Fig. 5: Exchange of lipid-exposed amino acids of Hs-A1 trans-IMM helix with At-A1 trans-IMM helix triggers in-cellulo incompatibility. | Nature Communications

Fig. 5: Exchange of lipid-exposed amino acids of Hs-A1 trans-IMM helix with At-A1 trans-IMM helix triggers in-cellulo incompatibility.

From: Kingdom-specific lipid unsaturation calibrates sequence evolution in membrane arm subunits of eukaryotic respiratory complexes

Fig. 5

a Cartoons showing Ndufa1 and ND1 when visualized by PyMol. Left: Human Ndufa1 (blue) and ND1 of CI (PDB: 5XTD). Middle: Human ND1 and Hs-A1Ex-switch (blue-green) where exposed amino acids of Hs-A1 replaced with corresponding amino acids of At-A1 (AlphaFold modeling). Right: Superimposed image. Hs-A1: blue; Hs-A1Ex-switch: teal; Hs-ND1: light gray. RMSD indicated out of 388  aligned amino acids. Template modeling score > 0.9. b Bar-graph showing total contacts of individual amino acids of Hs-A1 and At-A1 trans-IMM helices with the acyl chains of different lipid species in Model 1 (metazoan mimic) simulation experiment. Total contacts averaged over final ~500 ns of 1 µs simulation time (mean). Error bar: 1 SD from mean. Model 1 (metazoan mimic): PC, PE, CL, PS, and PI composition of metazoan IMM. Details in Source Data. c Immunoblot indicating Ndufa1-EGFP in mitochondrial fractions of Hs-A1, Ch-A1, Hs-A1FP/LL, Hs-A1LA/NS probed with EGFP antibody. Hsp60 as loading control. * Cleaved band of A1-EGFP. d Native-PAGE processed for in-gel EGFP fluorescence (IGF) and western blot (WB) using digitonin solubilised mitochondrial fractions from Hs-A1, Ch-A1, and Hs-A1LA/NS. WB sequentially probed with Ndufs1 (Supplementary Fig. 6f) and Sdha antibodies. e Native-PAGE processed for in-gel EGFP fluorescence (IGF) and western blot (WB) using digitonin solubilised mitochondrial fractions from Hs-A1, Ch-A1, and Hs-A1FP/LL. WB sequentially probed with Ndufs1 (Supplementary Fig. 6g) and Sdha antibodies. f Immunoblot indicating Ndufa1-EGFP in mitochondrial fractions of Hs-A1, Ch-A1, and Hs-A1Ex-switch expressing cells with EGFP antibody. *Cleaved band of A1-EGFP. g Native-PAGE processed for in-gel EGFP fluorescence (IGF) and western blot (WB) using digitonin solubilised mitochondrial fractions from Hs-A1, Ch-A1, and Hs-A1Ex-switch. WB sequentially probed with Ndufs1 (Supplementary Fig. 6h) and Sdha antibodies. All uncropped gels and blots in Source Data. d–g SC- supercomplex; HC- holocomplex; SubC- subcomplex. Sdha*- HC; Sdha**- SubC. c–g Data were verified in three independent experiments.

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