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

Rebsamen M, Pochini L, Stasyk T, de Araújo ME, Galluccio M, Kandasamy RK, Snijder B, Fauster A, Rudashevskaya EL, Bruckner M, Scorzoni S, Filipek PA, Huber KV, Bigenzahn JW, Heinz LX, Kraft C, Bennett KL, Indiveri C, Huber LA, Superti-Furga G - Nature (2015)

Bottom Line: 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, whereas 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.

View Article: PubMed Central - PubMed

Affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

ABSTRACT
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 responses. 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. 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, whereas 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.

Show MeSH

Related in: MedlinePlus

Biochemical and functional characterisation of SLC38A9a-b, Where indicated, HEK293T cells were transfected with the tagged SLC38A9 constructs (+) or empty vector (−). Cell lysates were left untreated (Untr.) or incubated 1 hour at 37 °C in presence or absence of PNGase and analysed by immunoblot. Results are representative of two independent experiments (n=2) c, Cell size measurements of HEK293T cells after short-hairpin (shRNA) mediated knockdown against GFP (control, dashed black line) or SLC38A9 (grey line), measured by automated microscopy and image analysis. Sparse and interphase cells were selected using image analysis and machine learning, and nucleus diameter was used as robust proxy for cell size38. Smoothed distributions of 2400 and 4165 cells, respectively, are shown. d, Cell proliferation measurement of HEK293T cells transduced with lentivirus-encoded shRNA against SLC38A9 or GFP. 105 cells were seeded and counted every 24 h. Mean values ± s.d. from triplicates. Results are representative of two independent experiments (n=2). e-f, Where indicated, HEK293T cells were transfected with the tagged SLC38A9. Cell lysates were prepared and left untreated (Untr.) or incubated 1 hour at 37 °C in presence or absence of PNGase and analysed by immunoblot, Where indicated, cell lysates were boiled for 5 min at 95 °C after PNGase treatment. g-h, Lysates from murine NIH/3T3 (g) or Raw 264.7 (h) cells were subjected to immunoprecipitation with the indicated antibodies, treated with PNGase and analysed by immunoblot. Results are representative of two independent experiments (n=2). <: SLC38A9; *: non-specific band.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4376665&req=5

Figure 6: Biochemical and functional characterisation of SLC38A9a-b, Where indicated, HEK293T cells were transfected with the tagged SLC38A9 constructs (+) or empty vector (−). Cell lysates were left untreated (Untr.) or incubated 1 hour at 37 °C in presence or absence of PNGase and analysed by immunoblot. Results are representative of two independent experiments (n=2) c, Cell size measurements of HEK293T cells after short-hairpin (shRNA) mediated knockdown against GFP (control, dashed black line) or SLC38A9 (grey line), measured by automated microscopy and image analysis. Sparse and interphase cells were selected using image analysis and machine learning, and nucleus diameter was used as robust proxy for cell size38. Smoothed distributions of 2400 and 4165 cells, respectively, are shown. d, Cell proliferation measurement of HEK293T cells transduced with lentivirus-encoded shRNA against SLC38A9 or GFP. 105 cells were seeded and counted every 24 h. Mean values ± s.d. from triplicates. Results are representative of two independent experiments (n=2). e-f, Where indicated, HEK293T cells were transfected with the tagged SLC38A9. Cell lysates were prepared and left untreated (Untr.) or incubated 1 hour at 37 °C in presence or absence of PNGase and analysed by immunoblot, Where indicated, cell lysates were boiled for 5 min at 95 °C after PNGase treatment. g-h, Lysates from murine NIH/3T3 (g) or Raw 264.7 (h) cells were subjected to immunoprecipitation with the indicated antibodies, treated with PNGase and analysed by immunoblot. Results are representative of two independent experiments (n=2). <: SLC38A9; *: non-specific band.

Mentions: Amino acids are essential for mTORC1 activity, as growth factors cannot efficiently activate mTOR in their absence5,9. 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 elusive1,2,4,9-13. 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 activity14, 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 transport15 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 staining16 and had been associated to lysosomes by proteomic analysis17 (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 families18 (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).


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

Rebsamen M, Pochini L, Stasyk T, de Araújo ME, Galluccio M, Kandasamy RK, Snijder B, Fauster A, Rudashevskaya EL, Bruckner M, Scorzoni S, Filipek PA, Huber KV, Bigenzahn JW, Heinz LX, Kraft C, Bennett KL, Indiveri C, Huber LA, Superti-Furga G - Nature (2015)

Biochemical and functional characterisation of SLC38A9a-b, Where indicated, HEK293T cells were transfected with the tagged SLC38A9 constructs (+) or empty vector (−). Cell lysates were left untreated (Untr.) or incubated 1 hour at 37 °C in presence or absence of PNGase and analysed by immunoblot. Results are representative of two independent experiments (n=2) c, Cell size measurements of HEK293T cells after short-hairpin (shRNA) mediated knockdown against GFP (control, dashed black line) or SLC38A9 (grey line), measured by automated microscopy and image analysis. Sparse and interphase cells were selected using image analysis and machine learning, and nucleus diameter was used as robust proxy for cell size38. Smoothed distributions of 2400 and 4165 cells, respectively, are shown. d, Cell proliferation measurement of HEK293T cells transduced with lentivirus-encoded shRNA against SLC38A9 or GFP. 105 cells were seeded and counted every 24 h. Mean values ± s.d. from triplicates. Results are representative of two independent experiments (n=2). e-f, Where indicated, HEK293T cells were transfected with the tagged SLC38A9. Cell lysates were prepared and left untreated (Untr.) or incubated 1 hour at 37 °C in presence or absence of PNGase and analysed by immunoblot, Where indicated, cell lysates were boiled for 5 min at 95 °C after PNGase treatment. g-h, Lysates from murine NIH/3T3 (g) or Raw 264.7 (h) cells were subjected to immunoprecipitation with the indicated antibodies, treated with PNGase and analysed by immunoblot. Results are representative of two independent experiments (n=2). <: SLC38A9; *: non-specific band.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4376665&req=5

Figure 6: Biochemical and functional characterisation of SLC38A9a-b, Where indicated, HEK293T cells were transfected with the tagged SLC38A9 constructs (+) or empty vector (−). Cell lysates were left untreated (Untr.) or incubated 1 hour at 37 °C in presence or absence of PNGase and analysed by immunoblot. Results are representative of two independent experiments (n=2) c, Cell size measurements of HEK293T cells after short-hairpin (shRNA) mediated knockdown against GFP (control, dashed black line) or SLC38A9 (grey line), measured by automated microscopy and image analysis. Sparse and interphase cells were selected using image analysis and machine learning, and nucleus diameter was used as robust proxy for cell size38. Smoothed distributions of 2400 and 4165 cells, respectively, are shown. d, Cell proliferation measurement of HEK293T cells transduced with lentivirus-encoded shRNA against SLC38A9 or GFP. 105 cells were seeded and counted every 24 h. Mean values ± s.d. from triplicates. Results are representative of two independent experiments (n=2). e-f, Where indicated, HEK293T cells were transfected with the tagged SLC38A9. Cell lysates were prepared and left untreated (Untr.) or incubated 1 hour at 37 °C in presence or absence of PNGase and analysed by immunoblot, Where indicated, cell lysates were boiled for 5 min at 95 °C after PNGase treatment. g-h, Lysates from murine NIH/3T3 (g) or Raw 264.7 (h) cells were subjected to immunoprecipitation with the indicated antibodies, treated with PNGase and analysed by immunoblot. Results are representative of two independent experiments (n=2). <: SLC38A9; *: non-specific band.
Mentions: Amino acids are essential for mTORC1 activity, as growth factors cannot efficiently activate mTOR in their absence5,9. 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 elusive1,2,4,9-13. 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 activity14, 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 transport15 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 staining16 and had been associated to lysosomes by proteomic analysis17 (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 families18 (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).

Bottom Line: 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, whereas 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.

View Article: PubMed Central - PubMed

Affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

ABSTRACT
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 responses. 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. 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, whereas 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.

Show MeSH
Related in: MedlinePlus