MINOS1 and Mio10 are not associated with the F₁F₀ATPase. (A) Coimmunoprecipitation of cytochrome c oxidase (COX) and F₁F_oATPase from isolated HEK293T mitochondria after digitonin solubilization. Eluates were analyzed by SDS–PAGE and Western blotting. Total, 1.5%; eluate, 100%. (B) Isolated wild-type (WT) and Atp20^ZZ mitochondria were solubilized and subjected to IgG chromatography. Total and eluate fractions were analyzed by SDS–PAGE, followed by immunoblot analyses with indicated antibodies. Total, 3%; eluate, 100%. (C) WT, mio10Δ, atp20Δ, and atp2Δ yeast cells were spotted in serial 10-fold dilutions on fermentable (glucose) and nonfermentable (glycerol) media. (D) Mitochondria (Mito) from WT, mio10Δ, and fcj1Δ were separated by SDS–PAGE and analyzed by Western blotting. (E) Solubilized mitochondria from WT and mio10Δ were separated by BN-PAGE and analyzed by Western blotting. Asterisk indicates nonspecific cross-reaction.

Mio10 and MINOS1 are part of the Fcj1/Mitofilin (MINOS) complex. (A) Isolated mitochondria from WT and Mio10-Streptavidin-FLAG (Mio10^SF) were solubilized and subjected to Strep-Tactin-Sepharose chromatography. Eluates were separated on SDS–PAGE and stained with colloidal Coomassie. Each gel lane was cut in 23 equal slices and proteins digested with trypsin. Peptides were analyzed by MS. (B) Total extracts and eluate fractions of Strep-Tactin-Sepharose chromatography using wild-type or Mio10^SF mitochondrial extracts were separated by SDS–PAGE and analyzed by Western blotting. Total, 3.0%; eluate, 100%. (C) Solubilized mitochondria of WT and Mio10^SF were subjected to separation by size exclusion chromatography and analyzed by Western blotting. (D) WT, mio10Δ, and fcj1Δ yeast cells were spotted in serial 10-fold dilutions on synthetic fermentable (glucose) and synthetic nonfermentable (glycerol) media. (E) Schematic overview of SILAC approach analyzing MINOS1-containing complexes. Mitochondria from HEK293T cells grown either in light or heavy isotope–containing medium were solubilized and subjected to coimmunoprecipitation (Co-IP) with MINOS1 antibodies or control antibodies. Eluates were mixed, separated by SDS–PAGE, and stained with colloidal Coomassie. The gel lane was cut into 23 equal slices and proteins digested with trypsin. Peptides were analyzed by LC-MS/MS. (F) Identification of MINOS10-associated proteins by Co-IP and SILAC-MS. RAW MS files from LC-MS/MS were analyzed by MaxQuant and Mascot using the IPI human protein database. Results from Maxquant were analyzed and visualized with R. Red dots indicate enriched proteins characterized by a high normalized ratio of heavy over light values (H/L). Forward, F (H, MINOS1; L, control); reverse, R (H, control; L, MINOS1; reverse ratios were inverted for plotting). (G) Isolated mitochondria from HEK293T cells were solubilized and subjected to Co-IP with MINOS1 and control antibodies Total, 1.5%; eluate 100%. Eluates were separated by SDS–PAGE and analyzed by Western blotting. (H) Solubilized mitochondria of HEK239T cells were subjected to separation by size exclusion chromatography and analyzed by Western blotting. Asterisks indicate nonspecific cross-reaction.

MINOS1 and Mio10 are conserved mitochondrial inner membrane proteins. (A) Alignment of Mio10 homologues (ClustalW 2.0.11). Black boxes indicate identical residues in at least four species; gray boxes indicate similar amino acids (Erdmann et al., 1991). Sc, S. cerevisiae; Ag, Ashbya gossypii; Nc, Neurospora crassa; Mm, Mus musculus; Hs, Homo sapiens. (B) Organization of S. cerevisiae Mio10. Black boxes, predicted transmembrane domains (TMs). (C) Subcellular localization of MINOS1 and cyclophilin D in Vero cells by immunofluorescence. Bars, 10 μm. (D) Western blot analyses of the submitochondrial localization and (E) membrane association of MINOS1 as described in Materials and Methods. (F) Analyses of the submitochondrial localization and (G) membrane association of Mio10 by Western blotting. T indicates the total; S and P indicate the supernatant and pellet after ultracentrifugation. TX-100, Triton X-100; PK, proteinase K.

Mitochondrial morphology is altered in mio10Δ. Fluorescence microscopy analysis of S. cerevisiae wild-type, atp20Δ, mio10Δ, and fcj1Δ (A–D) cells transformed with plasmid pVT100U-mitoGFP (Westermann and Neupert, 2000) to visualize mitochondrial morphology. Cells were analyzed using a DeltaVision Spectris fluorescence microscope equipped with a 100× objective and a fluorescein isothiocyanate filter. For each image 10–15 Z-section images were taken at 0.5-μm intervals after focusing on the middle plane of the cell. Images were deconvoluted using softWoRx. Bars, 2.5 μm.

Cristae morphology is defective in mio10Δ. (A) Electron microscopy of S. cerevisiae WT, atp20Δ, mio10Δ, and fcj1Δ cells after high-pressure freezing (HPF). (B) Statistical analysis of different types of mitochondria based on electron microscopy images from HPF fixed WT (n = 49), atp20Δ (n = 51), mio10Δ (n = 61), and fcj1Δ (n = 57) cells. Detailed view of normal, intermediate, and onion-like mitochondria types. (C) Electron microscopy of WT, atp20Δ, mio10Δ, and fcj1Δ cells after KMnO₄ fixation. (D) Detailed view of WT, atp20Δ, mio10Δ, and fcj1Δ mitochondria shown in C. Bars, 1 μm (A, C), 200 nm (B, D).