Fig. 1: MFSD7C interacts with heme and ETC components in the mitochondria.
From: MFSD7C switches mitochondrial ATP synthesis to thermogenesis in response to heme
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.
