Extended Data Fig. 7: Prohibitin structures.
(a) IMOD slicer view of a representative particle recorded on mitochondria of wildtype U2OS cells. 1: Side view shows a convex profile of the particle, tilted to 10° (x-rotation). 2: Top view exhibits a ring-like appearance of the same particle, tilted to 90° (x-rotation) with respect to (1). (b) Western blot analysis. RNAi-mediated knockdown of PHB1 and PHB2 showed that depletion of one prohibitin leads also to the reduction of the other paralog. (c) Immunofluorescence imaging of PHB1KD and PHB2KD cells using an antibody against PHB1 and PHB2, respectively. Mitochondrial network labelled with Tomm20 antibodies (cyan). (d) Representative central tomographic slices of U2OS cells. Prohibitin structures were marked with green spheres. PHB1KD and PHB2KD cells exhibit reduced prohibitin abundances compared to wildtype cells. (e) AlphaFold-predicted structure of the PHB2-Dreiklang fusion protein (PHB2 in blue, Dreiklang in green). (f) Central slices of tomograms of individual prohibitin complexes. PHB2-DK cells show additional densities at the top of the structures, indicating PHB2 molecules fused to Dreiklang. (g) Left: Subtomogram average of PHB-DK particles (U2OS PHB2-DK cells). STA confirms the presence of an additional density at the bell top, indicating the presence of DK (white arrowheads). Right: Isosurface rendering in ChimeraX (transparent) with the AlphaFold-predicted structure of Dreiklang fitted into the density. (h) Central tomographic slice of a prohibitin assembly. Image overlay with a volume representation of the prohibitin cryo-EM map (orange) and a Dreiklang barrel (green). The additional density at the bell top matches the size of the Dreiklang barrel. Scale bars in A, 20 nm. Scale bars in C, 50 µm. Scale bars in D, 500 nm. Scale bars in F, G, 10 nm. Scale bar in H, 20 nm. Source data and blots are available.
