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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.

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SLC38A9 is a lysosomal component of the amino acid-sensing machinery controlling mTORC1a, Interactors of SLC38A9 identified by TAP–LC-MS/MS. Data shown are based on two independent experiments (n=2), each analysed in two technical replicates. b-g, Lysates from HEK293T cells transfected with the indicated tagged constructs or empty vector (−) (b, c, g), control (empty vector or shGFP) or shSLC38A9 transduced HEK293T (d) and HeLa (e), or K562 (f) cells were subjected to immunoprecipitation. PNGase-treated immunoprecipitates (IP) and protein extracts (XT) analysed by immunoblot with the indicated antibodies. Results are representative of two independent experiments (n=2) <: ST-HA-SLC38A9; *: non-specific band h-j, Confocal microscopy images of HeLa cells transfected with tagged SLC38A9 construct and immunostained with the anti-HA and LAMP1(h), EEA1(i) or Giantin (j) antibodies. Representative cells are shown. Scale bar, 10 μm.
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Figure 1: SLC38A9 is a lysosomal component of the amino acid-sensing machinery controlling mTORC1a, Interactors of SLC38A9 identified by TAP–LC-MS/MS. Data shown are based on two independent experiments (n=2), each analysed in two technical replicates. b-g, Lysates from HEK293T cells transfected with the indicated tagged constructs or empty vector (−) (b, c, g), control (empty vector or shGFP) or shSLC38A9 transduced HEK293T (d) and HeLa (e), or K562 (f) cells were subjected to immunoprecipitation. PNGase-treated immunoprecipitates (IP) and protein extracts (XT) analysed by immunoblot with the indicated antibodies. Results are representative of two independent experiments (n=2) <: ST-HA-SLC38A9; *: non-specific band h-j, Confocal microscopy images of HeLa cells transfected with tagged SLC38A9 construct and immunostained with the anti-HA and LAMP1(h), EEA1(i) or Giantin (j) antibodies. Representative cells are shown. Scale bar, 10 μm.

Mentions: To test whether SLC38A9 would associate with the complex regulating mTORC1, we engineered HEK293 cells to express tagged SLC38A9 in an inducible fashion and verified the localisation of the protein to lysosomes (Extended Data Fig. 3a-c). We purified endogenously assembled protein complexes using tandem affinity purification (TAP) coupled to one-dimensional gel-free liquid chromatography tandem mass spectrometry (LC–MS/MS). The gel-free approach was critical as upon boiling SLC38A9 formed insoluble aggregates that failed to enter SDS-polyacrylamide gels (Extended Data Fig. 2e-f). The analysis identified all the five members of the Ragulator/LAMTOR complex and the four RAG GTPases as specific interactors of SLC38A9 (Fig. 1a, Extended Data 3d). Such collective high sequence coverage of all components of the Ragulator/RAG GTPases complex strongly indicated that SLC38A9 was an additional uncharacterized member. When co-expressed in HEK293T cells, SLC38A9 co-immunoprecipitated with LAMTOR1 and overexpressed LAMTOR1 bound endogenous SLC38A9 (Fig. 1b-c). We validated complex membership entirely at the endogenous level in different cell lines. Immunoprecipitation of SLC38A9 resulted in the specific recruitment of endogenous RAGA and LAMTOR1 and, conversely, immunoprecipitated RAGA bound SLC38A9 (Fig. 1d). This association was not observed when SLC38A9 was silenced, confirming specificity. Association of endogenous SLC38A9 and RAGA was demonstrated in HeLa and K562 cells (Fig. 1e-f) and in murine NIH/3T3 fibroblasts and RAW 264.7 macrophages (Extended Data Fig. 2g-h). To further challenge specificity, we applied the identical proteomic strategy to the two highest expressed members of the SLC38 family, SLC38A1 and SLC38A2, and SLC36A1/PAT1, which has been previously associated with the Ragulator/RAG GTPase complex19. Despite very high bait recovery, none of the Ragulator/RAG GTPase complex members was identified among the interactors, highlighting that the association of SLC38A9 with this complex is a unique property of this family member (Extended Data 3d). Moreover, when we immunoprecipitated SLC38A9, SLC38A1, SLC38A2, SLC36A1/PAT1 as well as a lysosomal member of the SLC38 family, SLC38A720, and a second member of the SLC36 family SLC36A4/PAT4, only SLC38A9 co-immunoprecipitated endogenous LAMTOR1, LAMTOR3, RAGA and RAGC, with both low and high expression levels (Fig. 1g).


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)

SLC38A9 is a lysosomal component of the amino acid-sensing machinery controlling mTORC1a, Interactors of SLC38A9 identified by TAP–LC-MS/MS. Data shown are based on two independent experiments (n=2), each analysed in two technical replicates. b-g, Lysates from HEK293T cells transfected with the indicated tagged constructs or empty vector (−) (b, c, g), control (empty vector or shGFP) or shSLC38A9 transduced HEK293T (d) and HeLa (e), or K562 (f) cells were subjected to immunoprecipitation. PNGase-treated immunoprecipitates (IP) and protein extracts (XT) analysed by immunoblot with the indicated antibodies. Results are representative of two independent experiments (n=2) <: ST-HA-SLC38A9; *: non-specific band h-j, Confocal microscopy images of HeLa cells transfected with tagged SLC38A9 construct and immunostained with the anti-HA and LAMP1(h), EEA1(i) or Giantin (j) antibodies. Representative cells are shown. Scale bar, 10 μm.
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Figure 1: SLC38A9 is a lysosomal component of the amino acid-sensing machinery controlling mTORC1a, Interactors of SLC38A9 identified by TAP–LC-MS/MS. Data shown are based on two independent experiments (n=2), each analysed in two technical replicates. b-g, Lysates from HEK293T cells transfected with the indicated tagged constructs or empty vector (−) (b, c, g), control (empty vector or shGFP) or shSLC38A9 transduced HEK293T (d) and HeLa (e), or K562 (f) cells were subjected to immunoprecipitation. PNGase-treated immunoprecipitates (IP) and protein extracts (XT) analysed by immunoblot with the indicated antibodies. Results are representative of two independent experiments (n=2) <: ST-HA-SLC38A9; *: non-specific band h-j, Confocal microscopy images of HeLa cells transfected with tagged SLC38A9 construct and immunostained with the anti-HA and LAMP1(h), EEA1(i) or Giantin (j) antibodies. Representative cells are shown. Scale bar, 10 μm.
Mentions: To test whether SLC38A9 would associate with the complex regulating mTORC1, we engineered HEK293 cells to express tagged SLC38A9 in an inducible fashion and verified the localisation of the protein to lysosomes (Extended Data Fig. 3a-c). We purified endogenously assembled protein complexes using tandem affinity purification (TAP) coupled to one-dimensional gel-free liquid chromatography tandem mass spectrometry (LC–MS/MS). The gel-free approach was critical as upon boiling SLC38A9 formed insoluble aggregates that failed to enter SDS-polyacrylamide gels (Extended Data Fig. 2e-f). The analysis identified all the five members of the Ragulator/LAMTOR complex and the four RAG GTPases as specific interactors of SLC38A9 (Fig. 1a, Extended Data 3d). Such collective high sequence coverage of all components of the Ragulator/RAG GTPases complex strongly indicated that SLC38A9 was an additional uncharacterized member. When co-expressed in HEK293T cells, SLC38A9 co-immunoprecipitated with LAMTOR1 and overexpressed LAMTOR1 bound endogenous SLC38A9 (Fig. 1b-c). We validated complex membership entirely at the endogenous level in different cell lines. Immunoprecipitation of SLC38A9 resulted in the specific recruitment of endogenous RAGA and LAMTOR1 and, conversely, immunoprecipitated RAGA bound SLC38A9 (Fig. 1d). This association was not observed when SLC38A9 was silenced, confirming specificity. Association of endogenous SLC38A9 and RAGA was demonstrated in HeLa and K562 cells (Fig. 1e-f) and in murine NIH/3T3 fibroblasts and RAW 264.7 macrophages (Extended Data Fig. 2g-h). To further challenge specificity, we applied the identical proteomic strategy to the two highest expressed members of the SLC38 family, SLC38A1 and SLC38A2, and SLC36A1/PAT1, which has been previously associated with the Ragulator/RAG GTPase complex19. Despite very high bait recovery, none of the Ragulator/RAG GTPase complex members was identified among the interactors, highlighting that the association of SLC38A9 with this complex is a unique property of this family member (Extended Data 3d). Moreover, when we immunoprecipitated SLC38A9, SLC38A1, SLC38A2, SLC36A1/PAT1 as well as a lysosomal member of the SLC38 family, SLC38A720, and a second member of the SLC36 family SLC36A4/PAT4, only SLC38A9 co-immunoprecipitated endogenous LAMTOR1, LAMTOR3, RAGA and RAGC, with both low and high expression levels (Fig. 1g).

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