Amino Acid-Dependent mTORC1 Regulation by the Lysosomal Membrane Protein SLC38A9

Abstract

The serine/threonine kinase mTORC1 regulates cellular homeostasis in response to many cues, such as nutrient status and energy level. Amino acids induce mTORC1 activation on lysosomes via the small Rag GTPases and the Ragulator complex, thereby controlling protein translation and cell growth. Here, we identify the human 11-pass transmembrane protein SLC38A9 as a novel component of the Rag-Ragulator complex. SLC38A9 localizes with Rag-Ragulator complex components on lysosomes and associates with Rag GTPases in an amino acid-sensitive and nucleotide binding state-dependent manner. Depletion of SLC38A9 inhibits mTORC1 activity in the presence of amino acids and in response to amino acid replenishment following starvation. Conversely, SLC38A9 overexpression causes RHEB (Ras homolog enriched in brain) GTPase-dependent hyperactivation of mTORC1 and partly sustains mTORC1 activity upon amino acid deprivation. Intriguingly, during amino acid starvation mTOR is retained at the lysosome upon SLC38A9 depletion but fails to be activated. Together, the findings of our study reveal SLC38A9 as a Rag-Ragulator complex member transducing amino acid availability to mTORC1 activity.

FIG 1

Interaction proteomics of regulatory mTORC1 components. (A to C) Grouped interaction network schemes obtained by IP-MS analysis of 12 baits consisting of the Rag GTPases (A), all Ragulator subunits (B), and TSC (C) and their high-confidence candidate interacting proteins (HCIPs; average APSM of ≥3 and WD^N score of ≥1). All interactions are color-coded according to functional group (red, mTORC1; blue, Ragulator; green, Rag proteins; orange, TSC; black, SLC38A9). Line thickness indicates interactions with WD^N scores between 1 and 10. Differential lysis with MCLB NP-40 (solid lines) or MCLB CHAPS (dashed lines) buffer is indicated. See Table S1A and B and Fig. S1 in the supplemental material for complete proteomic data.

FIG 2

SLC38A9 associates with the Rag-Ragulator complex. (A) Overview of the regulatory mTORC1 interaction network obtained by IP-MS analysis of 14 baits consisting of Raptor, TSC, LAMTOR1-5, and Rag GTPases, as well as SLC38A9. Functional groups and lysis buffer are coded as described in the legend to Fig. 1. SLC38A9 HCIPs are marked by black edging. Interactions with full-length SLC38A9 or its N-terminal fragment (aa 1 to 119) are indicated in black and gray, respectively. Schematic representation of the domain structure of SLC38A9 is provided. See Table S1A and B and Fig. S1 in the supplemental material for complete proteomic data. (B) 293T-REx cells inducibly expressing HA-tagged components of the Rag-Ragulator complex or TSC2 were transfected with GFP-SLC38A9 or left untransfected (mock). NP-40 cell lysates were subjected to immunoprecipitation with anti-GFP or anti-HA beads as indicated and analyzed by SDS-PAGE and immunoblotting. *, IgG heavy chain. (C and D) 293T cells were lysed with HEPES buffer followed by immunoprecipitation with anti-SLC38A9, anti-RRAGC, or anti-myc as a control. Coimmunoprecipitated proteins were separated by SDS-PAGE and analyzed by immunoblotting (C) or subjected to in-gel trypsin digestion and mass spectrometric analysis (D). The number of peptides is shown from 0 to 15 peptides per protein. Immunoprecipitated bait proteins are marked by red edging. See Table S1C in the supplemental material for complete proteomic data.

FIG 3

Cytosolic N terminus of SLC38A9 mediates association with the Rag-Ragulator complex. 293T-REx cells inducibly expressing the indicated HA-tagged Rag or LAMTOR proteins were transfected with GFP-SLC38A9 full length, GFP-SLC38A9 aa 1 to 119, or GFP alone. NP-40 cell lysates were subjected to immunoprecipitation with anti-GFP beads, and coimmunoprecipitated proteins were detected by SDS-PAGE and immunoblotting.

FIG 4

SLC38A9 localizes to lysosomes. (A) 293T cells transfected with GFP-SLC38A9 full length or GFP-SLC38A9 aa 1 to 119 were fixed and stained with anti-LAMP2 antibody. Scale bar, 10 μm. Pearson's correlation coefficient (R) was calculated in Excel after measuring pixel intensity in different channels of the overlay image along a line in ImageJ. A.U., arbitrary units. (B) 293T cells transfected with nontargeting control (sicon) or SLC38A9 siRNA were fixed and labeled with anti-SLC38A9 and anti-LAMP2 antibodies. Scale bar, 10 μm. R was calculated as described for panel A.

FIG 5

Amino acid-independent colocalization of SLC38A9 with the Rag-Ragulator complex. 293T or 293T-REx cells inducibly expressing HA-tagged components of the Rag-Ragulator complex were transfected with control (sicon) or SLC38A9 siRNA. Prior to fixation and labeling with anti-HA or anti-LAMP2 and anti-SLC38A9 antibody, cells were cultured for 50 min in full RPMI growth medium (fed) or amino acid starved. Scale bar, 10 μm. Pixel intensity was measured in different channels of the overlay image along a line in ImageJ, and Pearson's correlation coefficient (R) was calculated in Excel.

FIG 6

Association of SLC38A9 with Rag GTPases is amino acid and nucleotide dependent. (A) 293T-REx cells inducibly expressing HA-tagged Rag GTPase proteins were transfected with GFP-SLC38A9 full length, GFP-SLC38A9 aa 1 to 119, or GFP alone. Cells were either grown for 50 min in full RPMI growth medium (fed) or were amino acid (aa) starved and subsequently lysed with MCLB NP-40 for GFP immunoprecipitation. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting. (B) 293T cells were transfected with nontargeting control (sicon) or SLC38A9 siRNAs and myc-tagged LAMTOR2, as well as wild-type (RRAGB wild-type [RB wt], RRAGC wild-type [RC wt], RRAGA wild type [RA wt]) or mutant (RRAGB Q99L [RB QL], RRAGB T75L [RB TL], RRAGC Q120L [RC QL], RRAGC S54L [RC SL]) HA-tagged Rag GTPase proteins, as indicated. Cells were lysed in HEPES buffer, subjected to HA-immunoprecipitation, and analyzed as described above. (C) 293T-REx cells inducibly expressing HA-tagged RRAGC, LAMTOR1, or Raptor (marked with red edging) were transfected with control (sicon) or SLC38A9 siRNA as indicated, followed by lysis in MCLB CHAPS buffer and immunoprecipitation with HA beads. Eluates were analyzed by MS. The number of peptides is shown from 0 to 100 peptides per protein. See Table S1D in the supplemental material for complete proteomic data. (D) Cells like those described for panel C were lysed in HEPES buffer followed by immunoprecipitation, SDS-PAGE, and immunoblotting. (E) Raptor overexpressing 293T-REx cells were transfected as described for panel C, lysed in MCLB CHAPS buffer, and analyzed as described for panel D.

FIG 7

SLC38A9 regulates mTORC1 activity. (A and B) 293T cells were transfected with different nontargeting control (sicon) or SLC38A9 siRNAs as indicated. Cell lysates were subjected to SDS-PAGE and immunoblotting. The phosphorylation status of S6K (A), ULK1 (A), and 4EBP1 (A and B) was determined using phospho-specific antibodies. PCNA served as a loading control. exp., exposure. (C) SLC38A9 knockdown in panels A and B additionally was confirmed by RT-qPCR. geomean, geometrical means. (D) 293T cells were transfected with HA-SLC38A9 full length, HA-SLC38A9 aa 1 to 119, or empty vector. Forty-eight hours posttransfection, cells were lysed and the phosphorylation status of mTORC1 substrates was determined using phospho-specific antibodies. PCNA served as a loading control. (E) Cells transfected as described for panel D were treated with Torin1 (1 h, 250 nM) or dimethyl sulfoxide (DMSO) as a control and analyzed as described for panel D. (F) Stable shRNA-mediated RHEB GTPase depletion or control 293T cells were transfected with HA-SLC38A9 full length or empty vector, respectively, followed by SDS-PAGE and immunoblotting. mTORC1 activity was assessed by determining the phosphorylation status of S6K. (G) RHEB GTPase knockdown shown in panel F was confirmed by RT-qPCR.

FIG 8

SLC38A9 regulates mTORC1 activity upon amino acid availability. (A and B) Seventy-two hours after transfection with nontargeting control (sicon) or SLC38A9 siRNA, 293T cells were treated with CHX (2 h, 2 μg/ml) or DMSO as a control (A) or were fed or amino acid starved for 50 min (B), as indicated. Cell lysates were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. PCNA served as the loading control. (C) Lysates from 293T cells transfected with HA-SLC38A9 or empty vector were fed or amino acid starved for 50 min and analyzed by immunoblotting with the indicated antibodies. PCNA served as a loading control. (D) 293T cells transfected with nontargeting control (sicon) or SLC38A9 siRNA were either subjected to amino acid starvation for 50 min or amino acid starved for 50 min and then replenished for 10 min with the indicated amino acids. The phosphorylation status of ULK1 was determined using a phospho-specific antibody. PCNA served as the loading control. An asterisk indicates a nonspecific band.

FIG 9

SLC38A9 depletion affects mTOR relocation from lysosomes to the cytosol. (A to D) 293T cells transfected with nontargeting control (sicon) or SLC38A9 siRNA were fixed and labeled with anti-mTOR and anti-LAMP2 antibodies. Scale bar, 10 μm. (A) Example images. Quantification of mTOR (B and C)- and LAMP2 (D)-positive spots for a total of at least 40,000 (n = 4) (B), 110,000 (n = 5) (C), or 19,000 (n = 3) (D) cells was performed. norm to sicon/fed, normalized to sicon under fed conditions. Data represent means ± SEM, and statistical analysis was performed using GraphPad Prism. Statistical significance was determined with two-way ANOVA followed by Bonferroni's post hoc test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (E) 293T cells were transfected with nontargeting control (sicon) or SLC38A9 siRNA. Seventy-two hours posttransfection, cells were lysed and LAMP2 protein levels were analyzed by SDS-PAGE and immunoblotting. PCNA served as a loading control. (F) SLC38A9 interacts with the Rag-Ragulator complex via its N-terminal cytosolic domain depending on the cellular amino acid status, thereby regulating amino acid-dependent mTOR localization and mTORC1 activity.