Fig. 4
From: Structural insights of human mitofusin-2 into mitochondrial fusion and CMT2A onset
Characterization of MFN2IM dimeric interface. a Dimerization property of the G interface mutants in the presence of \({\mathrm{GDP}} \bullet {\mathrm{BeF}}_{3}^{-}\) was assayed by analytical gel filtration coupled to RALS. Calculated molecular masses at the absorption peaks of 280 nm are plotted in red. mAU milli-absorption units. b AUC results of MFN2IM and MFN1IM (theoretical molecular mass 52.6 and 50.9 kDa, respectively) in the presence of \({\mathrm{GDP}} \bullet {\mathrm{BeF}}_{3}^{-}\). The estimated molecular masses determined by sedimentation velocity are given in kilodaltons (kDa) above the peaks. c Liposome tethering assay for wild-type MFN2IM-TM/MFN1IM-TM and mutants. A representative plot from three independent experiments is shown. d Dimerization via the G interface slows down the nucleotide exchange of MFN2IM and MFN1IM. The initial part of each fluorescence trace was magnified. e Effect of MFN2IM(T129I) and MFN1IM(I108T) on the nucleotide exchange efficiency in the dimeric state. The initial part of each fluorescence trace was magnified and fitted to exponential function as shown in a white curve. f Surroundings of MFN2-Thr129 and MFN1-Ile108 in the dimerization form. g Model for comparison between MFN2 and MFN1 in G domain dimerization. Note the tighter G interface of MFN2 and its sustained dimerization in the GDP-bound state. Pi denotes phosphate group. h Mitochondrial elongation assay with quantification for wild-type MFN2 and G interface mutants. Representative images are shown. Mfn1/2-null MEFs were transduced with retrovirus expressing 16× Myc-tagged WT Mfn2 or G interface mutants. Actin was used as a loading control. A lysate from WT MEFs is shown for comparison. For each construct, 100 cells were scored in biological triplicate. Error bars indicate s.e.m. Scale bars, 10 μm. Source data are provided as a Source Data file
