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Manganese regulation of COPII condensation controls circulating lipid homeostasis

Nature Cell Biology volume 25, pages 1650–1663 (2023)Cite this article

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Abstract

Precise control of circulating lipids is instrumental in health and disease. Bulk lipids, carried by specialized lipoproteins, are secreted into the circulation, initially via the coat protein complex II (COPII). How the universal COPII machinery accommodates the abundant yet unconventional lipoproteins remains unclear, let alone its therapeutic translation. Here we report that COPII uses manganese-tuning, self-constrained condensation to selectively drive lipoprotein delivery and set lipid homeostasis in vivo. Serendipitously, adenovirus hijacks the condensation-based transport mechanism, thus enabling the identification of cytosolic manganese as an unexpected control signal. Manganese directly binds the inner COPII coat and enhances its condensation, thereby shifting the assembly-versus-dynamics balance of the transport machinery. Manganese can be mobilized from mitochondria stores to signal COPII, and selectively controls lipoprotein secretion with a distinctive, bell-shaped function. Consequently, dietary titration of manganese enables tailored lipid management that counters pathological dyslipidaemia and atherosclerosis, implicating a condensation-targeting strategy with broad therapeutic potential for cardio-metabolic health.

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Fig. 1: Self-constrained condensation of COPII differentiates cellular transport.
Fig. 2: Adenovirus hijacks COPII condensation to selectively control lipid delivery.
Fig. 3: A manganese messenger that enables viral hijacking of COPII condensation.
Fig. 4: The manganese signal directly controls COPII condensation in vitro.
Fig. 5: Balanced COPII condensation selectively supports lipoprotein secretion.
Fig. 6: Mitochondrial manganese store regulates the COPII machinery for lipid control.
Fig. 7: Manganese titration enables tailored treatment of dyslipidaemia and ASCVD.

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Data availability

Raw data for ICP-MS are provided in Supplementary Table 1. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank Z. Jiang (PKU) and J. Xiao (PKU) for helpful advice. The work is supported by National Key R&D Program grant no. 2018YFA0506900, National Science Foundation of China (NSFC) grants nos. 32125021, 92254308 and 91957119, National Key R&D Program grant no. 2021YFA0804802, NSFC grants nos. 91954001 and 31571213 to X.-W.C. and NSFC grant 32100947 to X.W. X.W. was supported by a Postdoctoral Fellowship of Peking-Tsinghua Center for Life Science and the China Postdoctoral Science Foundation (2021M700239). We thank the Imaging Core Facility of the State Key Laboratory of Membrane Biology at Peking University, Y. Liang for technical assistance with two-photon microscopy, the Core Facility of Life Science College at Peking University for assistance with confocal imaging and EM, Y. Hu for technical help, the Flow Cytometry Core at the National Center for Protein Sciences at Peking University and the Proteomics Core, H. Li for technical help, the Analytical Instrumentation Center of Peking University and J. Liu for assistance with ICP-MS.

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Author notes

  1. These authors contributed equally: Xiao Wang, Runze Huang.

Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, Peking University, Beijing, China

    Xiao Wang, Runze Huang, Yawei Wang, Wenjing Zhou, Yating Hu, Yuanhang Yao, Kunlun Cheng, Xin Li, Bolin Xu, Yuangang Zhu & Xiao-Wei Chen

  2. Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing, China

    Xiao Wang, Runze Huang, Wenjing Zhou, Yating Hu, Yuanhang Yao, Kunlun Cheng, Xin Li, Bolin Xu, Yuangang Zhu & Xiao-Wei Chen

  3. PKU-THU Joint Center for Life Sciences, Peking University, Beijing, China

    Xiao Wang, Yawei Wang & Xiao-Wei Chen

  4. Department of Clinical Research Center, Dazhou Hospital, Dazhou, Sichuan, China

    Jie Zhang, Yaowen Xu & Fanxin Zeng

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Contributions

Conceptualization was provided by X.-W.C and X.W., methodology by X.W., R.H., Y.W., W.Z. and Y.Z., investigations by X.W., R.H., Y.W., W.Z., Y.H., Y.Y., K.C., X.L., B.X. and X.-W.C. and the collection of blood samples and basic parameters of human participants by J.Z., Y.X. and F.Z. The paper was written by X.-W.C. and X.W. Supervision was provided by X.-W.C. Review and editing was carried out by all authors.

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Correspondence to Xiao Wang or Xiao-Wei Chen.

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X.-W.C., X.W. and Y.W. have filed a patent application related to this study. The remaining authors declare no competing interests.

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Nature Cell Biology thanks Elizabeth Miller 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 Condensation of COPII differentiates cellular transport.

a. FIB-SEM tomography of mouse liver revealing robust ER-Golgi transport of lipoproteins. Representative view reconstructed by FIB-SEM from 3 C57BL/6 J mice is shown. b, IB of mouse liver lysates untreated or treated with EndoH. c, Quantification of sizes of SEC24A puncta in Fig. 1b. n = 842, 900 and 989 puncta for CTL, LMAN1 and APOBN, respectively, pooled from 10 cells for each group. Representative of 3 biological independent experiments is shown. Lines represent median; edges represent the interquartile range; whiskers represent the 10% percentile and 90% percentile. P values were determined by unpaired two-sided Student’s t-test. d, Co-localization of EGFP that was knocked in the SEC24A locus (EGFPSEC24A) and endogenous SEC31A visualized by immunostaining in Huh7. Representative of 3 independent experiments is shown. e, Confocal images of EGFPSEC24A in Huh7 cells. Middle: fold of SEC24A fluorescence between puncta and diffusive cytosol. Right: sizes of EGFPSEC24A puncta. Lines represent median; edges represent the interquartile range; whiskers represent the 10% percentile and 90% percentile. n=531puncta from 5 cells. Representative of 3 biological independent experiments is shown. f, FRAP of EGFP-knock-in SEC24A coalescence in cells. 0 s indicates the time of photobleaching. Lower: quantification of FRAP. Data are shown as mean ± SEM. n = 19 puncta from 10 cells. Representative of 3 biological independent experiments is shown. g, IB of cell lysates from mouse primary hepatocytes treated with 1,6-HD for 3 hours. n = 3 biological independent experiments. h, Time-lapse confocal analysis of the ER (SEC61β-GFP) and Golgi (GM130-GFP) in primary hepatocytes treated with 1,6-HD. Representative of 3 independent experiments is shown. Scale bars, 200 nm (a), 2.5 μm (f), 5μm (d-e, h). Source numerical data and unprocessed blots are available in Source Data.

Source data

Extended Data Fig. 2 Evolutionarily conserved IDRs in SEC24 drive concentration-dependent condensation of SEC23/SEC24 in vitro.

a, IDRs are present in SEC24 of different organisms over evolution. The multiple sequence alignment was performed using Clustal W. b, The probability of spontaneous liquid-liquid phase separation (pLLPS) of SEC24A predicted by FuzDrop. c, Structural simulation of SEC24A disorder and order regions. SEC23A/SEC24A structure is obtained from PDB (2nut). The IDR region is simulated by the AlphaFold algorithm. d, Quantification of hepatic concentration of SEC24A, SEC24D, and SEC23A. Lower: representative IB results of specific proteins. e, Concentration of hepatic COPII subunits quantified using six different C57BL/6 J mice. Data are shown as mean ± SEM. For d and e, n = 6 male mice. f, IDRs of SEC24A-GFP undergo concentration-dependent liquid-liquid phase separation after MBP removal by protease cleavage in vitro. g, Homotypic fusion of IDR_SEC24A-GFP liquid droplets, indicated by the arrows. h, FRAP of IDR_SEC24A-GFP liquid droplets. Lower: Quantification of FRAP. Data are shown as mean ± SEM. n = 9 droplets. i, IDRs_SEC24A (Cy5 labeled) undergo liquid-liquid phase separation in a similar manner with SEC24A-GFP. j, Co-phase separation of IDR_SEC24A and IDR_SEC24A-GFP. MBP- IDR_SEC24A (Cy5 labeled) are mixed with MBP- IDR_SEC24A-GFP at 5 μM of each, followed by MBP removal by protease cleavage. For f-j, data are shown representative of 3 independent experiments. Scale bars, 5μm (f, g, h, i, j). Source numerical data and unprocessed blots are available in Source Data.

Source data

Extended Data Fig. 3 The selective and self-constrained nature of COPII condensation in vitro.

a, SEC31A/SEC13 alone doesn’t condense into protein droplets. SEC31A/SEC13 (10 μM) was assayed in 150 mM NaCl. b, Representative images of SEC23/24 droplets (10 μM) constrained with increasing SEC31A/SEC13 in 150 mM NaCl. c, Quantification of the sizes of SEC23A/SEC24A droplets in (b). n = 161, 183, 169, 117 SEC23/24 droplets for the presence of 0, 5, 10, 20 μM SEC31/13, respectively. Lines represent median; edges represent the interquartile range; whiskers represent the 5% percentile and 95% percentile. *P values are determined by one-way ANOVA test. #P values are determined by the posthoc test of Tukey. d, Size dependent exclusion of dextran by the SEC23/24 droplets. 1 μM of TAMRA-labeled dextran are mixed with 10 μM of SEC23A/SEC24A in 150 mM NaCl. Lower: relative fluorescence intensity along the dashed line. For a-d, Representative of 3 biological independent experiments is shown. Scale bars, 5μm (a, b, d). Source numerical data are available in Source Data.

Source data

Extended Data Fig. 4 Selective, binary regulation of lipoprotein transport by adenovirus.

a, Representative EM negative staining images of plasma lipoproteins purified from mice with indicated genotypes and treatments. b, IB of liver samples from mice with the indicated genotypes and treatments. c, Quantification of IB signals of APOB and ALB in (b). n = 3 biological independent sample. Data are shown as mean ± SEM. P values were determined by unpaired two-sided Student’s t-test. d, Confocal images of SEC31A in primary hepatocytes treated with or without AdV. The nucleus is highlighted with a yellow dashed line. e, Confocal images of SEC24A and SURF4 in primary hepatocytes treated with or without AdV. The nucleus is highlighted with a white dashed line. For a-e, Representative of 3 biological independent experiments is shown. Scale bars, 200 nm (a), or 5μm (d-e). Source numerical data and unprocessed blots are available in Source Data.

Source data

Extended Data Fig. 5 A manganese messenger that enables viral hijacking of COPII condensation.

a, The CP-factor in cytosol extracted from Adv infected liver is resistant to Benzonase cleavage or Proteinase K (PK) digestion. b, Representative images in Fig. 3c. Each fraction was incubated with 6 μM of SEC23A/SEC24A, followed by microscopy imaging. c, Ca2+ concentrations in cytosol extracted from CTL or Adv infected mouse liver were estimated by o-cresolphthalein complexone based colorimetric assay. n = 6 biological independent samples for each group. P values were determined by unpaired two-sided Student’s t-test. d, IB analysis of TMEM165 in mouse primary hepatocytes treated with 500 μM of indicated metal for 4 hours. e, Extracellular EDTA minimally restores TMEM165 levels in cells infected with AdV. 12 hours post infection, primary hepatocytes were treated with the indicated reagents, followed by another 12 hours-culture. f, Confocal images of endogenous SEC24A treated with or without 500 μM of MnCl2. EGFP is knocked in at the N terminus of SEC24A in Huh7 cells. g, Confocal images of endogenous SEC24A and SURF4 treated with or without 500 μM of MnCl2. For a-b and d-g, Representative of 3 biological independent experiments is shown. Scale bars, 5 μm (a-b, f-g). Source numerical data and unprocessed blots are available in Source Data.

Source data

Extended Data Fig. 6 Manganese directly interacts with SEC23/SEC24.

a. Fe2+ (1 mM) induces SEC23/24 (8 μM) aggregation in 150 mM NaCl. b. Absence of detectable binding between the indicated regent pairs measured by ITC. c. The binding of Mn2+ with the indicated SEC23A/SEC24D proteins was measured by ITC. d, Absence of detectable binding between SEC31A/SEC13 and Mn2+ measured by ITC. a-d, Representative of 3 biological independent experiments is shown. Scale bars, 5μm. Unprocessed blots are available in Source Data.

Source data

Extended Data Fig. 7 COPII condensation is selectively regulated by Manganese.

a. The binding of Zn2+ with SEC23A/ΔIDRSEC24A proteins was measured by ITC. b, Absence of detectable binding between SEC23A/ΔIDRSEC24A and Cu2+ measured by ITC. c, Zn2+ shows no effect on the interaction of the indicated SEC23A/SEC24A and Mn2+ immobilized on the IMAC beads. d, Confocal images of SEC24A coalescence in primary hepatocytes treated with indicated metals for 8 hours. a-d, Representative of 3 independent experiments is shown. Scale bars, 5μm.

Extended Data Fig. 8 Mn2+-induced COPII condensation promotes enrichment of the transport machinery.

a, Dose-dependent promotion of cellular COPII condensation by the manganese signal. Primary hepatocytes were treated with 5 μM of TPEN for 30 min, followed by treatments of MnCl2 at the indicated doses for 8 hours and stained for endogenous SEC24A. b, Quantification of the integrated signals of SEC24A puncta in (a). n = 11248 puncta for 0 μM, 8280 puncta for 1 μM, 8942 puncta for 5 μM, 4678 puncta for 25 μM, 4478 puncta for 125 μM, and 4566 puncta for 500 μM pooled from 10 cells for each group. For a and b, representative of 3 biological independent experiments is shown. Lines represent median; edges represent the interquartile range; whiskers represent the 5% percentile and 95% percentile. *P values were determined by one-way ANOVA test. #P values were determined by the posthoc test of Tukey. c, Mn2+ enhances cellular interaction of SEC16A and SEC23/24. HEK293A cells expressing FLAG-SEC16A treated with or without 500 μM of MnCl2 were lysed and subjected to anti-FLAG IP. Representative of 3 independent experiments is shown. d, Quantification of the IB signals in (c). n = 3 biological independent experiments. Data are shown as mean ± SEM. P values were determined by unpaired two-sided Student’s t-test. Scale bars, 5μm. Source numerical data and unprocessed blots are available in Source Data.

Source data

Extended Data Fig. 9 Mitochondrial manganese store regulates the COPII machinery for lipid control.

a, Confocal analysis of the ER (SEC61β-RFP), Golgi (anti-GM130 antibody) and lysosome (LysoTrackerTM Deep Red) in primary hepatocytes infected with or without AdV. Representative of 3 independent experiments is shown. b, Gating strategy for analyzing mitochondrial membrane potential of primary hepatocytes with indicated treatments by TMRM staining. Singlets were selected by FCS/SSC, and the boundaries of TMRM positive (TMRM+) was defined by the signal of non-stained primary hepatocytes. TMRM+ cells were used for histogram plotting in Fig. 6b. c, FIB-SEM tomography of Mitochondria-ER contact sites (MECS) in murine hepatocytes and the lipoproteins harbored in the smooth end of the ER that exhibits close contacts with the mitochondria. Representative view reconstructed by FIB-SEM from 3 C57BL/6 J mice is shown. Lipoproteins are pseudo-colored in yellow. Scale bars, 100 nm (c) or 5μm (a).

Extended Data Fig. 10 Manganese titration enables tailored treatment of dyslipidemia and ASCVD.

a, Bell-shape regulation of plasma triglyceride levels by increasing Mn2+ dose in drink. n = 15, 15, 8, 14, 9, and 6 mice for 0 g/L, 0.02 g/L, 0.06 g/L, 0.2 g/L, 1 g/L, and 5 g/L, respectively. Data are presented as mean ± SEM. *P values were determined by one-way ANOVA test. #P values were determined by the posthoc test of Tukey. Representative of 3 independent experiments is shown. b, c, Plasma samples in Fig. 7a were fractionated into VLDL, LDL and HDL by FPLC, prior to cholesterol measurement (b) and triglyceride measurement (c). Representative of 3 independent experiments is shown. d, Bell-shape regulation of plasma LDL-TG levels by increasing Mn2+ dose in drink. Representative of 3 independent experiments is shown. e, IB of plasma samples from Mn-supplemented hyperlipidemia mice in Fig. 7a. Representative of 3 independent experiments is shown. f, g, Mn2+ supplement fails to restore plasma triglycerides (f) and plasma cholesterol (g) in Surf4 LKO mice. n = 10 mice for CTL, 9 mice for Surf4 LKO. Data are presented as mean ± SEM. Statistical significance was determined by unpaired two-sided Student’s t-test. h, Plasma AST and ALT levels in mice from Fig. 7a. n = 7, 9, 8, 9, 9, and 6 mice for 0 g/L, 0.02 g/L, 0.06 g/L, 0.2 g/L, 1 g/L, 5 g/L, respectively. Data are presented as mean ± SEM. P values were determined by one-way ANOVA test. For a, d, and f-h, X-axis represent manganese doses in the drink. i, Summary of basic parameters in human samples j, k, Lack of correlation between plasma manganese concentrations and total circulating triglyceride levels (N = 297, j) or the HDL-cholesterol levels (N = 246, k) in healthy humans. Data are presented as scatter plots with the fit line and the two-sided Pearson’s correlation coefficient. Source numerical data and unprocessed blots are available in Source Data.

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Supplementary Video 1

FIB-SEM tomography of mouse liver reveals robust ER–Golgi transport of lipoproteins. Yellow, lipoproteins; blue, ER; purple, Golgi; cyan, lipid droplets.

Supplementary Table 2

Raw data of ICP-MS measurement and primers for genotyping.

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Wang, X., Huang, R., Wang, Y. et al. Manganese regulation of COPII condensation controls circulating lipid homeostasis. Nat Cell Biol 25, 1650–1663 (2023). https://doi.org/10.1038/s41556-023-01260-3

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