Ancestral sequence reconstruction of the Mic60 Mitofilin domain reveals residues supporting respiration in yeast

Figures

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  FIGURE 1. Boundaries of Mic60's C‐terminal Mitofilin domain defined based on its structural conservation. (a) AlphaFold2‐predicted S. cerevisiae Mic60 structure (AF‐P36112‐F1) and linear Mic60 scheme showing different definitions of Mitofilin domain boundaries. Blue (residues 318–540) denotes more generous domain boundaries based on structural features and alignments, which we used in all experiments in this study and which include all other domain definitions. Previously published experimentally determined Mitofilin domain boundaries are colored in salmon (*, residues 473–536 by Körner et al., 2012), light purple (**, residues 490–536 by Rabl et al., 2009), and dark purple (***, residues 490–533 by Hessenberger et al., 2017). MTS: mitochondrial target sequence; TM: transmembrane region; LBS 1 and LBS 2: lipid binding sites 1 and 2 (light blue) (Hessenberger et al., 2017). (b) Superposition of AlphaFold2‐predicted structures of the yeast (blue; residues 318–540), human (gold; residues 562–758) and alphaproteobacterial (purple; residues 270–433) Mitofilin domains. Root‐mean‐square deviation by residue values for superposition on the yeast Mitofilin domain: 1.04 Å for human Mitofilin…

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  FIGURE 2. Respiratory growth phenotypes for yeast Mic60 Mitofilin chimeras. (a) Spot assay of yeast Mic60 chimeric strains on YPEG media, which is non‐fermentable for yeast, after 5 days at 25°C. Deletion of the Mitofilin domain (mitofilinΔ and mitofilinΔ #2, which is the same strain as mitofilinΔ but a different colony) in yeast Mic60 or substituting it with the H. sapiens (mitofilinΔ::Mitofilin(H. sapiens)) and the C. sphaeroides (mitofilinΔ::Mitofilin(C. sphaeroides)) Mitofilin domain results in knockout‐like (mic60Δ) colony density and sizes. Yeast Mic60 with the Komagataella phaffi Mitofilin domain (mitofilinΔ::Mitofilin(K. phaffi)) exhibits wildtype‐like growth. (b) Immunoblots of the cytosolic and the mitochondrial fractions of yeast Mic60 wildtype and chimeras after mitochondrial isolation against antibodies for Mic60 (top), cytochrome c oxidase subunit 1 (MTCO1, mitochondrial marker, center), and phosphoglycerate kinase (PGK1, cytosolic marker, bottom). All yeast chimeras except for hLOCA and hLBnaCA are expressed and localized to the mitochondria. Strains were grown for 28 h in YPEG and the cell density was adjusted to an OD600 of 11 for each strain. The custom‐made (Cusabio)…

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  FIGURE 3. Ancestral sequence reconstruction of Mitofilin domains. (a) Yeast‐focused (left) and animal‐focused (right) phylogenies of the Mitofilin domain used for ancestral sequence reconstruction. Reconstructed nodes are designated by colored spheres (blue: yeast‐derived ancestors; gold: animal‐derived ancestors). Trees are scaled to identical branch lengths. Some clades are collapsed for clarity. Full trees with branch supports can be found in Figures S5 and S6. yLxCA: yeast‐derived last common ancestor, hLxCA: human‐derived last common ancestor, LSCA: last Saccharomycotina common ancestor, LACA: last Ascomycota common ancestor, LFCA: last fungi common ancestor, LOCA: last ophisthokont common ancestor, LVCA: last vertebrate common ancestor, LBnaCA: last Bilateria non‐arthropod common ancestor. (b) Table showing pairwise sequence identity (in %) between each extant or reconstructed ancestral Mitofilin domain. Pairwise sequence identity is colored from red (low) to blue (high). (c) Linear topology schemes of reconstructed ancestors color‐coded per residue by posterior probability. Posterior probability is colored from purple (low) to orange (high). Gray residues indicate absent residues in…

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  FIGURE 4. Four residues explain respiratory rescue in Mitofilin ancestors. (a) Contextualized regions of multiple sequence alignment between S. cerevisiae, K. phaffi, H. sapiens, and resurrected ancestor Mitofilin domains. Respiration functional and non‐functional sequences are highlighted as such and separated by a horizontal black line. The four residues that explain respiratory growth phenotypes (G421, L425, D497, R522 in S. cerevisiae numbering) are indicated by arrows with S. cerevisiae and yLOCA numbering listed above the alignment. Chemical properties of the four ASR‐identified residues in yeast and yeast‐derived ancestors relative to human and animal‐derived ancestors are denoted by colored spheres below the alignment (red sphere: change in charge; red/purple sphere: changes in charge and polar nature; yellow sphere: change in hydrophobicity). Colored boxes in alignment correspond to colored boxes in full alignment in Figure S9. (B) The ASR‐identified residues are mapped onto the AlphaFold2 predicted yLOCA model. Residues are numbered by yLOCA nomenclature (see (A) for conversion to S. cerevisiae numbering) and colored by their chemical properties as described in (A). All four…

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  FIGURE 5. Transmission electron micrographs suggest a lack of correlation between respiratory function and cristae attachment. (a) Representative transmission electron microscopy micrographs of mitochondria in wildtype (WT) S. cerevisiae (top left), mic60Δ (bottom left), mitofilinΔ::YLOCA (top right), mic60 G421T,L425Y,D497L,R522M (bottom right). Scale bar: 200 nm. (b) Percentage of attached cristae per mitochondrion. N equals the total number of mitochondria counted in separate cells: Wildtype = 43, mic60Δ = 23, mitofilinΔ = 14, mitofilinΔ::YLOCA = 22, mitofilinΔ::YLSCA = 33, mitofilinΔ::Mitofilin(H. sapiens) = 15, mic60 G421T,L425Y,D497L,R522M = 38. Significance of difference is tested relative to wildtype using Welch's t test; ****p < 0.0001. (c) Quantification of cristae length in indicated yeast strains shown in (a). Numbers of measured cristae in mitochondria from separate cells are represented as scatter plots with mean ± scanning electron microscopy (SEM). N refers to the number of cristae: WT = 212, mic60Δ = 75, mic60 G421T,L425Y,D497L,R522M = 137. Significance of difference is tested relative to wildtype using Welch's t test; ****p < 0.0001; ***p = 0.0008. (d) Quantification of…