Identification of the MiD51 DRR. (A) Structure of the DRR (green) displaying residues deleted in MiD51^ΔPEYFP (purple) and side chains of salt-bridging residues. (B) MiD51^TALEN MEFs expressing GFP-tagged MiD51, MiD51^ΔNBD, MiD51^ΔPEYFP, and MiD51^R235A (green) were fixed and immunostained for Drp1 (red) along with the mitochondrial marker Tom20 (gray). Bars, 20 µm. (C) Pearson correlation analysis of Drp1 colocalization on mitochondria with GFP-tagged MiD51^WT, MiD51^ΔNBD, MiD51^ΔPEYFP, MiD51^R234A, MiD51^R235A, and Miro1 (control). n = 3; mean ± SEM; >20 linescans/experiment. (D) MiD51^ΔPEYFP redirected to lysosomes cannot recruit Drp1. In the presence of the A/C Heterodimerizer (A/C Het), cytosolic FKBP-MiD51^ΔTM-GFP, FKBP-MiD51^ΔN118-GFP, and FKBP-MiD51^ΔTM/ΔPEYFP-GFP are targeted to lysosomes by binding to lysosomal-targeted FRB (lyso-FRB). Bars: (merge) 20 µm; (inset) 5 µm. Cells were immunostained for Drp1 (red) and mitochondria (Tom20, gray). Images are shown in the merged panel and the inset is magnified in the rightmost panel, with GFP and Drp1 (red) shown. (E, top) Detailed view of the DRR (green) including key residues deleted in MiD51^ΔPEYFP (purple), with black dotted lines indicating ionic interactions in a salt bridge and bond distances. (bottom) Crystal structure of the constricted DRR in MiD51^ΔN118/PEYFP displaying salt-bridging residues and bond distances.