Fig. 5. Comparison of activity of Mfn1 and Mfn2 for GTP-dependent complex formation. (A,B) HA-tagged or FLAG-tagged Mfn1 or Mfn2 was separately expressed in HeLa cells, and the intact mitochondria (A) or digitonin-solubilized mitochondria (B) were mixed in the indicated combinations and incubated with 0.4 mM GTP at 30°C for the indicated times. The complex formation was analyzed by immunoprecipitation using anti-HA antibodies and subsequent immunoblotting by anti-FLAG or anti-HA antibodies as in Fig. 2. The immunoprecipitated bands were quantified. (C) The solubilized (+dig) or intact (–dig) mitochondria from the HeLa cells expressing Mfn1-FLAG or Mfn2-FLAG were mixed, and incubated with or without GTP. The incubation mixtures with intact mitochondria were solubilized by digitonin. All the reaction mixtures were then analyzed by sucrose density gradient centrifugation in the presence of digitonin as in Fig. 4. (D) The solubilized (+dig) or untreated (–dig) rat liver mitochondria were incubated with or without GTP. The reaction mixtures were analyzed by sucrose density gradient centrifugation in the presence of digitonin as in Fig. 4. Recovery (%) of Mfn proteins in the peak fractions is shown.