Figure 3.

GSSG-induced Mfn2 oligomers require GTP hydrolysis and occur primarily in cis. (A) Basal mitochondrial fusion reactions (without cytosol) were performed in the presence of the indicated combinations of 1 mM GSSG and/or 0.5 mM GTP-γS. Amount of fusion, as determined by luciferase activity, is normalized to the basal reaction set at 100%. Error bars show mean+s.e. from the replicates of three independent experiments, and statistical significance was determined by paired t-test analysis; **P<0.005, ****P<0.0001. (B) A model highlighting the different types of Mfn2 oligomers that might be occurring, either in cis or in trans. Blue regions highlight the heptad repeats, the GTP binding domain is shown in green, and a potential disulphide is shown as a red line. (C) Decreasing concentrations of mitochondria (10, 4, 1 μg/μl) were incubated under basal fusion conditions without an initial pelleting step and analysed for fusion as determined by luciferase activity. Data are normalized to the 4 μg/μl reaction set at 100%, with error bars showing mean+s.e. from the replicates of three independent experiments. (D) Western blot of Mfn2 oligomers from basal, 1 mM GSSG or 1 mM GSSG+0.5 mM GTP-γS-treated fusion reactions containing decreasing concentrations of mitochondria as in B. GSSG, oxidized glutathione; Mfn2, Mitofusin 2; Mt, mitochondria.