Figure 2

Complementation of Renilla luciferase demonstrates the core requirements for mitochondrial fusion. (a) The relative luciferase units (RLU) from the Renilla luciferase assay demonstrates that mitochondrial fusion requires physiological temperature and energy. Sonication of the basal fusion containing 5 mg/mL cytosol for 5 min, performed in the absence of trypsin, provided a maximum complementation of the reporter constructs to determine the efficiency of the reaction. Negative controls performed in the presence of apyrase or on ice are shown. The error bars in the histogram (as in all experiments) show the standard deviation of two independent reactions per condition. All conditions were repeated at least three times in duplicate. (b) Time course of the fusion reaction, as indicated. (c) Nucleotide dependence of the fusion reaction in the presence of 1 mM ATPγS, 1 mM GTPγS and in the absence of exogenous GTP, as indicated. (d) Mitochondrial fusion in the presence of 2 μM CCCP, 1 μM Valinomycin and 2 μM nigiricin demonstrates the dependence of the reaction on the charge gradient across the inner membrane.