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Cholesterol sensing by the SCAP–FAM134B complex regulates ER-phagy and STING innate immunity

Nature Cell Biology (2025)Cite this article

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Abstract

The endoplasmic reticulum (ER) is central to cholesterol biosynthesis and trafficking, yet paradoxically maintains low cholesterol levels, enabling it to sense fluctuations that impact various signalling pathways. However, the role of ER cholesterol in cellular signalling remains unclear. Here we show that the ER-phagy receptor FAM134B interacts directly with both cholesterol and SCAP, a key regulator of cholesterol biosynthesis. When ER cholesterol is high, FAM134B and SCAP are sequestered by cholesterol-tightened interactions, halting ER-phagy, STING activation and cholesterol synthesis. Under low cholesterol conditions, FAM134B dissociates from SCAP, allowing SCAP to activate SREBP2 and upregulate cholesterol synthesis, while FAM134B either facilitates ER-phagy through oligomerization or aids STING trafficking to activate innate immune responses. These findings reveal that the SCAP–FAM134B complex senses ER cholesterol levels, regulating both ER-phagy and immune signalling, with implications for diseases linked to cholesterol imbalance.

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Fig. 1: Cholesterol fluctuations alter ER-phagy flux in vivo and in vitro.
Fig. 2: Cholesterol stimulates the interaction between FAM134B and SCAP.
Fig. 3: FAM134B is a cholesterol-binding protein.
Fig. 4: Cholesterol regulates ER-phagy via the SCAP–FAM134B complex.
Fig. 5: FAM134B is essential for STING-mediated innate immunity.
Fig. 6: FAM134B has a crucial role in host defence against DNA viruses.
Fig. 7: FAM134B coordinates with COPII to escort STING to the ERGIC for activation.
Fig. 8: Cholesterol sensing by the SCAP–FAM134B complex regulates STING innate immunity.

Data availability

There are no restrictions on data availability in this article, and all data supporting the findings of this study are provided. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank B. Song, M. S. Brown and J. L. Goldstein for providing the SRD-13A cells. We also thank Z. Jiang for sharing the VACV virus, P. Xu for providing the HSV-1 virus, VSV-GFP virus, STING-HA inducible stable HeLa cell lines and the STING KO HeLa cell lines. We thank H. Xu for providing the SLC38A9 KO and TFEB/TFE3 DKO HeLa cell lines, Z. Zhong for providing the BJ cells and X. Shen for providing the BV2 cells. We thank J. Xuan from the core facilities, Zhejiang University School of Medicine for her help with spinning disk confocal. This study received funding from the National Natural Science Foundation of China (grants 92254307 and 32025012 to Q.S.) and the Ministry of Science and Technology of the People’s Republic of China (grant 2024YFA1803003 to Q.S.) and the National Natural Science Foundation (grants 3227050033 and 32270793 to X.J.; 32200600 to X.W.; 32400612 to Y.S.; 31970695 and 31771525 to Q.S.; 32230023 and 92057203 to W.L.) and the China Postdoctoral Science Foundation (grant 2024M752771 to B.L.).

Author information

Author notes

  1. These authors contributed equally: Boran Li, Dongheng Zhou, Xinyi Wang, Xiao Jiang.

  2. These authors jointly supervised this work: Wei Liu, Qiming Sun.

Authors and Affiliations

  1. Department of Respiratory and Critical Care Medicine, Center for Metabolism Research, Fourth Affiliated Hospital, Zhejiang University School of Medicine and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China

    Boran Li, Yongjuan Sang, Youjing Dai, Yu Yao, Aifu Lin, Dante Neculai, Wei Liu & Qiming Sun

  2. Departments of Biochemistry and Cardiology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Dongheng Zhou, Xinyi Wang, Xiao Jiang, Xinyang Hu, Dante Neculai, Wei Liu & Qiming Sun

  3. Department of Medical Genetics and Center for Rare Diseases, Second Affiliated Hospital, Zhejiang University School of Medicine, and Zhejiang Key Laboratory of Rare Diseases for Precision Medicine and Clinical Translation, Hangzhou, Zhejiang, China

    Yi Zhang, Wang Ni & Zhiying Wu

  4. MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China

    Chen Chen & Pinglong Xu

  5. State Key Laboratory of Membrane Biology, Beijing, China

    Shulin Li & Liang Ge

  6. Tsinghua-Peking Center for Life Sciences, Beijing, China

    Shulin Li & Liang Ge

  7. School of Life Sciences, Tsinghua University, Beijing, China

    Shulin Li & Liang Ge

  8. Department of Surgical Oncology, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China

    Quan Zhou

  9. MOE Laboratory of Biosystem Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou, China

    Aifu Lin

  10. Cancer Center, Zhejiang University, Hangzhou, China

    Aifu Lin

  11. Key Laboratory for Cell and Gene Engineering of Zhejiang Province, Hangzhou, China

    Aifu Lin

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  1. Boran Li
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  2. Dongheng Zhou
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Contributions

Q.S. conceptualized the project. Q.S. and W.L. designed the experiments. B.L., D.Z., X.W., X.J., Y.S., Y.D., Y.Y., Y.Z., C.C. and S.L. performed the experiments. W.N., Q.Z., A.L., X.H., L.G., Z.W., P.X. and D.N. contributed reagents. Q.S., W.L. and B.L. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Wei Liu or Qiming Sun.

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The authors declare no competing interests.

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Nature Cell Biology thanks Dan Kober, Carmine Settembre and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Cholesterol fluctuations alter ER-phagy flux in vivo and in vitro.

A, Eight-week-old ER-TRG mice were fed with either a control diet or a high-cholesterol diet for 8 weeks. Confocal images of hepatocytes exhibit RFP-EGFP fluorescence. Scale bars represent 20 μm (insets: magnified views). B, Quantification was performed on RFP-positive but EGFP-negative puncta (n = 4-6 mice per group, 30 cells per group). Data are shown as mean ± SEM and analyzed with Student’s t test (two-tailed, unpaired). C, Immunoblotting analysis showing the cleavage of ER-TRG in primary hepatocytes isolated from transgenic mice. D, ER-TRG mice primary hepatocytes were treated with 0.1% Cholesterol-MβCD for 24 h or subjected to Cholesterol-Depletion for 24 h. Confocal images of RFP-EGFP fluorescence. Scale bars, 20 µm (insets: magnified views). F, Immunoblotting analysis showing the cleavage of ER-TRG in primary hepatocytes isolated from transgenic mice. G, ER-TRG mice primary monocytes were treated with 0.1% Cholesterol-MβCD for 24 h or subjected to Cholesterol-Depletion for 24 h. Confocal images of RFP-EGFP fluorescence. Scale bars, 10 µm (insets: magnified views). I, Immunoblotting analysis showing the cleavage of ER-TRG in primary monocytes isolated from transgenic mice. J, ER-TRG mice primary MEFs were treated with 0.1% Cholesterol-MβCD for 24 h or subjected to Cholesterol-Depletion for 24 h. Confocal images of RFP-EGFP fluorescence. Scale bars, 10 µm (insets: magnified views). L, Immunoblotting analysis showing the cleavage of ER-TRG in primary MEF isolated from transgenic mice. M, ER-TRG stable HepG2 cells were subjected to treatment with EBSS, CQ, 0.1% Cholesterol-MβCD or Cholesterol-Depletion for 24 h. Confocal images of RFP-EGFP fluorescence. Scale bars, 10 µm (insets: magnified views). E, H, K, RFP-positive but EGFP-negative puncta were quantified (n = 20 cells per group) and N, (n = 10 cells per group). Data are shown as mean ± SEM and analyzed with one-way ANOVA.

Extended Data Fig. 2 Cholesterol fluctuations alter ER-phagy flux in vivo and in vitro.

A, B, C, Cells were treated with 1.5% Cholesterol-MβCD treatment for 3 h or Cholesterol Depletion treatment for 24 h. After cell lysis, the intracellular cholesterol content was measured using the Amplex™ Red Cholesterol Assay Kit and normalized to the Ctrl. D, HEK293T cell lysis was incubated with the indicated dose of CHO-Biotin and cholesterol, followed by IP with streptavidin beads. E, GFP cleavage in HepG2 cells transfected with GFP-SEC61B and FAM134B-Flag was examined by Western blot after treatment with 1.5% Cholesterol-MβCD or Cholesterol Depletion. F, G, RFP cleavage in SLC38A9 knockout or LYCHOS knockdown HeLa cells transfected with ER-TRG was examined by Western blot after treatment with 1.5% Cholesterol-MβCD or Cholesterol Depletion. H, RFP cleavage in TFEB knockout HeLa cells transfected with ER-TRG was examined by Western blot after treatment with 1.5% Cholesterol-MβCD or Cholesterol Depletion. I, HeLa cells inducibly express RFP-EGFP-FAM134B were subjected to treatment with 0.1% Cholesterol-MβCD for 24 h, with or without BafA1 as indicated or Cholesterol-Depletion for 24 h. Scale bars, 10 µm (insets: magnified views). J, RFP-positive but EGFP-negative puncta were quantified (n = 20 cells per group). K, FAM134B-Flag stable cells treated with 50 μg/ml CHX were analyzed by immunoblot at various time points. L, GFP cleavage in HepG2 cells transfected with GFP-SEC61B and Flag-tagged ER-phagy receptors was analyzed. M, HEK293T cells transfected with HA-NPC1-WT and HA-NPC1 mutant (I1061T) were treated with 1.5% Cholesterol-MβCD for 6 h or subjected to Cholesterol Depletion for 24 h. O, MEF cells were treated with 1.5% Cholesterol-MβCD for 6 h or subjected to Cholesterol Depletion for 24 h. N, P, Grayscale ratio of HA to tubulin (N) and Collagen I to actin (P) were quantified and normalized to Ctrl. A, B, C, J, N, P, Data are presented as mean ± SEM and analyzed using one-way ANOVA or two-way ANOVA.

Extended Data Fig. 3 Characterization of the interaction between FAM134B and SCAP.

A, Flag-tagged ER-phagy receptors, including FAM134B, ATL3, CCPG1, RTN3L, SEC62, and TEX264, were individually expressed in HEK293T cells, which concomitantly expressed HA-SREBP1. IP was performed with anti-Flag beads, which was followed by Western blot. B, Flag-tagged ER-phagy receptors, including FAM134B, ATL3, CCPG1, RTN3L, SEC62, and TEX264, were individually expressed in HEK293T cells, which concomitantly expressed HA-SREBP2. IP was performed with anti-Flag beads, which was followed by Western blot. C, Flag-tagged SCAP mutants were expressed individually in HEK293T cells, which simultaneously expressed HA-FAM134B. IP was performed with anti-Flag beads, which was followed by Western blot. D, Flag-tagged FAM134B mutants were expressed individually in HEK293T cells, which simultaneously expressed HA-SCAP. IP was performed with anti-Flag beads, which was followed by Western blot. E, F, Purified recombinant protein SCAP 1-734 and Flag-FAM134B for in vitro micelles assay. G, WT and SCAP knockdown HEK293 cells were treated with Torin1 (100 nM) for 3 h or subjected to Cholesterol-Depletion for 24 h. Immunoprecipitants of Flag from HEK293 cells were immunoblotted. H, HeLa cells co-expressing VC155-FAM134B and VN173-SCAP were stained for endogenous BAP31. Cells were treated with 0.1% Cholesterol-MβCD for 24 h. I, The Venus fluorescence intensity was quantified (n = 10 cells per group). Scale bars, 10 µm. Data are shown as mean ± SEM and analyzed with Student’s t test (two-tailed, unpaired).

Extended Data Fig. 4 FAM134B is a cholesterol-binding protein.

A, HeLa cells inducibly express RFP-EGFP-FAM134B were stained for endogenous BAP31 and cholesterol. Cells were treated with 0.1% Cholesterol-MβCD for 24 h or subjected to Cholesterol depletion for 24 h Scale bars, 10 µm (insets: magnified views). B, HEK293T cell lysis was incubated with the indicated dose of CHO-Biotin and cholenic acid for 2 h at 4 °C. IP was performed with streptavidin-conjugated beads, which was followed by Western blot. C, HEK293T cell lysis was incubated with the indicated dose of CHO-Biotin, cholesteryl methyl ether or cholesterol for 2 h at 4 °C. IP was performed with streptavidin-conjugated beads, which was followed by Western blot. D, Schematic diagram of molecular formulas of all the sterols used. E, Measurement of interaction between AcCHO-Biotin, FAM134B (WT)-Flag in SRD-13A cells. Cell lysis was incubated with the indicated dose of AcCHO-Biotin and CHO-Biotin for 2 h at 4 °C. IP was performed with streptavidin-conjugated beads, which was followed by Western blot.

Extended Data Fig. 5 Cholesterol regulates ER-phagy via the SCAP-FAM134B complex.

A, WT or SCAP knockdown HepG2 stable cell lines transfected with HA-NPC1 mutant (I1061T) were treated with 1.5% Cholesterol-MβCD for 6 h or subjected to Cholesterol Depletion for 24 h. B, Comparing the grayscale ratio of HA to tubulin, and normalized using Ctrl as the standard. C, Purified recombinant protein FAM134B for liposome fragmentation assay. D, ER-TRG HeLa stable cell lines and SCAP knockdown ER-TRG HeLa stable cell lines were treated with 0.1% Cholesterol-MβCD for 24 h or subjected to Cholesterol-Depletion for 24 h. Confocal images of RFP-EGFP fluorescence. Scale bars, 10 µm (insets: magnified views). E, RFP-positive but EGFP-negative puncta were quantified (n = 10 cells per group). F, Lysosomal cleavage of GFP was analyzed by Western blot in HepG2 cells transfected with GFP-SEC61B, Vector, FAM134B-Flag or FAM134B-E259A-Flag. Cells were treated with 1.5% Cholesterol-MβCD for 3 h or Cholesterol-Depletion 24 h. G, Measure the grayscale value and statistically analyze the relative ratio of free GFP/total GFP, and normalized using Ctrl as the standard. H, FAM134B knockout U2OS cell lines were transfected with HA-NPC1 mutant (I1061T), Vector, EGFP-FAM134B, or EGFP-FAM134B-E259A. Cells were then treated with 1.5% Cholesterol-MβCD for 6 h or subjected to Cholesterol Depletion for 24 h. I, Comparing the grayscale ratio of HA to actin, and normalized using Ctrl as the standard. B, E, G, I, Data are shown as mean ± SEM and analyzed using two-way ANOVA.

Extended Data Fig. 6 Characterization of the interaction between FAM134B and STING.

A, BV2 cells were treated with DMXAA for 1 h and overlaying with 1.5% Cholesterol-MβCD treatment for 6 h or Cholesterol Depletion treatment for 24 h. B, BJ cells were treated with diABZI for 1 h and overlaying with 1.5% Cholesterol-MβCD treatment for 6 h or Cholesterol Depletion treatment for 24 h. C, the interaction between FAM134B-Flag and STING investigated in vitro. IP was performed with anti-Flag beads, which was followed by Western blot. D, Purified recombinant protein STING for in vitro micelles assay. E, N-terminal of STING interacts with FAM134B. Flag-tagged STING mutants were expressed individually in HEK293T cells, which simultaneously expressed HA-FAM134B. IP was performed with anti-Flag beads, which was followed by Western blot. F, Flag-tagged FAM134B mutants were expressed individually in HEK293T cells, which simultaneously expressed STING-HA. IP was performed with anti-Flag beads, which was followed by Western blot. G, Schematic showing domain structure of FAM134B and SCAP or STING as indicated in the text.

Extended Data Fig. 7 FAM134B mediates STING trafficking with COPII.

A, Measurement of interaction between HA-FAM134B and SEC23A-Flag by Western blot in HEK293T cells treated with 1.5% Cholesterol-MβCD for different time points. IP was performed with anti-Flag beads, which was followed by Western blot. B, Measurement of interaction between SEC23A-HA, FAM134B-Flag, and FAM134B-E259A-Flag by Western blot in HEK293T cells treated with 1.5% Cholesterol-MβCD 0.5 h. IP was performed with anti-HA beads, which was followed by Western blot. C, D, E, HeLa cells inducibly express STING-HA treated with cGAMP for 0.5 h. Cells were homogenized and the lysates were subjected to differential centrifugations with indicated G forces. The 25000x g (25K) pellet, which had the most STING, FAM134B and p-STING was selected and a sucrose gradient ultracentrifugation was performed to separate the 25K pellet to L (light) and P (pellet) fractions. The L fraction, which contained the majority of these proteins, was further resolved on an Opti-Prep gradient after which ten fractions from the top were collected. Membrane fractionation scheme.

Extended Data Fig. 8 FAM134B regulates STING-mediated innate immunity by affecting its subcellular localization.

A-H, HeLa cells inducibly express STING-HA and EGFP-FAM134B treated with cGAMP for the indicated time. Cells were immunostained with an antibody against GM130 (cis-Golgi), TGN38 (trans-Golgi), RAB7 (late endosomes) or LAMP1 (lysosomes), followed by immunofluorescence microscopy. Quantification of the percentage of cells in which STING and FAM134B colocalized with different organelle markers (n = 20 cells per group). Scale bars, 10 µm. Data are shown as mean ± SEM and analyzed with Student’s t test (two-tailed, unpaired) (insets: magnified views). I, the colocalization of FAM134B and STING was analyzed by Pearson’s correlation coefficient (n = 20 cells per group). Data are shown as mean ± SEM and analyzed with one-way ANOVA. J, Schematic showing working model. Panel J created with BioRender.com.

Extended Data Fig. 9 Chemical synthesis and characterization.

A, The synthetic chemical formulas of CHO-Biotin and AcCHO-Biotin.

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Unprocessed western blots and gels for main figures and extended data figures.

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Statistical source data for main figures and extended data figures.

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Li, B., Zhou, D., Wang, X. et al. Cholesterol sensing by the SCAP–FAM134B complex regulates ER-phagy and STING innate immunity. Nat Cell Biol (2025). https://doi.org/10.1038/s41556-025-01766-y

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