A putative amphipathic alpha-helix is required for Mdi1 function. (A) Helical wheel projection plots generated by HeliQuest (Gautier et al., 2008) of the second α-helical region of the indicated fungal orthologues of Mdi1. Valine 56 of S. cerevisiae Mdi1 is circled in black. (B) Maximum intensity projections of deconvolved images of the indicated yeast strains expressing mito-dsRed. Mdi1*-2xFLAG is expressed with the MDI1 promoter. Mdi1*(V56A)-2xFLAG and Mdi1*(V56E)-2xFLAG are driven by an estradiol-controlled GalL promoter and were grown in the presence of 3 nM β-estradiol. (C) A graph depicting the categorization of mitochondrial morphology of cells as in B. Data shown represent at least 75 cells per strain in each of three independent experiments and bars indicate SEM. Asterisks (***P < 0.001) represent unpaired two-tailed t tests. (D) A model for a potential role of Mdi1 in facilitating mitochondrial fission. Cell boundaries are indicated with dotted lines. Scale bar = 3 µm. See also Fig. S3.