Figure 5.
C20orf24 is Required for Respirasome Assembly
(A) Model of C20orf24-respirasome interplay in healthy individuals versus patients with respiratory chain defects (i). Sub-network of C20orf24 link with CI/III/IV subunits in DNLCs (gray lines; ii). CI/III/IV-specific antibody immunoprecipitates and input from the mt extracts of ECSCs, DNLCs, and mouse brain immunoblotted (IB; iii) with antibody raised against C20orf24 and its interacting proteins; protein G beads alone (without antibody) served as negative control.
(B) Docking of C20orf24 (yellow) on the human respirasome CI (green), CIII (cyan), and CIV (orange) structures (i) is shown as a zoom-in where C20orf24 can be seen at the interface with indicated structures of respiratory complex subunits (ii–iv).
(C) Endogenous C20orf24 level in ECSCs and DNLCs with anti-C20orf24 (i). Band intensities normalized to HSPD1 loading control (LC). t-SNE (t-distributed stochastic neighbor embedding) plot (ii; number of single cells shown in parentheses) and log2 UMI (unique molecular identifier) counts (ii; *p value by Wilcoxon rank-sum test) of the C20orf24 transcript in DNLCs over ECSCs.
(D) Respirasome activity (fold change, FC) of C20orf24 KO or complemented with FLAG-tagged C20orf24 (rescue) versus control (ctrl) single-guide RNA (sgRNA) in DNLCs (i). Respirasome complexes in control and C20orf24 KO DNLCs by BN-PAGE (ii); percent reduction of respirasome complex subunits in KO over control is shown after subtracting background signals from each band.
(E) Oxygen consumption rate (OCR) measured in control and KO DNLCs under basal conditions followed by the sequential addition of oligomycin (1 μM), FCCP (0.25 μM; carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), as well as rotenone and antimycin A (0.5 μM).
(F) C20orf24 mRNA or respirasome activity measured by qRT-PCR (i; FC, fold change), RNA sequencing (ii), or colorimetric assay (iii) in the CIV-deficient fibroblasts (CDFs) of two subjects (S1, S2) versus healthy fibroblasts (HFs). Immunoblotting (i) of endogenous C20orf24 level in HFs or CDFs (S2) with anti-C20orf24. Band intensities normalized to HSPD1 loading control (LC).
(G) C20orf24 mRNA or respirasome activity measured by qRT-PCR or colorimetric assay in C20orf24 knockdown (KD) versus control (ctrl) of HFs (i) or CDFs of S2 complemented with C20orf24-FLAG (rescue) versus GFP-FLAG control (ii). Data for (D, E, F, and G) are mean ± SD (n = 3 technical replicates; *p ≤ 0.05 by Student's t test).