Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Sep 24;11(1):4837.
doi: 10.1038/s41467-020-18607-1.

MFSD7C switches mitochondrial ATP synthesis to thermogenesis in response to heme

Yingzhong Li #  1 , Nikola A Ivica #  1 , Ting Dong #  1 , Dimitrios P Papageorgiou  2 , Yanpu He  1   3 , Douglas R Brown  1 , Marianna Kleyman  1 , Guangan Hu  1 , Walter W Chen  4   5 , Lucas B Sullivan  1 , Amanda Del Rosario  1 , Paula T Hammond  1 , Matthew G Vander Heiden  1   6 , Jianzhu Chen  7
Affiliations

MFSD7C switches mitochondrial ATP synthesis to thermogenesis in response to heme

Yingzhong Li et al. Nat Commun. .

Abstract

ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. MFSD7C interacts with heme and ETC components in the mitochondria.
a Superdex 75 gel filtration chromatograms of human NTD and heme. NTD was incubated with heme and run on Superdex 75 gel filtration column. The flow through was measured for absorbance at 230 nm (gray), 380 nm (blue), and 415 nm (green). Absorbance intensity was normalized to maximum value. b Changes in absorption spectrum intensity of heme incubated with different concentrations of wild-type (red) or mutant (gray) NTD (see color scale). Heme (100 µM) absorption was set to zero. c Changes in absorption spectrum intensity of heme (100 µM) incubated with different concentrations of wildtype (blue) or mutant (gray) HP motif peptide (see color scale). Wildtype (WT) and mutant (Mut) peptide sequences are shown. d Co-IP and immunoblotting analysis of HA-tagged MFSD7C and FLAG-tagged CYC1, NDUFA4, COX4I1, ATP5h, ATP5c1, or HMOX1. Shown are representative data from five separate experiments. e Co-IP and immunoblotting analysis of endogenous MFSD7C with ATP5h, SERCA2b and HMOX1 in bone marrow-derived macrophages from Mfsd7cfl/fl or Mfsd7c−/− C57BL/6 mice (see “Methods” section for details). Shown are immunoblots of MFSD7C, ATP5h, SERCA2b, and HMOX1 on whole lysates or immunoblots of MFSD7C on anti-ATP5h, anti-SERCA2b and anti-HMOX1 immunoprecipitates. Representative data from one from three experiments are shown. f Mouse whole brain extract was fractionated using differential centrifugation to enrich for mitochondria and analyzed by immunoblotting against the indicated proteins. Shown are representative data from three separate experiments. WCE: whole cell extract, Sup: supernatant, Mito: 10,000×g mitochondrial fraction. g Immunofluorescent localization of MFSD7C in mitochondria. THP-1 cells were stained with MitoTracker (green), fixed and permeabilized, and then stained with rabbit polyclonal antibody specific for the C-terminus of MFSD7C, followed with Alexa Fluor® 594-labeled goat anti-rabbit antibody (red). Nuclei were labeled using DAPI (blue). Co-localization between MFSD7C and MitoTracker appears as yellow in the merged images. Scale bar in d and f: 10 µm.
Fig. 2
Fig. 2. MFSD7C and heme regulate coupling of mitochondrial respiration.
a–f Comparison of mitochondrial respiratory activities between parental THP-1 cells and four Mfsd7c knockout clones (A11, B11, 3D12, and 4B8). Parental and knockout THP-1 cells were cultured in the presence of either vehicle or 40 µM heme for 1 h. a Representative OCR measurements of THP-1 cells and 4B8 knockout clone under the indicated conditions. Comparison of basal OCR (b), maximal OCR (c), and ECAR (d), between THP-1 cells and 7CKO clones from three separate experiments. Each dot represents a technical replicate (b–d, n = 18 independent experiments). e MMP was measured using TMRE (200 nM) by flow cytometry (n = 3 independent experiments). f Cellular ATP/ADP ratio (n = 5 independent experiments). g, h Comparison of thermogenesis between parental THP-1 cells and two 7CKO clones (A11 and 4B8) by microscopy using FPT. Green channel detects the FPT intensity and the red channel distinguishes knockout cells, which expressed mCherry, from the parental THP-1 cells. Shown are representative images (g) and FPT intensity of A11 and 4B8 relative to THP-1 cells from three separate experiments (h). i Comparison of thermogenesis between THP-1 and 7CKO cells by flow cytometry. FPT fluorescence intensity was quantified by flow cytometry. Shown are representative plots from three separate experiments. j Comparison of temperature changes in THP1 (n = 4 independent samples) and 7CKO (n = 3 independent samples) culture media (ΔT) with or without heme treatment as measured by thermocouples. k and l Cells were not treated or treated with heme for 1 h before lysis. Cell lysates were precipitated with anti-FLAG antibody, eluted with FLAG peptide, then precipitated with anti-HA antibody, and eluted with HA peptide, and finally subjected to immunoblotting with anti-HA and anti-FLAG antibodies. Input is the total cell lysate. Shown are representative co-IP/immunoblots (k) and average band intensities with standard deviation from three independent experiments (l). p values were calculated using two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.005). Data are presented as mean value ± standard deviation.
Fig. 3
Fig. 3. Loss of Mfsd7c stimulates OCR, ECAR, and thermogenesis in BMDM.
Bone marrow-derived macrophages (BMDM) were derived from exon 2 floxed (Mfsd7cfl/fl) C57BL/6 mice and C57BL/6 mice with exon 2 deletion in macrophages (Mfsd7c−/−). a, b Representative OCR analysis from Mfsd7cfl/fl and Mfsd7c−/− macrophages as measured by Seahorse XF96e Analyzer and their responses to oligomycin (oligo), FCCP, and rotenone plus antimycin A (Rot/AA) (n = 3 technical replicates) (a), and their average basal and maximal OCR (b) (n = 9 independent experiments from 3 biological replicates). c Representative ECAR analysis of Mfsd7cfl/fl and Mfsd7c−/− macrophages and their responses to glucose, Rot/AA, and 2-deoxyglucose (2-DG) (n = 3 technical replicates). d Mitochondrial membrane potential of Mfsd7cfl/fl and Mfsd7c−/− macrophages analyzed with TMRE staining followed by flow cytometry (1 representative histogram picked from 3 biological replicates). e Cellular ATP/ADP ratio, (n = 4 biological replicates from average of 5 technical replicates). f Estimates of cellular temperature of Mfsd7cfl/fl and Mfsd7c−/− macrophages as measured by fluorescent thermoprobe (n = 3 biological replicates). p values were calculated using unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.005). Data are presented as mean value ± standard deviation.
Fig. 4
Fig. 4. NTD of MFSD7C mediates heme effect on mitochondrial respiration.
a–c Comparison of basal OCR (a), maximal OCR (b), and ECAR (c) of THP-1, 4B8, and 4B8FL, and 4B8ΔN cells treated with vehicle or heme for 1 h (n = 18 separate experiments). d Comparison of FPT intensity of THP-1, 4B8, 4B8FL, and 4B8ΔN cells. Cells were incubated with FPT for 6 h, washed and reseeded in poly-lysine coated glass bottom dishes. After cells attached to the glass, medium containing 40 μM heme was added. Cells in the same field were imaged immediately and again 1 h later. Relative FPT fluorescent intensities, normalized to THP-1, from three experiments are shown. Representative images are shown in Supplementary Fig. 9e. (n = 3 independent experiments). e Restoration of thermogenesis in 7CKO cells by expression of MFSD7CFL and MFSD7CΔN. THP-1 cells were co-cultured with 4B8, 4B8FL, or 4B8ΔN in FPT solution for 6 h, washed and reseeded in poly-lysine coated glass bottom dishes. Cells were imaged by confocal laser-scanning microscopy. Green channel shows the FPT intensity and the red channel (mCherry) distinguishes knockout cells from the parental THP-1 cells. Representative images from three experiments are shown. Scale bar: 10 µm. f Heme treatment reduces MMP in 4B8 cells complemented with MFSD7CFL but not MFSD7CΔN. THP-1, 4B8, 4B8FL, and 4B8ΔN cells were treated with vehicle or heme for 1 h and MMP was measured by using JC-10 Mitochondrial Membrane Potential Assay Kit followed by flow cytometry. Representative mitochondrial staining profiles from three experiments are shown. g and h Heme does not disrupt MFSD7CΔN interactions with ETC components. Co-IP was performed as in Fig. 2k, except HA-tagged MFSD7CΔN was used. Shown are representative Western blotting data (g) and quantification of band intensities from n = 3 independent experiments (h). p values were calculated using two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.005). Data are presented as mean value ± standard deviation.
Fig. 5
Fig. 5. MFSD7C and heme regulate thermogenesis through SERCA2b in THP-1 cells.
a 293FT cells were transfected with HA-tagged murine MFSD7C and FLAG-tagged murine SERCA2b. Thirty hours later, MG132 was added into half of the cells and the other half was not treated. Another 35 h later, some cells were treated with 40 μM heme for 1 h before lysis. Cell lysates were immunoprecipitated with anti-FLAG antibody, and eluted, followed by anti-HA immunoprecipitation and elution. Total cell lysate and elute were subjected to Western blotting and probed with anti-SERCA2b or anti-MFSD7C antibodies. Shown are representative data from one of three experiments. b 293T cells were co-transfected with HA-MFSD7C and FLAG-SERCA2b. Twenty-four hours later, cells were either not treated or treated with 10 μM MG132 for either 6 or 12 h. The cells were lysed and subjected to FLAG pull-down and blotted with anti-MFSD7C, anti-SERCA2b and anti-ubiquitin antibodies. Shown are representative data from one of three experiments. c SERCA2b protein levels in parental THP-1 cells, 7CKO cells (3D12, A11, B11, and 4B8) or SERCA2b−/− (#1 and #3) cells. Parental THP-1 cells were either not treated or treated with heme before lysis. Shown are representative data from one of four experiments. d THP-1, 4B8, 4B8FL, and 4B8ΔN cells were either not treated or treated with 40 μM heme for 1 h, lysed and subjected to Western blotting with anti-MFSD7C (top), anti-SERCA2b (middle), and anti-®-tubulin (bottom). Shown are representative data from one of two experiments. e THP-1 cells were incubated with FPT for 6 h, washed, treated with or without 4 μM thapsigargin for 2 h. Cells were washed and treated with 40 μM heme for 1 h before flow cytometry. Representative FPT histograms from one of three experiments are shown. f Parental and Serca2b−/− THP-1 cells were incubated with FPT for 6 h, washed and treated with or without 40 μM heme for 1 h, followed by flow cytometry. Shown are representative FPT histograms from one of three experiments.
Fig. 6
Fig. 6. Proposed MFSD7C-regulated cellular thermogenesis model.
a, b Proposed models of MFSD7C regulation of mitochondrial respiration in response to heme, with MFSD7C residing in the inner (a) or the outer (b) mitochondrial membrane. When heme level is low, MFSD7C interacts with ETC components and SERCA2b, leading to SERCA2b ubiquitination and degradation and coupled mitochondrial respiration: increased ATP synthesis and reduced thermogenesis. When heme level is high, heme binding to the NTD of MFSD7C disrupts its interactions with ETC components and SERCA2b, leading to SERCA2b stabilization and uncoupled mitochondrial respiration: increased thermogenesis and reduced ATP synthesis.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

  • Dysregulation of FLVCR1a-dependent mitochondrial calcium handling in neural progenitors causes congenital hydrocephalus.
    Bertino F, Mukherjee D, Bonora M, Bagowski C, Nardelli J, Metani L, Zanin Venturini DI, Chianese D, Santander N, Salaroglio IC, Hentschel A, Quarta E, Genova T, McKinney AA, Allocco AL, Fiorito V, Petrillo S, Ammirata G, De Giorgio F, Dennis E, Allington G, Maier F, Shoukier M, Gloning KP, Munaron L, Mussano F, Salsano E, Pareyson D, di Rocco M, Altruda F, Panagiotakos G, Kahle KT, Gressens P, Riganti C, Pinton PP, Roos A, Arnold T, Tolosano E, Chiabrando D. Bertino F, et al. Cell Rep Med. 2024 Jul 16;5(7):101647. doi: 10.1016/j.xcrm.2024.101647. Cell Rep Med. 2024. PMID: 39019006 Free PMC article.
  • Molecular mechanism of choline and ethanolamine transport in humans.
    Ri K, Weng TH, Claveras Cabezudo A, Jösting W, Zhang Y, Bazzone A, Leong NCP, Welsch S, Doty RT, Gursu G, Lim TJY, Schmidt SL, Abkowitz JL, Hummer G, Wu D, Nguyen LN, Safarian S. Ri K, et al. Nature. 2024 Jun;630(8016):501-508. doi: 10.1038/s41586-024-07444-7. Epub 2024 May 22. Nature. 2024. PMID: 38778100 Free PMC article.
  • Structural and molecular basis of choline uptake into the brain by FLVCR2.
    Cater RJ, Mukherjee D, Gil-Iturbe E, Erramilli SK, Chen T, Koo K, Santander N, Reckers A, Kloss B, Gawda T, Choy BC, Zhang Z, Katewa A, Larpthaveesarp A, Huang EJ, Mooney SWJ, Clarke OB, Yee SW, Giacomini KM, Kossiakoff AA, Quick M, Arnold T, Mancia F. Cater RJ, et al. Nature. 2024 May;629(8012):704-709. doi: 10.1038/s41586-024-07326-y. Epub 2024 May 1. Nature. 2024. PMID: 38693257
  • A positive feedback inhibition of isocitrate dehydrogenase 3β on paired-box gene 6 promotes Alzheimer-like pathology.
    Wang X, Liu Q, Yu HT, Xie JZ, Zhao JN, Fang ZT, Qu M, Zhang Y, Yang Y, Wang JZ. Wang X, et al. Signal Transduct Target Ther. 2024 Apr 29;9(1):105. doi: 10.1038/s41392-024-01812-5. Signal Transduct Target Ther. 2024. PMID: 38679634 Free PMC article.
  • MFSD7c functions as a transporter of choline at the blood-brain barrier.
    Nguyen XTA, Le TNU, Nguyen TQ, Thi Thuy Ha H, Artati A, Leong NCP, Nguyen DT, Lim PY, Susanto AV, Huang Q, Fam L, Leong LN, Bonne I, Lee A, Granadillo JL, Gooch C, Yu D, Huang H, Soong TW, Chang MW, Wenk MR, Adamski J, Cazenave-Gassiot A, Nguyen LN. Nguyen XTA, et al. Cell Res. 2024 Mar;34(3):245-257. doi: 10.1038/s41422-023-00923-y. Epub 2024 Feb 2. Cell Res. 2024. PMID: 38302740 Free PMC article.
See all "Cited by" articles

References

    1. Mishra P, Chan DC. Mitochondrial dynamics and inheritance during cell division, development and disease. Nat. Rev. Mol. Cell Biol. 2014;15:634–646. doi: 10.1038/nrm3877. - DOI - PMC - PubMed
    1. Bennett AF, Ruben JA. Endothermy and activity in vertebrates. Science. 1979;206:649–654. doi: 10.1126/science.493968. - DOI - PubMed
    1. Brand MD, et al. The significance and mechanism of mitochondrial proton conductance. Int. J. Obes. Relat. Metab. Disord. 1999;23:S4–S11. doi: 10.1038/sj.ijo.0800936. - DOI - PubMed
    1. Jastroch M, Divakaruni AS, Mookerjee S, Treberg JR, Brand MD. Mitochondrial proton and electron leaks. Essays Biochem. 2010;47:53–67. doi: 10.1042/bse0470053. - DOI - PMC - PubMed
    1. Ricquier D, Bouillaud F. Mitochondrial uncoupling proteins: from mitochondria to the regulation of energy balance. J. Physiol. 2000;529:3–10. doi: 10.1111/j.1469-7793.2000.00003.x. - DOI - PMC - PubMed

Publication types

MeSH terms

-