Extended Data Fig. 4: Yeast assays.

a, Schematic overview of yeast complementation experiments. In the presence of 2% glucose, expression of chromosomally encoded Mgm1 from the GAL1 promoter is suppressed. Yeast cells irreversibly lose the mitochondrial genome in the absence of Mgm1 (that is, become ρ⁰) and cannot switch to respiratory growth upon glucose depletion (as shown by the shift from low glucose conditions to the oxidation of ethanol produced during the fermentation of glucose). By co-expressing wild-type yeast Mgm1 or the corresponding Mgm1 mutants, functionality of the Mgm1 variants is assessed through various rescue parameters. b, Representative growth curve for the unmodified yeast strain transformed with an empty vector (e.v.), the engineered yeast strain (P_GAL1-MGM1) complemented with yeast Mgm1 or an empty vector control (n = 3 independent experiments). c, Time-dependent expression of Mgm1, mitochondrially encoded cytochrome c oxidase subunit 1 (Cox1) and the nuclear-encoded mitochondrial heat shock protein Ssc1 (loading control) was assessed by western-blot analysis of isolated mitochondria upon transfer of yeast cells from a glucose-rich to a glucose-depleted medium containing 2% ethanol as the carbon source. = marks the long and short isoforms of Mgm1, ∼ is an unspecific band and * marks an Mgm1 degradation product (n = 2 independent experiments). Uncropped blots are shown in Supplementary Fig. 2. d, Western-blot analysis of isolated mitochondria from P_GAL1-MGM1 yeast grown in glucose-containing medium containing plasmids that encode the respective mutant (n = 3 independent experiments). e, f, Yeast growth complementation assays with Mgm1 mutants containing mutations in the dimer interface and the paddle–paddle contacts. F805D in yeast corresponds to F840D in C. thermophilum and N675A corresponds to I700D. F779D/S780D in C. thermophilum corresponds to M745D/S746D in yeast. Representative growth curves are shown from n = 3 independent experiments. Data in Fig. 3b and Extended Data Fig. 4e are derived from the same experiment; the controls are shown in all graphs as a reference. g, Mitochondrial morphology of the indicated yeast strains was assessed by fluorescence microscopy. DNA and mitochondria were stained with DAPI and DiOC₆, respectively. Three representative images from n = 2 independent cultures are shown. Dimensions of the images are 7.5 µm × 7.5 µm. h, Overexpression of Mgm1(Y520A) (with a mutation in the tetramer interface) leads to a strong dominant-negative effect on respiratory yeast growth (in media containing 3% glycerol as the carbon source). Representative growth curves are shown from n = 3 independent experiments. i, Overexpression of Mgm1(Y520A) leads to only a partial loss of mitochondrial DNA, as assayed by Cox1 expression. n = 3 independent experiments. j, k, Overexpression of dominant-negative Mgm1(Y520A) leads to a fragmentation of the mitochondrial network. Representative images and quantification of mitochondrial morphology in cells from n = 3 independent cultures, data displayed as mean ± s.d. l, Representative electron micrographs of yeast ultrathin sections assaying mitochondrial ultrastructure. Compared to mitochondria in wild-type yeast transformed with empty vector or pMgm1, mitochondria from cells expressing Mgm1(Y520A) showed a substantial loss of cristae and altered crista shape, as indicated by an increased diameter of the crista junctions and lumen and shorter crista length. Scale bars, 70 nm. m, Quantification of cristae morphology. WT+pMgm1: n_mito = 208, n_cristae = 132; WT+e.v.: n_mito = 201, n_cristae = 135; WT+pMgm1(Y520A): n_mito = 202, n_cristae = 81; 2 independent experiments. ***P < 0.0001 (Gaussian approximation); Mann–Whitney U-test (two-sided, 95% confidence interval); cristae number graph shows mean ± s.e.m.: WT+pMgm1: (4.8 ± 0.2) nm; WT+e.v.: (3.8 ± 0.2) nm; WT+pMgm1(Y520A): (1.4 ± 0.2) nm; crista length graph shows mean ± s.e.m.: WT+pMgm1: (153 ± 5) nm; WT+e.v.: (147 ± 5) nm; WT+pMgm1(Y520A): (115 ± 5) nm; crista diameter graph shows mean ± s.e.m.: WT+pMgm1 junction: (19.9 ± 0.5) nm; WT+e.v. junction: (21.0 ± 0.5) nm; WT+pMgm1(Y520A) junction: (26 ± 1) nm; WT+pMgm1 lumen: (24.7 ± 0.6) nm; WT+e.v. lumen: (25.8 ± 0.7) nm; WT+pMgm1(Y520A) lumen: (35 ± 2) nm.