repeated more than three times with similar results, and a representative example is shown. (B) In vitro SLC38A9 transports leucine with a K_m of ∼90 μM. Experiment was repeated more than three times with similar results, and a representative example is shown. (C) Steady-state kinetic analysis of SLC38A9-mediated leucine transport in the presence of 200 μM arginine, but not glycine, reveals an arginine-induced increase in V_max from ∼220 to ∼470 pmol min^-1. Velocity, as shown, was calculated as a function of the leucine concentration. The experiment was repeated three times, with a representative example shown.

Figure 5. see also Figure S5: Arginine regulates the lysosomal concentrations of many essential amino acids via SLC38A9

(A) Arginine, but not leucine, deprivation increases the lysosomal concentrations of many of the same amino acids that are affected by loss of SLC38A9. Fold changes are relative to concentrations in cells cultured in RPMI and bar graphs show mean ± SEM (n=3, *p<0.05). HEK-293T cells were incubated in full RPMI media or in RPMI lacking the indicated amino acid for 60 minutes and lysosomes were purified and analyzed as described in methods. Cystine was used as a control metabolite. (B) Deprivation of arginine or leucine activates autophagy to similar extents. HEK-293T cells were treated as in (A) in the absence or presence of chloroquine and lysates were analyzed for LC3B processing. (C) Arginine re-addition time-dependently reverses the increase caused by arginine starvation in the lysosomal concentrations of the indicated amino acids. HEK-293T cells deprived of arginine for 50 minutes were re-stimulated with arginine for the indicated times. Fold changes are relative to concentrations in cells cultured in RPMI and bar graphs show mean ± SEM (n=3, *p<0.05). Cystine served as a control metabolite. (D) In cells lacking SLC38A9 or expressing the transport-deficient T133W mutant, arginine deprivation does not further increase the already high lysosomal concentrations of the SLC38A9-regulated amino acids. Wild-type and SLC38A9-null HEK-293T cells were analyzed as in (A) and fold changes are relative to cells cultured in RPMI. Bar graphs show mean ± SEM (n=3, *p<0.05). Cystine served as a control metabolite. (E-F) In vitro, arginine, but not several other amino acids, promotes the release of leucine from lysosomes in a fashion that requires SLC38A9 and its transport function. (E) Purified lysosomes still attached to beads were loaded with [³H]Leucine in vitro for 15 minutes and then stimulated with 500 μM of the indicated amino acid for 10 minutes. The amount of [³H]Leucine released was quantified and normalized to the total amount of [³H]leucine in lysosomes. This amount was obtained in two ways that gave the same value: by measuring the [³H]Leucine in the bead-bound lysosomes not simulated with an amino acid after the 15 minute loading period or in the supernatant of lysosomes lysed with distilled water (dH₂0 lysis). (F) Arginine does not induce leucine release in lysosomes lacking SLC38A9 or containing the T133W mutant. The experiment was performed as in (E) using lysosomes from the appropriate cell lines.

Figure 6. see also Figure S6: 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 HEK-293T cells were deprived of leucine for either 50 minutes or the indicated time points, and where specified, restimulated for 10 minutes 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 HEK-293T cells were deprived of leucine for 50 minutes or the indicated time points and, where indicated, re-stimulated with leucine for 10 minutes. Cell lysates were analyzed by immunoblotting for the levels and phosphorylation states of 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 HEK-293T cells stably expressing the indicated proteins were starved for leucine for 50 minutes or 8 hours and, where indicated, re-stimulated with leucine for 10 minutes. 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. Fold changes are relative to concentrations of cells in cultured in RPMI. Bar graphs show mean ± SEM (n=3).

Figure 7. SLC38A9 and its transport function are required for albumin to activate mTORC1 and support cell proliferation and for pancreatic tumor growth

(A-B) Loss of SLC38A9 or just its transport capacity prevents the activation of mTORC1 induced by extracellular protein. Murine KRAS^G12D/+P53^-/- pancreatic cancer cells that are wild-type, null for SLC38A9, or SLC38A9-null and expressing T133W SLC38A9, were deprived of leucine for 50 minutes and re-stimulated with leucine for 10 minutes or 3% albumin for the times indicated. Cell lysates were analyzed by immunoblotting for the levels or phosphorylation states of indicated proteins. (C) Loss of SLC38A9 inhibits the proliferation of pancreatic cancer cells cultured in 3% albumin as the leucine source. Wild-type and SLC38A9-null murine KRAS^G12D/+P53^-/- pancreatic cancer cells were cultured for 3 days in media lacking leucine, and supplemented, where indicated, with 3% albumin. Cells were counted every 24 hours and bar graphs show mean ± SD (n=3; *p<0.05). (D) KRAS^G12D/+P53^-/- pancreatic cancer cells lacking SLC38A9 or expressing its transport deficient T133W mutant have a severe defect in forming tumors in an orthotropic allograft model of pancreatic cancer. SLC38A9-null KRAS^G12D/+P53^-/- PaCa cells expressing the control protein metap2, SLC38A9, or SLC38A9 T133W were used to generate tumors. Each dot represents the calculated tumor volume (mm³) of an individual tumor. The mean ± SEM (n= 9-11, *p<0.0001) is shown. (E) Tumors formed by KRAS^G12D/+P53^-/- pancreatic cancer cells lacking SLC38A9 or expressing its transport deficient T133W mutant have decreased mTORC1 signaling. Tumors were analyzed by immunohistochemistry for S6 pS235/S236 levels and stained with hematoxyline and eosin (H&E) (10× and 40× magnifications are shown, arrow defines pancreatic cancer cells shown in 40× insets). Scale bars represent 100 μM (10×) and 20 μM (40×). (F) A model depicting how arginine signals through SLC38A9 to promote mTORC1 activation as well as the lysosomal efflux of essential amino acids like leucine.