Figure S2. Mdi1 is a soluble... | Rockefeller University Press

Figure S2.

$Mdi1 is a soluble intermembrane space protein that concentrates at discrete submitochondrial foci. (A) AlphaFold2 predicted structure of Mdi1 (Jumper et al., 2021; Varadi et al., 2022). Arrow marks the site of insertion of the internal 2xFLAG tag. (B) Left: Representative deconvolved maximum intensity projections of the indicated yeast strains expressing mito-DsRed. Right: A graph depicting categorization of mitochondrial morphology of cells as shown on left. 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 test. NS indicates not statistically significant. (C) Western analysis with the indicated antibodies of mitochondria isolated from a ∆mdi1 strain expressing Mdi1*-2xFLAG and subjected to alkaline extraction. Crude membranes were incubated with 0.1 M Na2CO3 pH11.5, and total (T), pellet (P), and supernatant (S) fractions were collected after centrifugation. (D) Western analysis with the indicated antibodies of mitochondria isolated from a ∆mdi1 strain expressing Mdi1*-2xFLAG and subjected to protease protection analysis. Mitochondria were treated where indicated with proteinase K. Mitoplast sample indicates selective disruption of the OMM by a combination of osmotic swelling and mechanical disruption. (E) Western analysis of whole-cell lysates of the indicated yeast strains grown in SCD media and expressing Mdi1*-2xFLAG driven by the indicated promoter and treated as indicated with β-estradiol. (F) Maximum intensity projections of deconvolved images of cells expressing mito-dsRed and grown as in E. (G) A graph depicting categorization of mitochondrial morphology from the indicated strains as in E and F. 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. (H) Maximum intensity projections of confocal images of chromosomally tagged Mdi1-GFP11x7 haploid cells (left) or heterozygous diploid cells (right) cells coexpressing IMS-targeted GFP1-10 (green) and mito-dsRed (magenta). Arrows mark focal assemblies of Mdi1-GFP11x7. (I) A graph depicting the categorization of mitochondrial morphology from the indicated strains as in H. 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 test. NS indicates not statistically significant. (J) Maximum intensity projections of confocal images of wild-type cells co-expressing mito-dsRed, IMS-targeted GFP1-10, and plasmid-borne IMS-GFP11x7 (left) or chromosomally integrated Mdi1-GFP11x7 (right). Arrows mark focal accumulations of Mdi1-GFP11x7. Cell boundaries are indicated with dotted lines. Scale bar = 3 µm. Source data are available for this figure: SourceData FS2.$

Mdi1 is a soluble intermembrane space protein that concentrates at discrete submitochondrial foci. (A) AlphaFold2 predicted structure of Mdi1 (Jumper et al., 2021; Varadi et al., 2022). Arrow marks the site of insertion of the internal 2xFLAG tag. (B) Left: Representative deconvolved maximum intensity projections of the indicated yeast strains expressing mito-DsRed. Right: A graph depicting categorization of mitochondrial morphology of cells as shown on left. 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 test. NS indicates not statistically significant. (C) Western analysis with the indicated antibodies of mitochondria isolated from a ∆mdi1 strain expressing Mdi1*-2xFLAG and subjected to alkaline extraction. Crude membranes were incubated with 0.1 M Na₂CO₃ pH11.5_, and total (T), pellet (P), and supernatant (S) fractions were collected after centrifugation. (D) Western analysis with the indicated antibodies of mitochondria isolated from a ∆mdi1 strain expressing Mdi1*-2xFLAG and subjected to protease protection analysis. Mitochondria were treated where indicated with proteinase K. Mitoplast sample indicates selective disruption of the OMM by a combination of osmotic swelling and mechanical disruption. (E) Western analysis of whole-cell lysates of the indicated yeast strains grown in SCD media and expressing Mdi1*-2xFLAG driven by the indicated promoter and treated as indicated with β-estradiol. (F) Maximum intensity projections of deconvolved images of cells expressing mito-dsRed and grown as in E. (G) A graph depicting categorization of mitochondrial morphology from the indicated strains as in E and F. 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. (H) Maximum intensity projections of confocal images of chromosomally tagged Mdi1-GFP11_x7 haploid cells (left) or heterozygous diploid cells (right) cells coexpressing IMS-targeted GFP1-10 (green) and mito-dsRed (magenta). Arrows mark focal assemblies of Mdi1-GFP11_x7. (I) A graph depicting the categorization of mitochondrial morphology from the indicated strains as in H. 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 test. NS indicates not statistically significant. (J) Maximum intensity projections of confocal images of wild-type cells co-expressing mito-dsRed, IMS-targeted GFP1-10, and plasmid-borne IMS-GFP11x7 (left) or chromosomally integrated Mdi1-GFP11_x7 (right). Arrows mark focal accumulations of Mdi1-GFP11_x7. Cell boundaries are indicated with dotted lines. Scale bar = 3 µm. Source data are available for this figure: SourceData FS2.