Fig. 4: Incompatibility of A. thaliana trans-IMM helix in human cells. | Nature Communications

Fig. 4: Incompatibility of A. thaliana trans-IMM helix in human cells.

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

Fig. 4

a Cartoon showing Ndufa1 and ND1 of human (PDB: 5XTD), A. thaliana (PDB: 7ARB) and Chimera (AlphaFold modelling) as visualized by PyMol. Chimera-Ndufa1 (Ch-A1) – A. thaliana Ndufa1 (At-A1) trans-IMM helix in Human Ndufa1 (Hs-A1). b Superimposed images of Hs-A1, At-A1, and Ch-A1. Hs-ND1: light gray; At-ND1: dark gray. RMSD indicated out of 388 aligned amino acids. (a–b) Black dotted lines: trans-IMM regions. c Microscopy of cells expressing EGFP-tagged Hs-A1, At-A1, and Ch-A1. Single Z-section of 0.3 μm. DAPI: nucleus. Tom20: mitochondrial marker. Scale bar: 10 μm. Colocalization coefficients in Supplementary Table 1. d Immunoblots of EGFP-tagged Hs-A1, At-A1, and Ch-A1 expressing cells probed with EGFP-antibody in mitochondrial fraction. Hsp60 as loading control. e Immunoblots indicating Ndufa1-EGFP in mitochondrial fractions and mitoplast fractions of Hs-A1, At-A1, Ch-A1 and A1(G8R) expressing cells probed with EGFP-antibody. Hsp60 as loading control. Tom40 - Outer mitochondrial membrane marker. f Immunoblot indicating Ndufa1-EGFP in total cell lysates of Hs-A1, Ch-A1, and Δ27Hs-A1-EGFP expressing cells, probed with EGFP-antibody. Δ27Hs-A1-EGFP was treated with 2.5 μM MG132 for 24 h. β-actin as loading control. * Cleaved bands of A1-EGFP and full-length Δ27Hs-A1-EGFP. g Immunoblot indicating cleaved product of Ndufa1-EGFP in total cell lysates of Ch-A1, Ch-A1-N21L, and Ch-A1-Q23T expressing cells probed with EGFP-antibody. β-actin as loading control. d–e, g * Cleaved band of A1-EGFP. h in-gel EGFP fluorescence (IGF) and western blot (WB) of digitonin solubilised mitochondrial fractions on native-PAGE from cells shown in 4 d. WB sequentially probed with anti-Ndufs1 (Supplementary Fig. 6d) and anti-Sdha. i Top: Pair-wise sequence alignment of trans-IMM helix of Hs-A1 and Mm-A1. *– identical;: – strongly similar; as per Clustal Omega convention. 1st and 23rd amino acids represented with number at top. Red font: mismatched amino acids in Mm-A1. Bottom: IGF and WB of digitonin solubilised mitochondrial fractions on native-PAGE from Hs-A1-EGFP and Ch -A1Mm-switch-EGFP. WB sequentially probed with anti-Ndufs1 (Supplementary Fig. 6e) and anti-Sdha. h–i SC - supercomplex; HC - holocomplex; SubC - subcomplex. Sdha*- HC; Sdha**- SubC. All uncropped gels and blots in Source Data. c–i Data verified in three independent experiments.

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