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SAR1B senses leucine levels to regulate mTORC1 signalling

Abstract

The mTOR complex 1 (mTORC1) controls cell growth in response to amino acid levels1. Here we report SAR1B as a leucine sensor that regulates mTORC1 signalling in response to intracellular levels of leucine. Under conditions of leucine deficiency, SAR1B inhibits mTORC1 by physically targeting its activator GATOR2. In conditions of leucine sufficiency, SAR1B binds to leucine, undergoes a conformational change and dissociates from GATOR2, which results in mTORC1 activation. SAR1B–GATOR2–mTORC1 signalling is conserved in nematodes and has a role in the regulation of lifespan. Bioinformatic analysis reveals that SAR1B deficiency correlates with the development of lung cancer. The silencing of SAR1B and its paralogue SAR1A promotes mTORC1-dependent growth of lung tumours in mice. Our results reveal that SAR1B is a conserved leucine sensor that has a potential role in the development of lung cancer.

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Fig. 1: SAR1B senses intracellular leucine levels to regulate mTORC1 signalling through GATOR2.
Fig. 2: SAR1B directly binds leucine.
Fig. 3: sar-1 has a conserved role in C. elegans.
Fig. 4: SAR1B acts as a potential tumour suppressor in lung cancer.

Data availability

All data that support the findings of this study are contained in the Article and its Supplementary Information; any further relevant data are available from corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank I. Hanson for reading and editing the manuscript. This work was supported by the National Natural Science Foundation of China (31925012 and 91854205), the National Key Research and Development Program of China (2017YFA0504000973) and an HHMI International Research Scholar Program (55008739) to Y. Liu. Y. Liu was also supported by Beijing Advanced Innovation Center for Genomics, Peking-Tsinghua Center for Life Sciences, and the Tencent Foundation through the XPLORER PRIZE.

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Contributions

J.C., Y.O. and Y. Liu conceived the studies. J.C. and Y.O. performed most of the experiments and analysed the data with help from R.L. and D.W. Y. Li and P.X. generated worm strains. J.G. and J.C. performed bioinformatic analysis. J.W. and P.R.C. synthesized the leucine probe. J.C., Y.O. and Y. Liu wrote the manuscript.

Corresponding authors

Correspondence to Jie Chen or Ying Liu.

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The authors declare no competing interests.

Additional information

Peer review information Nature thanks Tibor Vellai and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 SAR1B regulates leucine-dependent mTORC1 signalling.

a, Identification of amino-acid-sensitive GATOR2-interacting proteins by immunoprecipitation and mass spectrometry analysis. b, c, Knockdown of SAR1B specifically affects leucine-dependent mTORC1 signalling. AAs, amino acid mixture. d, SAR1B is not involved in insulin-dependent mTORC1 signalling. e, f, Knockdown of SAR1B suppresses the induction of autophagy under leucine starvation (e), which is altered by administration of the mTORC1 inhibitor rapamycin (f). Data are mean ± s.e.m., n = 3 biologically independent repeats. ***P < 0.001; unpaired, two-tailed Student’s t-test. g, Expression of Flag-tagged SAR1B restores leucine sensitivity in SAR1B-deficient cells. h, SAR1B functions upstream of Rag GTPase. RagDN, dominant-negative Rag GTPase. i, SAR1B is required for preventing mTORC1 lysosomal localization under leucine starvation. LAMP2, lysosome marker. j, k, SAR1A compensates for the loss of SAR1B in mTORC1 signalling. l, mTORC1 activity is not affected in SAR1B-knockout cells. m, n, Knockdown of SAR1B upregulates the transcription of SAR1A. Data are mean ± s.e.m., n = 3 biologically independent repeats. ns, no significant difference; **P < 0.01; ***P < 0.001; unpaired, two-tailed Student’s t-test. o, Simultaneous silencing of SAR1A and SAR1B persistently activates mTORC1. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 2 SAR1B regulates mTORC1 in a COPII- and GTPase activity-independent manner.

a–c, Locking SAR1A and SAR1B into GDP- or GTP-loaded states does not affect their roles in regulating mTORC1 activity (a, b) or mTORC1 localization (c). d, e, Knockdown of SAR1B, but not other COPII components, affects the leucine sensitivity of mTORC1. Knockdown efficiencies were quantified by qPCR. Data are mean ± s.e.m., n = 3 biologically independent repeats. **P < 0.01; ***P < 0.001; unpaired, two-tailed Student’s t-test. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 3 SAR1B modulates mTORC1 activity by interacting with GATOR2.

a, b, SAR1B interacts with the GATOR2 subunit SEH1L (a) but not the GATOR1 subunit DEPDC5 (b) in a leucine-sensitive fashion. MG132 was added to prevent proteasomal degradation of DEPDC520. c, The interaction of SAR1B with GATOR2 is sensitive to leucine starvation, but not to arginine starvation. The asterisks represent the IgG light chain. d, SAR1A also interacts with GATOR2 in a leucine-sensitive manner. e, Preparation of tag-free SAR1B recombinant proteins. GF, gel-filtration chromatography; Q, anion-exchange chromatography. f, SAR1B directly binds MIOS but not other GATOR2 subunits. g, h, Depletion of MIOS abolishes the interaction between SAR1B and GATOR2. i, SAR1B regulates mTORC1 signalling in a GATOR2-dependent manner.

Extended Data Fig. 4 The binding of SAR1B with GATOR2 is essential for the regulation of mTORC1 signalling.

a, Alignment of SAR1B protein sequences across species by Jalview. Identical residues (*) and similar (:) residues are indicated. b, Structure of hamster SAR1B (Protein Data Bank code 1F6B) displayed by PyMOL, with H58, E114 and E122 highlighted. c, SAR1B mutants that are incapable of binding GATOR2 are unable to prevent mTORC1 lysosomal localization under leucine deficiency. LAMP2, lysosome marker. mTOR–lysosome colocalization was quantified. Data are mean ± s.e.m., n = 3 biologically independent repeats. ns, no significant difference; ***P < 0.001; unpaired, two-tailed Student’s t-test. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 5 Leucine stimulates SAR1B dissociation from GATOR2 and lysosomes in vitro.

a, Isolation of the GATOR2 protein complex from cells by anti-Flag immunoprecipitation. b, c, Leucine, but not any other amino acid, stimulates SAR1B dissociation from the GATOR2 protein complex in vitro. The asterisks represent the IgG light chain. d, Intracellular distribution of SAR1B in the presence or absence of leucine. e, SAR1B associates with lysosomes isolated from leucine-starved cells. The addition of leucine to the lysosomes stimulates SAR1B dissociation in vitro. f, SAR1B mutants that are incapable of binding to MIOS cannot associate with lysosomes. Twenty μM of leucine was used in this in vitro assay. g, SAR1B disassociates from GATOR2 complex derived from cells that are triple knockout for sestrin 1, sestrin 2 and sestrin 3 upon leucine stimulation in vitro. h, SAR1B does not interact with sestrin 2. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 6 SAR1B undergoes a conformational change and dissociates from GATOR2 upon binding to leucine.

a, b, SAR1B recombinant proteins purified from bacteria do not bind to radiolabelled leucine. Sestrin 2, positive control. Rap2a, negative control. Data are mean ± s.e.m., n = 3 repeated measurements for one biological sample. ns, no significant difference; ***P < 0.001; unpaired, two-tailed Student’s t-test. c, d, SAR1B recombinant proteins purified from HEK293T cells bind to radiolabelled leucine in vitro. Data are mean ± s.e.m., n = 3 repeated measurements for one biological sample. ns, no significant difference; ***P < 0.001; unpaired, two-tailed Student’s t-test. e, Leucine binding induces a conformational change of SAR1B. Data are mean ± s.e.m., n = 3 biologically independent repeats. f, g, SAR1A recombinant proteins purified from HEK293T cells bind to leucine. h, i, SAR1B mutants that are incapable of interacting with MIOS bind to leucine with an affinity similar to that of wild-type SAR1B. Data are mean ± s.e.m., n = 3 biologically independent repeats. j, k, SAR1B mutants with impaired leucine-binding ability undergo little or no conformational change upon leucine supplementation. Data are mean ± s.e.m., n = 3 biologically independent repeats. l, m, SAR1B mutants with impaired leucine-binding ability persistently associate with GATOR2 (l) and lysosomes (m) even in the presence of 20 μM leucine. n, Sestrin 2 directly binds SEH1L but not other subunits of GATOR2. o. SAR1B and sestrin 2 sequentially dissociate from GATOR2 in vitro upon stimulation with increasing concentrations of leucine. The asterisks represent the IgG light chain. p, q, Treatment of cells with increasing leucine concentrations stimulates a sequential dissociation of SAR1B and sestrin 2 from GATOR2 (p) and elicits a two-phase activation of mTORC1 (q). For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 7 SAR1B and sestrin 2 detect different structural features of leucine.

a, Leucine analogues with modifications at the amino group, the carboxyl group or the side chain. b, c, Modifications at the carboxyl group of leucine are tolerated by SAR1B. Sestrin 2 tolerates modifications at the side chain to some extent. Protein complexes were immunopurified from leucine-starved HEK293T cells stably expressing tagged GATOR2. Leucine analogues were directly added to immunoprecipitates in vitro to induce SAR1B or sestrin 2 dissociation. The asterisks represent the IgG light chain. d, The workflow for capture of receptor proteins by LeuProbe. LeuProbe has a modified carboxyl group compared to native leucine. e, LeuProbe has an ability to stimulate mTORC1 activation similar to that of native leucine. f, LeuProbe captures SAR1B, but not sestrin 2. g, Interaction of LeuProbe with SAR1B can be competed against by an excess amount of native leucine. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 8 Different leucine sensing events in myotubes and adipocytes.

a, SAR1B, but not other COPII components, is highly expressed in mouse muscle tissue. Expression, transcripts per million. b, Similar to sestrin 2, sestrin 1 partially tolerates modifications at the side chain of leucine. The asterisk represents the IgG light chain. c, Differentiation of C2C12 and 3T3-L1 cells into myotubes and adipocytes. d, SAR1B and sestrin 1 are expressed in myotubes and interact with GATOR2, whereas sestrin 2 is expressed in adipocytes and interacts with GATOR2. Protein–protein interactions were assayed by anti-WDR24 immunoprecipitation. e, Activation of mTORC1 in myotubes and adipocytes in response to leucine analogue treatments. f, g, The indicated leucine analogues can be transported into cells without being hydrolysed. Columns are data from n = 1 biological repeat. h, i, Leucine starvation induces a sequential binding of sestrin 1 and SAR1B to GATOR2 and a corresponding two-phase mTORC1 inactivation in myotubes. j, k, Leucine starvation induces the binding of sestrin 2 to GATOR2 and therefore mTORC1 inhibition in adipocytes. l, m, Leucine stimulation induces the dissociation of sestrin 2 from GATOR2 and therefore mTORC1 activation in adipocytes. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 9 SAR1B has a conserved role in regulating TORC1 signalling in C. elegans.

ac, sar-1, but not any other subunit of COPII, is involved in CeTORC1 signalling. HLH-30 nuclear localization is quantified to represent TORC1 inactivation. Data are mean ± s.e.m., n = 3 biologically independent repeats. ns, no significant difference; **P < 0.01; ***P < 0.001; unpaired, two-tailed Student’s t-test. dg, Expression of the codon-optimized human SAR1B::GFP restores CeTORC1 signalling in sar-1-deficient C. elegans. Data are mean ± s.e.m., n = 3 biologically independent repeats. ***P < 0.001; unpaired, two-tailed Student’s t-test. hj, sar-1 regulates CeTORC1 in a GATOR2-dependent manner. CeMios, F39C12.1; CeShe1L, npp-18. Data are mean ± s.e.m., n = 3 biologically independent repeats. **P < 0.01; ***P < 0.001; unpaired, two-tailed Student’s t-test. k, l, sar-1 and sesn-1 are not involved in arginine- or isoleucine-dependent TORC1 signalling in C. elegans. Data are mean ± s.e.m., n = 3 biologically independent repeats. ***P < 0.001; unpaired, two-tailed Student’s t-test. m, Tissue distribution of sar-1 and sesn-1 in C. elegans. n, sar-1 regulates worm lifespan in a GATOR2-dependent manner. n = 3 biologically independent repeats. Data are representative one repeat. log–rank test for survival comparisons. o, RNAi knockdown efficiency of let-363. Data are mean ± s.e.m., n = 3 biologically independent repeats. ***P < 0.001; unpaired, two-tailed Student’s t-test. p, q, sar-1 functions upstream of TORC1 to regulate C. elegans lifespan. n = 3 biologically independent repeats. Data are representative one repeat. log–rank test for survival comparisons. For gel source data, see Supplementary Fig. 1

Source data.

Extended Data Fig. 10 Deficiency of SAR1A and SAR1B correlates with mTORC1 activation in lung cancers and promotes anchorage-independent cell growth.

a, Tables summarizing the number and frequency of SAR1B alterations in human lung squamous cell carcinoma and lung adenocarcinoma datasets. b, SAR1A mRNA levels are downregulated in cases with (w/) SAR1B deletion compared to those without (w/o) SAR1B deletion. Box plots show the median and quartiles (boxes) and minimum to maximum (whiskers). ***P < 0.001; unpaired, one-tailed Student’s t-test. c, SAR1A transcription cannot be upregulated in human lung cancer cells with SAR1B copy loss, in contrast to wild-type lung fibroblasts. Data are mean ± s.e.m., n = 3 biologically independent repeats. ns, no significant difference; ***P < 0.001; unpaired, two-tailed Student’s t-test. d, mTORC1 is constitutively activated in human lung cancer cells with SAR1B copy loss. e, Lower expression level of SAR1B correlates with worse prognoses in patients with lung cancer. log–rank test. f, g, Silencing of SAR1A and SAR1B in human lung fibroblasts leads to constitutive mTORC1 activation even under leucine deprivation. h, i, SAR1A and SAR1B regulate mTORC1 signalling in a Rag-GTPase-dependent manner in human lung fibroblasts. RagDN, dominant-negative Rag GTPase. j. Deficiency of SAR1A and SAR1B promotes anchorage-independent cell growth in soft agar, and the cell growth is sensitive to treatment with 100 nM rapamycin (rapa.). Colony numbers were counted and indicated as columns. Data are mean ± s.e.m., n = 3 biologically independent repeats. ns, no significant difference; ***Padj < 0.001; one-way analysis of variance (ANOVA) test. k, Silencing of SAR1A and SAR1B increases the sensitivity of human lung fibroblasts to rapamycin. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 11 Silencing of SAR1A and SAR1B promotes subcutaneous xenograft tumour growth.

Silencing of SAR1A and SAR1B promotes mTORC1-dependent growth of subcutaneous xenograft tumours. Tumour growth was recorded every other day. Tumour weights were obtained on the last day. Data are mean ± s.e.m., n = 10 mice (with an expectation that n = 9 mice for HFL1 sgSAR1B plus shSAR1A), one biological repeat. ns, no significant difference; ***Padj < 0.001; One-way ANOVA test. For gel source data, see Supplementary Fig. 1

Source data.

Extended Data Fig. 12 Deficiency of SAR1A and SAR1B promotes orthotopic xenograft tumour growth in the lung of mice.

ac, Silencing of SAR1A and SAR1B promotes mTORC1-dependent growth of xenograft tumours in the lungs of mice. Lungs with tumours in situ (a, b) and representative haematoxylin and eosin staining of lung sections (c) are shown. Surface tumour lesion areas are calculated as mean ± s.d.. HFL1: n = 8 mice for sgCtrl and shCtrl, sgSAR1B-1 and shSAR1A + rapa., sgSAR1B-2 and shSAR1A − rapa.; n = 9 mice for sgSAR1B-1 and shSAR1A − rapa., sgSAR1B-2 and shSAR1A + rapa.; IMR-90, n = 8 mice for sgCtrl and shCtrl, sgSAR1B-1 and shSAR1A −rapa., sgSAR1B-2 and shSAR1A − rapa.; n = 9 mice for sgSAR1B-1 and shSAR1A + rapa., sgSAR1B-2 and shSAR1A + rapa. One biological repeat. d, Immunohistochemical staining shows that silencing of SAR1A and SAR1B activates protein synthesis, inhibits autophagy and promotes cell proliferation in orthotopic lung tumours. e, Deficiency of SAR1A and SAR1B hyperactivates mTORC1 in the orthotopic tumours. f, Deficiency of sestrins in HLF1 cells stimulates continuous mTORC1 activation. gi, Deficiency of sestrins promotes mTORC1-dependent growth of subcutaneous and lung orthotopic tumours. For subcutaneous tumours, data are mean ± s.e.m., n = 10 mice (with an expectation that n = 9 mice for sgCtrl), one biological repeat. **Padj < 0.01, ***Padj < 0.001; one-way ANOVA test. For orthotopic tumours, surface tumour lesion areas are calculated as mean ± s.d., n = 7 mice for sgCtrl; n = 9 mice for sgRNAs against sestrins; n = 8 mice for sgRNAs against sestrins + rapa.. For gel source data, see Supplementary Fig. 1

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Chen, J., Ou, Y., Luo, R. et al. SAR1B senses leucine levels to regulate mTORC1 signalling. Nature (2021). https://doi.org/10.1038/s41586-021-03768-w

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