Fig 1.
The cytoplasmic domain of MiD51 interacts with Drp1 is dependent on Drp1 oligomerization.
(A) Pull-down assays were performed to test the binding of purified Drp1 or mutants to GST-MiD51133-463 in the presence of different nucleotides. MiD51 and Drp1 or their mutants were mixed evenly before adding to the same amount of resin with the same volume to ensure equal amount of protein was used, and then 1 mM nucleotide at final concentration was added. (B) Quantification of the results in (A). (C) Pull-down assays were performed to demonstrate that the binding of Drp1 to MiD51 depends on Drp1 oligomerization. Purified GST, and GST-MiD51133-463 were loaded onto Glutathione Sepharose beads, and incubated with wild-type and mutated Drp1 to test their binding by SDS-PAGE. (D) Quantification of the results in (C). The binding affinity is expressed as molar ratio of Drp1 to MiD51 mutants. Data are shown as mean ± SEM of three independent experiments performed in triplicate, * P < 0.05; ** P < 0.005 compared to wild-type. (E) Size-exclusion chromatography profiles of Drp1 and Drp1 mutants as indicated. Size-exclusion chromatography was performed with the size-exclusion column Superdex 200 PC 3.2/20 (GE Healthcare). The elution peak at ~11 ml represents the Drp1 dimer.
Fig 2.
Two sites on MiD51 are involved in the interaction with Drp1.
(A) Crystal structure of MiD51133-463. The N domain is colored blue, and the C domain is colored green. Secondary structure elements are labeled. (B) MiD51 sites that bind to Drp1. Left: Overview of the two MiD51 sites, which are outlined in dotted rectangles. Middle: Close-up views show the two binding sites. Key residues are labeled. Right: Electrostatic surface representation of two binding sites, with blue coloring indicating positive charges and red coloring indicating negative charges. (C) WT and mutant GST-MiD51133-463 in vitro pull-down assays were performed with purified Drp1. (D) Quantitation of the results in (C). The binding affinity is expressed as molar ratio of Drp1 to MiD51 mutants. Data are shown as mean ± SEM of three independent experiments performed in triplicate, with ** P < 0.005 compared to wild-type. (E) Sequence alignment of MiD51 and MiD49 sequences. Strictly conserved residues are highlighted in red. Secondary structural elements are depicted on the top of the alignments. Residues involved in Drp1 interaction are marked with★ for DBS1 and ▲ for DBS2.
Fig 3.
MiD51133-463 forms a dimer via an interface close to residue C452 and is important for its interaction with Drp1.
(A) A time course experiment, where the level of dimer formation was quantified every twenty four hours, and non-reducing SDS-PAGE indicates that MiD51133-463 can form dimers in the air. (B) Quantification of the results in (A). The level of dimerization is expressed as the ratio of dimer to monomer. All error bars represent SD from three independent experiments. (C) Native PAGE analysis of monomeric and dimeric MiD51133-463. (D) Size-exclusion chromatography analysis of monomeric and dimeric MiD51133-463. Size-exclusion chromatography was performed with the size-exclusion column Superdex 75 PC 3.2/20 (GE Healthcare). The blue profile represents dimer and the green profile represents monomer. (E), Non-reducing SDS-PAGE of wild-type and mutant MiD51133-463 shows that the C452S mutant is not able to form dimers. (F) Native PAGE of wild-type and mutant MiD51133-463 also indicates that the C452S MiD51133-463 mutant does not form dimers. (G) Gel filtration analysis of MiD51133-463 and mutants shows that the dimer peak is missing in the C452S mutant. (H) Dimerization analysis of full-length MiD51 and mutants C165S and C452S in HeLa cells. Wild type or mutant MiD51-Myc was expressed in HeLa Cells and analyzed with Myc antibody. Actin is a loading control. (I) HeLa cells were transfected with wild type MiD51 or mutant C452S, and treated with Antimycin A. Transfection with wild type MiD51 results in mitochondrial fission, but no fission is observed in cells transfected with the C452S mutant, indicating that in the absence of dimer formation, MiD51 can not perform its function in mitochondrial fission. (J) Quantification of the results in (I). Mitochondrial morphology was scored as described previously [26]. Data were obtained from three independent experiments, with 100 cells per experiment, ** P < 0.005.
Fig 4.
A proposed model for MiD51-mediated recruitment of Drp1 and mitochondrial fission.