Skip to main content
. 2020 Mar 17;48(17):9762–9786. doi: 10.1093/nar/gkaa148

Figure 6.

Figure 6.

The YBEY loss results in uS11m-deficient mitoribosomes. (A) Western blot analysis of total protein from YBEY+ and YBEY KO cells reveals the selective depletion of mitoribosomal proteins uS11m and mS37. Two independent cell lines of each genotype are shown. VDAC1 is shown as loading control. (B) Quantitative immunofluorescence analysis of the uS11m protein levels in parental YBEY+ and three independent YBEY KO cell lines. Cytochrome c is shown as a mitochondrial marker. Means ± SEM for n = 6 (YBEY+ and YBEY KO #3), n = 5 (YBEY KO #1), and n = 7 (YBEY KO #2) frames are shown; P-values, two-tailed Welch's test. (C) Rescue of uS11m and COX2 levels in YBEY KO cells by complementation with WT or mutant YBEY genes. Western blotting of total cellular protein is shown; GAPDH is used as loading control. All lanes are cropped from the same membrane. (D) High-resolution 10–40% glycerol gradient analysis of total YBEY+ and YBEY KO cell lysates followed by western blotting reveals the absence of uS11m from the mitochondrial SSU in YBEY KO cells. (E) The structural context of the uS11m protein in the mammalian mitochondrial SSU (pdb: 3jd5). The primary uS11m-binding site (residues 320–440 of 12S rRNA) is shown in grey. The helix 45 contacted by the C-terminal extension of uS11m (‘C’) is shown in magenta. Proteins bS21m (yellow) and mS37 (blue) depend on extensive protein-protein contacts with uS11m and serve to sandwich the 3′-terminus of 12S rRNA (3′). (F) Transient knockdown of uS11m results in destabilization of mS37. A non-targetting siRNA with a ‘scrambled’ sequence was used as a control. (G) Quantitative mass spectrometry analysis of the levels of mitoribosomal and copurifying proteins from YBEY KO as compared to YBEY+ mitochondria. Particularly strongly downregulated proteins are named. See also Supplementary Table S7. (H) Mapping of the proteins particularly strongly depleted from the mitoribosomes of YBEY KO cells on the structure of the mammalian mitoribosome. (I) Transient overexpression of YBEY-3 × FLAG but not of uS11m in YBEY KO cells restores the translation of COX1 and rescues the stability of mS37. Western blotting of total cellular protein is shown.