Fig 2: OMA1 genetically interacts with supercomplex-stabilizing factors.(A) BN- and SDS-PAGE analysis of mitochondria from log-phase oma1Δ cells expressing OMA1, overexpressing RCF1 or COX5a. Source data (full-length blots) are available online in Supplementary information. (B) Mitochondria from log-phase wild-type (WT), oma1Δ rcf1Δ and rcf1Δ oma1Δ cells were analyzed by native (70 μg of mitochondria) or denaturing (20 μg of mitochondria) PAGE. (C) Indicated strains were cultured in YPD medium for 0.5 (12 hours) and 8 days at 28 °C, spotted onto YPD (Glucose) plates and incubated at 28 °C or 37 °C. Pictures were taken after 2 days of growth. (D) The indicated strains were dropped onto YPD (Glucose), YPGal (Galactose) and YPGL (Glycerol/Lactate) plates and incubated at 28 °C.

Fig 3: Respiratory supercomplexes are impaired in Oma1-deficient yeast cells.(A) Steady-state levels of the Cox1-Cox3 subunits of CcO (Complex IV), Cyt1 and Rip1 subunits of bc1cytochrome c reductase (Complex III), Sdh2 subunit of succinate dehydrogenase (Complex II), and Atp2 subunit of F1FO ATP synthase (Complex V), and porin were assessed by immunobloting of mitochondria (20 μg) from WT and oma1Δ cells. (B) BN-PAGE of individual ETC complexes from log and stationary phase WT and oma1Δ cells. Mitochondria (40 μg) were solubilized with 1% dodecyl maltoside (DDM). The complexes were visualized by blotting with indicated antibodies. (C) BN-PAGE of the above mitochondria lysed with 1.5% digitonin. (D) oma1Δ cells bearing vector, Myc-tagged Oma1 or its H203A variant were grown in synethetic galactose medium and used for mitochondrial isolation. Mitochondria (70 μg) were analyzed by BN-PAGE as in C. Another 20 μg of mitochondria were used for SDS-PAGE. Source data (full-length blots) are available online in Supplementary information.

Fig 4: Deletion of Oma1 leads to progressive mitochondrial dysfunction.(A) Electrophoretic analyses of mitochondria from wild-type (WT) strain with Oma1-13xMyc chromosomal tag isolated at exponential (12 h post-inoculation, A600 of 0.8; Log.) and stationary (48 h post-inoculation, A600 of 8.0; Stat.) phases of growth. Mitochondria (70 μg) were solubilized with 1.5% digitonin and subjected to BN-PAGE; 20 μg of mitochondria were used for SDS-PAGE. Oma1 was detected by immunoblotting with anti-Myc. Monomeric form of Complex V (V; BN-PAGE loading control) visualized with anti-F1 serum; porin was SDS-PAGE loading control. Source data (full-length blots) are available online in Supplementary information. (B) Respiratory growth of WT and oma1Δ strains. Synchronized cells were cultured in YPD medium for 0.5 (12 hours), 4 and 8 days at 28 °C and spotted onto YPD (Glucose) or YPGL (Glycerol/Lactate) plates. Pictures taken after 2 (YPD) or 4 (YPGL) days of growth at 28 °C. (C) WT and oma1Δ strains were grown in YPD medium for indicated number of days, diluted to 600 cells and plated on YPGL plates. Following a 4-days incubation at 28 °C, number of colony forming units was determined (n = 3 biologically independent experiments). The values were normalized to number of colonies that each strain formed when cultured on glucose-supplemented plates. (D) Endogenous superoxide levels in WT and oma1Δ cells. Log-phase cells stained with O2.−-specific dye dihydroethidium (DHE) were analyzed by flow cytometry (n = 4). (E) Oxygen consumption of synchronized WT and oma1Δ cells at log (A600 of 0.8) and stationary (A600 of 8.0) stages of growth; n = 3 independent cultures per each strain. (F) Mitochondrial membrane potential of WT and oma1Δ strains during log and stationary growth, assessed by flow cytometry analysis of JC-1-stained cells (n = 3). Data represent mean values ± S.D. **p < 0.01, *p < 0.05, n.s. = non-significant (t-test).

Fig 5: Impaired bioenergetic function in oma1−/− MEFs.(A) Schematic of cellular energy-converting pathways and inhibitors used to profile MEFs’ bioenergetics. FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; OLA, oligomycin A. (B,C) Oxygen consumption rates (OCR) in wild type (OMA1+/+) and oma1−/− MEFs under basal, OLA- and FCCP-stimulated conditions. Cells were cultured in the media containing 10 mM glucose (B) or 10 mM galactose (C). Data are shown as mean ± S.E.M. (n = 3 biological replicates); *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired t-test). (D,E) Respiratory control ratios (a ratio between FCCP-stimulated OCR and basal OCR) in OMA1+/+ and oma−/− cells cultured in 10 mM glucose (D) or 10 mM galactose (E). Error bars indicate S.D.; p values are relative to control.