Fig 2. mtUPR induces dynamic remodelling of the mitochondrial signature lipid cardiolipin.

(A) Lipidomic analysis of indicated phospholipids in highly purified wild-type (WT) and mas1^ts mitochondria isolated after growth at permissive temperature, or after induction of mtUPR for 2 and 4 hours. n = 3, data represent means ± SEM. PC, phosphatidylcholine; PE, phosphatidylethalonamine; PI, phosphatidylinositol; PS, phosphatidylserine; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethalonamine; LPI, lysophosphatidylinositol; LPS, lysophosphatidylserine. (B) Quantification of cardiolipin in WT and mas1^ts mitochondria isolated after growth under non-stress conditions, or after mtUPR induction for 2 or 4 hours. Non-standardized, absolute values in pmol measured in total lipid extracts from isolated, highly purified mitochondria are shown. n = 3, data represent means ± SEM. (C) Quantification of the CL-precursor CDP-diacylglycerol (CDP-DAG) in WT and mas1^ts mitochondria isolated after growth at permissive temperature, or after induction of mtUPR for 2 and 4 hours. Non-standardized, absolute values in pmol measured in total lipid extracts from isolated, highly purified mitochondria are shown. n = 3, data represent means ± SEM. ND, not detected. (D) Quantification of indicated CL subspecies in WT and mas1^ts mitochondria isolated after induction of mtUPR for 4 hours. Non-standardized, absolute values in pmol measured in total lipid extracts from isolated, highly purified mitochondria are shown. n = 3, data represent means ± SEM. (E) Heatmap of distribution of different CL subspecies standardized to total CL content per sample in WT and mas1^ts mitochondria isolated from cells shifted for 4 hours to non-permissive growth temperature. Heatmaps were generated using standardized values in mol% and thus total CL content in each individual sample was set to 100% to represent relative CL species distribution within each sample. Relative changes, scaled and centered for each CL species, are depicted. Shown are three biological replicates for each strain. (F) Schematic overview of the CL biosynthesis pathway in mitochondria. Adapted from [41]. PGP, phosphatidylglycerolphosphate; PG, phosphatidylglycerol; OM, outer mitochondrial membrane; IM, inner mitochondrial membrane. (G) Immunoblot analysis of WT and mas1^ts mitochondria isolated from cells shifted to non-permissive temperature for 10 hours. Tom22 serves as loading control.