Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1

Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase-dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid-sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.

Figure 1

Interaction of SLC38A9.1 with Ragulator and the Rag GTPases. (A) The spectral counts of SLC38A9-derived peptides detected by mass spectrometry in immunoprecipitates prepared from HEK-293T cells stably expressing the indicated FLAG-tagged proteins. (B) Schematic depicting SLC38A9 isoforms and truncation mutants. Transmembrane domains predicted by the TMHMM (transmembrane hidden Markov model) algorithm (http://www.cbs.dtu.dk/services/TMHMM/) are shown as blue boxes. (C) Effects of PNGase F treatment of SLC38A9.1 on its electrophoretic migration. (D) Interaction of full-length SLC38A9.1 or its N-terminal domain with endogenous Ragulator (p18 and p14) and RagA and RagC GTPases. HEK-293T cells were transfected with the indicated cDNAs in expression vectors and lysates were prepared and subjected to FLAG immunoprecipitation followed by immunoblotting for the indicated proteins. (E) Identification of key residues in the N-terminal domain of SLC38A9.1 required for it to interact with Ragulator and the Rag GTPases. Experiment was performed as in (D) using indicated SLC38A9.1 mutants. (F) Interaction of SLC38A9.1 with v-ATPase components V0d1 and V1B2. HEK-293T cells stably expressing the indicated FLAG-tagged proteins were lysed and processed as in (D).

Figure 2

Localization of SLC38A9.1 to the lysosomal membrane in an amino acid-independent fashion and requirement of SLC38A9 for mTORC1 pathway activation by amino acids. (A) SLC38A9.1 localization in cells deprived of or replete with amino acids. HEK-293T cells stably expressing FLAG-SLC38A9.1 were starved and stimulated with amino acids for the indicated times. Cells were processed and immunostained for LAMP2 and FLAG-SLC38A9.1. (B) Requirement of SLC38A9 for the activation of the mTORC1 pathway by amino acids. HEK-293T cells expressing indicated short hairpin RNAs (shRNAs) were deprived of amino acids for 50 min or deprived of and then re-stimulated with amino acids for 10 min. Cell lysates were analyzed for the levels of indicated proteins and the S6K1 phosphorylation state.

Figure 3

Stable overexpression of full-length SLC38A9.1 or its N-terminal Ragulator-binding domain makes the mTORC1 pathway insensitive to amino acid deprivation. (A) Stable overexpression of FLAG-SLC38A9.1 largely restores mTORC1 signaling during total amino acid starvation and completely restores it upon deprivation of leucine or arginine. HEK-293T cells transduced with lentiviruses encoding the specified proteins were deprived for 50 min of all amino acids, leucine, or arginine and, where indicated, re-stimulated for 10 min with the missing amino acid(s). Cell lysates were analyzed for the levels of the specified proteins and the phosphorylation states of S6K1, ULK1, and 4E-BP1. (B and C) Overexpression of neither SLC38A9.2 nor a point mutant of SLC38A9.1 that fails to bind Ragulator rescues mTORC1 signaling during amino acid starvation. Experiment was performed as in (A) except that cells were stably expressing SLC38A9.2 (B) or SLC38A9.1 I68A (C). (D) Stable overexpression of the Ragulator-binding domain of SLC38A9.1 largely restores mTORC1 signaling during total amino acid starvation and completely rescues it upon deprivation of leucine or arginine. Experiment was performed as in (A) except cells were stably expressing FLAG-SLC38A9.1 1-119. (E) The ability of SLC38A9.1 overexpression to rescue mTORC1 signaling during amino acid starvation is eliminated by co-expression of RagB^T54N-RagC^Q120L, a Rag heterodimer locked in the nucleotide configuration associated with amino acid deprivation. Effects of expressing the indicated proteins on mTORC1 signaling were monitored by the phosphorylation state of co-expressed FLAG-S6K1. (F) Effects of concanamycin A and Torin1 on mTORC1 signaling in cells stably expressing SLC38A9.1. HEK-293T cells stably expressing the indicated FLAG-tagged proteins were treated with the DMSO vehicle or the specified small molecule inhibitor during the 50 min starvation for and, where indicated, the 10 min stimulation with amino acids.

Figure 4

Modulation of the interaction between SLC38A9 and Ragulator and the Rag GTPases by amino acids. (A) Effects of amino acids on interaction between the Ragulator complex and endogenous SLC38A9. HEK-293T cells stably expressing the indicated FLAG-tagged Ragulator components were deprived of total amino acids, leucine, or arginine for 1 hour and, where indicated, re-stimulated with amino acids, leucine, or arginine for 15 min. After lysis, samples were subject to FLAG immunoprecipitation and immunoblotting for the indicated proteins. Quantification of SLC38A9 levels in the stimulated state relative to starved state, p14 IP: 0.75 (+AA), 0.79 (+L), 0.74 (+R); p18 IP: 0.56 (+AA), 0.57 (+L), 0.49 (+R). (B) Effects of amino acids on the interaction between full-length or truncated SLC38A9.1 and endogenous Ragulator and the Rag GTPases. Experiment was performed as in (A) except that cells stably expressed the indicated SLC38A9 isoforms or its N-terminal domain (SLC38A9.1 1-119). Quantification of indicated protein levels in the stimulated state relative to starved state, SLC38A9.1 IP: 0.43 (p18), 0.51 (p14), 0.61 (RagC), 0.58 (RagA); SLC38A9.1 1-119 IP: 0.99 (p18), 1.05 (p14), 1.04 (RagC), 1.09 (RagA). (C) Effects of the RagB^T54N mutation on association with endogenous SLC38A9. HEK-293T cells were transfected with the indicated cDNAs in expression vectors and lysates were prepared and subjected to FLAG immunoprecipitation followed by immunoblotting for the indicated proteins. Two different antibodies were used to detect endogenous SLC38A9.

Figure 5

SLC38A9.1 is a low affinity amino acid transporter and is necessary for mTORC1 pathway activation by arginine. (A) Time-dependent uptake of [³H]arginine at 0.5 μM by proteoliposomes containing 22.4 pmol of SLC38A9.1. To recapitulate the pH gradient across the lysosomal membrane, the lumen of the proteoliposomes is buffered at pH 5.0, while the external buffer is pH 7.4. (B) Steady-state kinetic analysis of SLC38A9.1 uptake activity reveals a Michaelis constant (K_m) of ~39mM and catalytic rate constant (k_cat) of ~1.8min⁻¹. (Left) Time course of [³H]arginine (R*) uptake, given fixed [³H]arginine (0.5 μM) and increasing concentrations of unlabeled arginine. (Right) Velocity, calculated from left panel, as a function of total arginine concentration. Data were fitted to the Michaelis-Menton equation. Experiment was repeated over 4 times with similar results and a representative one is shown. (C) Time-dependent efflux of SLC38A9.1 proteoliposomes following 1.5 hr loading with 0.5 μM [³H]arginine. (D) Competition of 0.5 μM [³H]arginine transport by SLC38A9.1 using 100 mM of indicated unlabeled amino acids. In A-D, error bars represent standard deviation derived from at least 3 measurements. (E) HEK-293T cells null for SLC38A9 were generated using CRISPR-Cas9 genome editing using two different guide sequences and isolated by single cell cloning. The AAVS1 locus was targeted as a negative control. (F) Impairment of arginine-induced activation of the mTORC1 pathway in SLC38A9-null HEK-293T cells. Cells were starved of the indicated amino acid for 50 minutes and stimulated for 10 minutes using the indicated amino acid concentrations. The leucine and arginine concentrations in RPMI are, respectively, 381 μM and 1.14 mM. (G) Model for distinct amino acid inputs to the Rag GTPases in signaling amino acid sufficiency to mTORC1.