Figure 4
Depletion of MIEF1/2 largely reduces Mff-Drp1 interaction and the binding preference of Drp1 to MIEFs versus Mff depends on the relative amounts of MIEFs and Mff. (A) Knockdown of MIEF1/2 severely reduced the binding of exogenous Mff to Drp1. 293T cells were treated with control, MIEF1 or MIEF2 siRNA alone, or MIEF1 plus MIEF2 siRNAs in two different combinations, and then transfected with Myc-Mff. Cell lysates were used for co-IP with anti-Myc beads. (B) Knockdown of MIEF1/2 severely reduced the endogenous interaction between Mff and Drp1. 293T cells were treated with control, or MIEF1 plus MIEF2 siRNAs. Cell lysates were used for co-IP at endogenous levels with goat normal IgG (negative control) or goat anti-Mff antibody. The ratio of co-IPed endogenous Drp1/Mff was analyzed by densitometry. *Represents protein G. (C) Knockout of MIEF1/2 severely reduced endogenous interaction of Mff with Drp1. Cell lysates from wild-type or MIEF1/2 DKO 293T cells were used for co-immunoprecipitation (IP) with Protein G beads connected to goat normal IgG (negative control) or goat anti-Mff antibody. The ratio of co-IPed endogenous Drp1/Mff was analyzed by densitometry. *Represents protein G. (D) Knockdown of Mff does not impair the association between MIEFs and Drp1. 293T cells were treated with control or Mff siRNA, and then transfected with MIEF1-V5 or MIEF2-V5. Cell lysates were used for co-IP with anti-V5 beads. The ratio of co-IPed Drp1/MIEF-V5 (IP) and total Drp1/GAPDH (Input) were analyzed by densitometry. The variation of Drp1 input signals between lanes is due to unequal protein loading (see also Fig. S4C and S4D). In (A–D), all co-IPs were analyzed by immunoblotting with indicated antibodies. (E) Elevated levels of MIEF1 or MIEF2 reduce the interaction of exogenous Flag-Mff with Drp1. 293T cells were co-transfected with Flag-Mff (0.5 µg) and either MIEF1-V5 or MIEF2-V5 in different amounts as indicated. Cell lysates were used for co-IP with anti-Flag beads followed by immunoblotting with indicated antibodies. The ratio of co-IPed Drp1/Mff is shown to the right. (F) Elevated levels of Mff reduce the MIEF-Drp1 interaction. 293T cells were co-transfected with 0.3 µg of either MIEF1-V5 or MIEF2-V5 and Flag-Mff in different amounts as indicated. The lysates were used for co-IP with anti-V5 agarose followed by immunoblotting with indicated antibodies. The ratio of co-IPed Drp1 to MIEF signal is shown to the right. (G) Elevated levels of MIEF1 or MIEF2 also reduce the interaction of endogenous Mff with Drp1. 293T cells were transfected with either MIEF1-V5 or MIEF2-V5 in different amounts as indicated. Cell lysates were used for co-IP with goat anti-Mff antibody followed by immunoblotting with indicated antibodies. Goat normal IgG was used as the negative control. The ratio of co-IPed Drp1/Mff is shown to the right. (H) Mitochondrial localization is required for the association of Mff with Drp1 but not for MIEFs to associate with Drp1. 293T cells were transfected with wild-type Flag-Mff, or transfected with the cytoplasmic mutants Flag-Mff∆C, MIEF1Δ1–48 or MIEF2Δ1–49. Cell lysates were used for co-IP with anti-Flag (for Mff) and anti-V5 (for MIEF) beads followed by immunoblotting with indicated antibodies.
