Abstract
Mechanistic target of rapamycin complex 1 (TORC1) integrates nutrient signals to control cell growth and organismal homeostasis across eukaryotes1,2,3,4. The evolutionarily conserved GATOR complex regulates mTORC1 signalling through Rag GTPases, and GATOR1 displays GTPase activating protein (GAP) activity for RAGA and RAGB (RAGA/B) and GATOR2 has been proposed to be an inhibitor of GATOR15,6. Furthermore, the metazoan-specific SESN proteins function as guanine nucleotide dissociation inhibitors (GDIs) for RAGA/B, and interact with GATOR2 with unknown effects7,8,9. Here we show that SZT2 (seizure threshold 2), a metazoan-specific protein mutated in epilepsy10,11,12,13, recruits a fraction of mammalian GATOR1 and GATOR2 to form a SZT2-orchestrated GATOR (SOG) complex with an essential role in GATOR- and SESN-dependent nutrient sensing and mTORC1 regulation. The interaction of SZT2 with GATOR1 and GATOR2 was synergistic, and an intact SOG complex was required for its localization at the lysosome. SZT2 deficiency resulted in constitutive mTORC1 signalling in cells under nutrient-deprived conditions and neonatal lethality in mice, which was associated with failure to inactivate mTORC1 during fasting. Hyperactivation of mTORC1 in SZT2-deficient cells could be partially corrected by overexpression of the GATOR1 component DEPDC5, and by the lysosome-targeted GATOR2 component WDR59 or lysosome-targeted SESN2. These findings demonstrate that SZT2 has a central role in dictating GATOR-dependent nutrient sensing by promoting lysosomal localization of SOG, and reveal an unexpected function of lysosome-located GATOR2 in suppressing mTORC1 signalling through SESN recruitment.
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Change history
16 May 2018
In the originally published version of this Letter, the actin blot in Extended Data Fig. 7e was a duplicate of the blot in Extended Data Fig. 7f. This has been corrected online.
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Acknowledgements
We thank H. Erdjument-Bromage at the Microchemistry and Proteomics Core for help with the mass spectrometry experiments, and J. Xie for the WDR59 antibody. This work was supported by a Leukemia & Lymphoma Society Scholar Award (M.O.L.), a Functional Genomics Initiative Grant from Memorial Sloan Kettering Cancer Center (M.O.L.), a Faculty Scholar grant from the Howard Hughes Medical Institute (M.O.L.), and the Memorial Sloan Kettering Cancer Center Support Grant/Core Grant (P30 CA008748).
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Contributions
M.P. and M.O.L. conceived the project. M.P. designed and performed most experiments with input from M.O.L. N.Y. performed experiments. M.P. and M.O.L. analysed the data. M.P. wrote and M.O.L. edited the manuscript.
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The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 SZT2 is essential for mTORC1 inactivation upon nutrient deprivation.
a, b, Control (sgGFP) or SZT2-deficient HeLa cells (a) or HEK293T (b) cells were deprived of amino acids and/or glucose for 1 h and stimulated with amino acids and/or glucose for 10 min where indicated. Total cell lysates were analysed by immunoblotting. c, The size of control and SZT2-deficient HEK293E cells was determined by flow cytometry. d, HEK293T cells stably overexpressing Flag–RAP2A or Flag–SZT2 cells were analysed as in a. e, Control or SZT2-deficient HeLa cells were deprived of amino acids for 1 h and stimulated with amino acids for 10 min where indicated. The localization of mTOR and LAMP2 was determined by immunostaining. f, Quantification of the co-localization between mTOR and LAMP2. Data represent mean ± s.d. 100 cells were analysed from each condition (two-tailed unpaired t-test). NS, not significant. g, Control or SZT2-deficient HEK293T cells were transfected with the indicated constructs and selected with puromycin. Cells were deprived of amino acids for 1 h, and stimulated with amino acids for 10 min where indicated. Total cell lysates were analysed by immunoblotting. Data (a–e, g) are representative of three independent experiments.
Extended Data Figure 2 SZT2 forms a complex with GATOR.
a, Mass spectrometry analysis of SZT2-interacting proteins in HEK293T cells stably overexpressing Flag–RAP2A or Flag–SZT2. Specific recovery of the GATOR complex components is shown. b, c, A diagram of the template (b) and sequence (c) used to introduce a Flag tag into the SZT2 locus. d, Total cell lysates from HEK293T cells, HEK293T cells stably expressing Flag–SZT2, or SZT2Flag/− cells were analysed by immunoblotting. e, Control or SZT2Flag/− cells were deprived of amino acids for 1 h and stimulated with amino acids for 10 min where indicated. Total cell lysates were analysed by immunoblotting. f, Size-exclusion chromatography of the SOG complex purified from SZT2Flag/− cells on a Superose 6 Increase 10/300 GL column. The insert shows the calibration of the column, and the equation used for calculating the size of the SOG complex. g, Peak fraction of SOG from f was analysed by silver staining and immunoblotting. h, Sucrose density gradient centrifugation analysis of the sedimentation pattern of GATOR components from control or SZT2-deficient HEK293T cells. Dashed box indicates fractions containing LAMP1, SZT2, WDR59, WDR24, NPRL3 and NPRL2. Data (a, d–h) are representative of three independent experiments.
Extended Data Figure 3 The integrity of the SOG complex is not regulated by amino acids or SESN2.
a, SZT2Flag/− cells were either untreated, deprived of amino acids for 60 min, deprived of amino acids for 60 min and stimulated with amino acids for 10 or 30 min, or treated with rapamycin (Rapa) for 60 min. Anti-Flag immunoprecipitates and total cell lysates were analysed by immunoblotting. HEK293T cells stably overexpressing Flag–RFP were used as a control. b, Anti-Flag immunoprecipitates prepared from SZT2Flag/− cells were washed three times with buffer containing sodium chloride, and analysed by immunoblotting. HEK293T cells stably overexpressing Flag–RFP were used as a control. c, The leucine-, arginine- or glutamine-binding activity of Flag–RFP, Flag–SZT2 and Flag–SESN2 was determined. Data represent mean ± s.d. (n = 3, two-tailed unpaired t-test). d, The leucine-binding activity of Flag–SESN2 expressed and purified from control or SZT2-deficient HEK293T cells. Data represent mean ± s.d. (n = 3, two-tailed unpaired t-test). e, Control or SZT2-deficient HEK293T cells stably overexpressing Flag–WDR24 were deprived of amino acids for 1 h and stimulated with amino acids for 10 min where indicated. Anti-Flag immunoprecipitates and total cell lysates were analysed by immunoblotting. f, HEK293T cells were transfected with the indicated constructs. Anti-HA immunoprecipitates and total cell lysates were analysed by immunoblotting. HA, haemagglutinin. Data (a, b, e, f) are representative of two independent experiments.
Extended Data Figure 4 An intact SOG complex is essential for mTORC1 regulation.
a, HEK293T cells were transfected with the indicated constructs. Anti-Flag immunoprecipitates and total cell lysates were analysed by immunoblotting. b, SZT2-deficient HEK293T cells were transfected with the indicated constructs, selected with puromycin, and deprived of amino acids for 1 h. Total cell lysates were analysed by immunoblotting. c, d, HEK293T cells were transfected with the indicated constructs. Anti-Flag immunoprecipitates and total cell lysates were analysed by immunoblotting. e, SZT2-deficient HEK293T cells were transfected with the indicated constructs, selected with puromycin, and deprived of amino acids for 1 h. Total cell lysates were analysed by immunoblotting. f, A diagram of SZT2 protein and its truncation mutants, and a summary of their NPRL3 and WDR24 binding capacity. Data (a–e) are representative of three independent experiments.
Extended Data Figure 5 SZT2 is localized on the lysosome, which together with GATOR1 controls lysosomal localization of WDR59.
a, HEK293T or SZT2Flag/− cells were immunostained with anti-Flag together with anti-LAMP1. b, Control or WDR59-deficient HEK293T cells were immunostained with anti-WDR59 together with anti-LAMP2. c–e, SZT2Flag/− cells were deprived of amino acids for 1 h and stimulated with amino acids for 20 min where indicated. Cells were immunostained with anti-Flag (c–e), together with anti-LAMP2 plus anti-WDR59 (c), or anti-LAMP2 plus anti-EEA1 (d), or anti-RAB7 plus anti-PMP70 (e). f, Quantification of the co-localization among SZT2, PMP70, EEA1, RAB7, LAMP2 and WDR59. Data represent mean ± s.d. with 100 cells analysed for each condition. g, DEPDC5-deficient HEK293T cells were deprived of amino acids for 1 h and stimulated with amino acids for 20 min where indicated. The localization of WDR59 and LAMP2 was determined by immunostaining. h, Control, SZT2-deficient, DEPDC5-deficient and NPRL3-deficient cells were deprived of amino acids for 1 h and stimulated with amino acids for 20 min where indicated. Total cell lysates were analysed by immunoblotting. i, mRNA levels of DEPDC5 in control and DEPDC5-deficient HEK293T cells were measured by qPCR and normalized to β-actin. AU, arbitrary units. j, Control or SZT2-deficient HEK293T were treated with rapamycin (100 nM), deprived of amino acids for 1 h and stimulated with amino acids for 20 min where indicated. The localization of WDR59 and LAMP2 was determined by immunostaining. Data (a, b, g–j) are representative of three independent experiments.
Extended Data Figure 6 The roles of GATOR2 components in SZT2 regulation of mTORC1 signalling.
a–c, Cells were deprived of amino acids for 1 h and stimulated with amino acids for 10 min where indicated. Total cell lysates were analysed by immunoblotting. d, Control or SZT2-deficient cells were transfected with LentiCrisprV2 plasmids targeting GFP or SEH1L, selected with puromycin, and analysed as in a. Data (a–d) are representative of three independent experiments.
Extended Data Figure 7 Lysosome-targeted WDR59 suppresses mTORC1 signalling in SZT2-deficient cells.
a–d, h, Control (a, b), SZT2-deficient (c, d), or SZT2- and WDR59-deficient (h) HEK293T cells were transfected with the indicated constructs and selected with puromycin. These cells were deprived of amino acids for 1 h and stimulated with amino acids for 20 min where indicated. Total cell lysates were analysed by immunoblotting. e, Control or WDR59-deficient HEK293T cells were analysed as in a. f, Primary Wdr59+/+ or Wdr59−/− MEFs were analysed as in a. g, mRNA levels of Wdr59 in Wdr59+/+ and Wdr59−/− MEFs were measured by qPCR and normalized to that of β-actin. AU, arbitrary units. HA, haemagglutinin. Data (a–h) are representative of three independent experiments.
Extended Data Figure 8 Lysosome-targeted WDR59 or SESN2 suppresses mTORC1 signalling in GATOR1-deficient cells.
a, b, d, Cells were transfected with the indicated constructs and selected with puromycin. Cells were deprived of amino acids for 1 h and stimulated with amino acids for 20 min where indicated. Total cell lysates were analysed by immunoblotting. c, e, Cells were deprived of amino acids for 1 h and stimulated with amino acids for 20 min where indicated. Total cell lysates were analysed by immunoblotting. Data (a–e) are representative of three independent experiments.
Extended Data Figure 9 SZT2 is essential for mTORC1 inactivation under nutrient-deprived conditions in mice and MEFs.
a, PCR genotyping of Szt2+/+, Szt2+/− and Szt2−/− mice. The expected sizes of wild-type (WT) or Szt2 mutant (KO) alleles are indicated by arrows. b, RT–PCR analysis of mRNA of Szt2 in Szt2+/+ and Szt2−/− MEFs. Primer pair 1 (Szt2-1) detects a region downstream of the gene trap, and primer pair 2 (Szt2-2) detects a region upstream of the gene trap. c, The birth weight of neonates from the indicated genotypes are shown. Data represent mean ± s.d., n = 10–25, two-tailed unpaired t-test. NS, not significant. d, e, Neonates were fasted for 2 h (d) or 10 h (e). Total cell lysates prepared from the indicated organs were analysed by immunoblotting. f, g, Szt2+/+ and Szt2−/− MEFs (<4 passages) were deprived of amino acids for 1 h (f), or amino acid plus glucose for 2 h (g) and stimulated with amino acids (f), or amino acid plus glucose (g) for 10 min where indicated. Total cell lysates were analysed by immunoblotting. h, Szt2+/+ and Szt2−/− MEFs (<4 passages) were cultured in complete medium or starved with amino acids for 16 h and analysed as in f. i, Szt2+/+ and Szt2−/− MEFs (<4 passages) were deprived of amino acids for 1 h and stimulated with amino acids for 10 min where indicated. The localization of mTOR and LAMP2 was determined by immunostaining. j, Szt2+/+ and Szt2−/− MEFs (<4 passages) were cultured in complete medium or serum-starved for 24 h and analysed as in f. Data (f–j) are representative of two independent experiments.
Extended Data Figure 10 SZT2–GATOR–SESN2 in nutrient sensing and mTORC1 signalling.
We propose that the SZT2-orchestrated GATOR (SOG) complex sets a platform for nutrient sensing and mTORC1 regulation in metazoans. In this model, GATOR1 not only functions as a GAP that inactivates RAGA and RAGB (not depicted), but also recruits GATOR2 to the lysosome through SZT2. Under nutrient-deprived conditions, SESN2 is further recruited to the lysosome, and represses RAGA/B through its GDI activity. Upon nutrient stimulation, the GAP activity of GATOR1 may be repressed by GATOR2 through unknown mechanisms, whereas the leucine-bound SESN2 dissociates from GATOR2. In line with the general mode of GDI regulation, leucine could be considered as a GDI-dissociation factor (GDF) for SESN2. In SZT2-deficient cells, GATOR1, GATOR2 and probably SESN2 dissociate from the lysosome resulting in constitutive RAGA/B activation and mTORC1 signalling, but the leucine-regulated SESN2 interaction with GATOR2 is not affected. a.a., amino acids; Leu, leucine.
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Peng, M., Yin, N. & Li, M. SZT2 dictates GATOR control of mTORC1 signalling. Nature 543, 433–437 (2017). https://doi.org/10.1038/nature21378
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DOI: https://doi.org/10.1038/nature21378
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