Fig 2: 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 oma1? cells. (B) BN-PAGE of individual ETC complexes from log and stationary phase WT and oma1? 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) oma1? 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 3: Loss of OMA1 impairs mammalian RSCs.(A) BN-PAGE of mitochondria from wild type (OMA1+/+) and oma1-/- MEFs. Mitochondria (80 µg) were solubilized with 2% digitonin. Protein complexes were visualized with antibodies to NDUFA9 (Complex I), CORE1 (Complex III), MTCO1 (Complex IV) and ATP5A (Complex V). (B) BN-PAGE of WT and oma1-/- mitochondrial lysates. Mitochondria (40 µg) were solubilized with 1% dodecyl maltoside (DDM). Individual ETC complexes were visualized by immunoblotting with indicated antibodies. (C) Steady-state levels of the indicated subunits of ETC complexes in WT and oma1-/- mitochondria. Ten and 15 µg of mitochondria were analyzed by SDS-PAGE. Source data (full-length blots) for key panels of this figure are available online in Supplementary information.

Fig 4: Truncated Sod2 protein accumulates in the cytosol. A, SOD2C cells supplemented with 1 mM BCS or 5 µM CuSO4 were assayed for Sod1 and Sod2 protein localization after subcellular fractionation of crude mitochondrial extracts from the cytosolic fraction and visualized by Western blotting. Control proteins specific to cytosol (GAPDH—glyceraldehyde-3-phosphate dehydrogenase) and mitochondria (MTCO1—mitochondrial COX1 subunit) were included to validate fractionation efficacy. B, Subcellular fractionation as in (A) but using the SOD2-CuRE1/2mut strain. BCS, bathocuproinedisulfonic acid.

Fig 5: Dynamics of non-OXPHOS RNAs through mitochondrial biogenesisa, b, c, RNA levels (reads per kb) normalized to spike-in controls and plotted as -fold change compared to levels in log phase glucose growth for (a) all nuclear-encoded structural components of the complexes shown, (b) intron-encoded maturases, and (c) nuclear and mitochondrial-encoded mitoribosome subunits. To calculate values for maturase transcripts, only reads not overlapping the main ORF (COX1 or COB) were considered. Group II intron splicing intermediates stably accumulate and may not represent translation-competent transcripts.