Fig. 2. GTP-dependent formation of trans-mitochondrial complex with Mfn1 protein. (A) Assay for the formation of Mfn1-mediated trans-mitochondrial complex. (B) Post nuclear supernatant (PNS), mitochondria (mit) or salt-washed mitochondria (wash mit) fractions were prepared from HeLa cells expressing HA-tagged or FLAG-tagged Mfn1. Fractions with HA-tagged and FLAG-tagged Mfn1 were mixed, and incubated with or without GTP at 0°C or 30°C for 30 minutes. In a reaction with PNS, mitochondria (p) and the supernatant (s) were separated by centrifugation after the reaction. All these fractions were solubilized with 1% digitonin and subjected to immunoprecipitation using anti-HA antibodies. The immunoprecipitates were subjected to SDS-PAGE and subsequent immunoblotting using anti-FLAG and anti-HA antibodies. As controls, the reaction mixtures using mock-transfected cells instead of Mfn1-HA (w/o HA), or immunoprecipitation without antibodies (w/o IgG) were analyzed. The reaction mixture prior to immunoprecipitation was also analyzed by immunoblotting by anti-FLAG (input). (C) Time course of co-immunoprecipitation. PNS or mitochondria as prepared in B were incubated for the indicated time intervals at 0°C or 30°C. The recovery of Mfn1-FLAG from the reaction mixture is shown. (D) The PNS fractions with HA-tagged or FLAG-tagged Mfn1 were mixed and incubated with 1 mM ATP, GTP, GTPS or 1 mM ATP plus 1 mM GTP at 30°C for 30 minutes, then subjected to immunoprecipitation and immunoblotting as in B. (E) The PNS fractions obtained from HeLa cells expressing wild-type Mfn1 (wt) or its mutant on the GTPase (K88T) with HA- or FLAG-tag were incubated with GTP in the indicated combinations, then analyzed as in B. (F) The PNS fractions obtained from HA-tagged or FLAG-tagged Mfn1-expressed cells were mixed and incubated at 30°C for 1 hour with or without GTP, in the presence of 5 mM NADH plus 20 mM sodium succinate (NaSuc), or 20 µM CCCP to dissipate membrane potential. In a separate experiment, PNS fractions with HA-Mfn1 or FLAG-Mfn1 were sonicated, then mixed and incubated as above (sonic). These samples were solubilized by digitonin and subjected to immunoprecipitation and subsequent immunoblot analysis.