Fig 1: Gt-hMIA40 regulates the ETC biogenesis and ROS production. Lysates of HEK293T transfected cells used as above. (A) Mitochondria were isolated, resolved on SDS-PAGE and western blotted. The blots probed with OXPHOS antibody that can detect significant components of ETC such as complex I (NDUFB8, 20 kDa), complex II (SDHB, 30 kDa), complex III (UQCRC2, 48 kDa), complex IV (MTCO1, 40 kDa) and complex V (ATP5A, 55 kDa) as shown (B) Quantification of blots from three independent experiments, (C, D, E & F) Activities of complexes I, II, III, and IV of ETC were analyzed spectrophotometrically as described in the Methods section and shown here. (G) ROS levels were measured in HEK293T transfected cells by H2DCFDA method, as described in the methods. All results plotted with mean ± S. E. (n = 3), *P ≤ 0.05. (H) Mitochondria of HEK293T cells over-expressing hMIA40 WT and QM mutant were used for immunocapture of complex III or pre-immune serum (PI) or no antibody (control) and binding to protein A sepharose beads. After resolution on SDS-PAGE and western blotting, the immunoblot probed with the Complex III antibody (H) and hMia40 and Cytochrome C (I).
Fig 2: Updated modular model of human cIV assembly. The present scheme reflects the novel findings presented by this work. It implies COX6B subunit involvement in MT-CO2 subunit maturation. Also, the COX6A subunit incorporates at the final assembly stages independent of the MT-CO3 module. cIV, complex IV; COX, cytochrome c oxidase.
Fig 3: R20C pathogenic variant of COX6B1 affects an early cIV assembly, unlike R20H that disrupts cIV stability. A, differential amount of cIV subunits associated in cIV forms between wt and 6B1KO (top left), 6B1KO + R20C (top right), 6B1KO + R20H (bottom right), and between 6B1KO + R20C and 6B1KO + R20H (bottom left). Volcano plots represent LFQ–MS analysis (n = 2) of all detected proteins (gray) and subunits of cIV modules (initial: orange, MT-CO1: magenta, MT-CO2: green, MT-CO3: yellow, and NDUFA4: blue). COX6B1 protein missing in 6B1KO was visualized; thanks to the imputation step (missing values replaced from a normal distribution) performed during the Perseus analysis of the LFQ–MS data. B, differential amount of cIV AFs associated with cIV forms between wt and 6B1KO (top left), 6B1KO + R20C (top right), 6B1KO + R20H (bottom right), and between 6B1KO + R20C and 6B1KO + R20H (bottom left). Volcano plots represent LFQ–MS analysis (n = 2) of all detected proteins (gray) and cIV assembly factors (MT-CO1 metalation: triangle in magenta, MT-CO1 maturation: circle in magenta, MT-CO2 metalation: triangle in green, MT-CO2 maturation: circle in green, MT-CO1 + MT-CO2 metalation: triangle in yellow, MT-CO1 + MT-CO2 association: circle in orange, and other: circle in blue). cIV, complex IV; COX, cytochrome c oxidase; LFQ–MS, label-free quantification–mass spectrometry.
Fig 4: COX6B subunit is indispensable for early human cIV assembly and function. A, modular model of monomeric cIV assembly. Initially, the MT-CO1 module is formed by the connection of the initiating COX4–COX5A module with MT-CO1, which maturates in the MITRAC complex (mitochondrial translation regulation assembly intermediates of cytochrome c oxidase) (15, 80, 81). The second module consists of MT-CO2, which undergoes copper insertion, to form the binuclear CuA site, assisted by SCO1, SCO2, COX16, COX17, and COA6 chaperones, and subunits COX5B, COX6C, COX7B, and COX8A (39, 82, 83, 84, 85). In the end, the third module containing MT-CO3, COX6A, COX6B, and COX7A subunits joins the nascent cIV under the assistance of HIGD2A, and the addition of NDUFA4 finalizes the assembly of a complete and fully functional cIV (12, 16, 34). cIV subunits are depicted in various colors and mentioned on the top; assembly factors (AFs) of individual modules are noted on the bottom. Cartoons are based on human cIV cryo-EM structure (PDB ID: 5Z62) (65). B, representative SDS-PAGE–WB analysis of the protein steady-state level of cIV subunits (MT-CO1, MT-CO2, COX4I1, COX5A, and COX6C) and citrate synthase (CS) as the loading control in whole-cell lysates of wt, 6B1KO clones (c.1, c.2, and c.3), and COX4 KO (4dKO). COX6C∗ represents an overexposed signal of COX6C from the same image. See also quantification in Figure S1A. C, differential content of cIV subunits between wt and 6B1KO cells. Volcano plot represents LFQ–MS analysis (wt: n = 4; 6B1KO combines c.1, c.2, and c.3: n = 2 per each/n = 6 in total) of all analyzed proteins (gray), and subunits of cIV modules (initial: orange, MT-CO1: magenta, MT-CO2: green, MT-CO3: yellow, and NDUFA4: blue). COX6B1 protein missing in 6B1KO was visualized; thanks to the imputation step (missing values replaced from normal distribution) performed during the Perseus analysis of the LFQ–MS data. For individual 6B1KO clone data, see Figure S1B. D, differential content of cIV assembly factors (AFs) between wt and 6B1KO cells. Volcano plot represents LFQ–MS analysis (wt: n = 4; 6B1KO combines c.1, c.2, and c.3: n = 2 per each/n = 6 in total) of all analyzed proteins (gray), and cIV AFs (MT-CO1 metalation: triangle in magenta, MT-CO1 maturation: circle in magenta, MT-CO2 metalation: triangle in green, MT-CO2 maturation: circle in green, MT-CO1 + MT-CO2 metalation: triangle in yellow, MT-CO1 + MT-CO2 association: circle in orange, and other: circle in blue). For individual 6B1KO clone data, see Figure S1C. E, 2D (BN/SDS)-PAGE–WB detection of cIV (MT-CO1, MT-CO2, COX4I1, COX5A, and COX6C antibodies), cIII (UQCRC2 antibody), and cI (NDUFC2 antibody) in wt (left) and 6B1KO (c.3, right) mitochondrial fraction. Antibody against cII (SDHA) was used as a loading control. F, representative SDS-PAGE–WB analysis of the protein steady-state level of cIV subunits (COX6B2, COX6B1, MT-CO2, COX4I1, and COX6C) and citrate synthase (CS) as the loading control in whole-cell lysates of wt, 6B1KO, 6B1KO + 6B1, 6B1KO + 6B2C-FLAG, and 6B1KO + 6B2N-FLAG. FLAG-tagged COX6B1 and COX6B2 proteins are marked by 6B1FLAG and 6B2FLAG, respectively; endogenous COX6B1 is labeled with 6B1endo (in italics). See also quantification in Figure S1D. G, respiratory rates representing maximal capacities of OXPHOS, ETS, and COX measured in digitonin-permeabilized cells (0.4–1.5 mg of protein) are plotted as the mean ± SD value for wt (n = 3), 6B1KO (n = 2), 6B1KO + 6B1 (n = 4), and 6B1KO + 6B2C-FLAG (n = 3). One-way ANOVA (∗∗∗p < 0.001) was performed. Representative traces with indicated additions of substrates and inhibitors are shown in Figure S1E. cI/III/IV, complex I/III/IV; COX, cytochrome c oxidase; ETS, electron transporting system; LFQ–MS, label-free quantification–mass spectrometry; OXPHOS, oxidative phosphorylation; PDB, Protein Data Bank.
Fig 5: Expression of alternative oxidase (AOX) restores cIV assembly intermediate formation with negligible cIV function in COX6B1-deficient cells. A, representative SDS-PAGE–WB analysis of the protein steady-state level of cIV subunits (MT-CO1, MT-CO2, COX4I1, COX5A, COX6B1, and COX6C), C-terminal FLAG-tagged COX6B1 (FLAG antibody), HA-tagged AOX (HA-tag antibody), and citrate synthase (CS) as the loading control in whole-cell lysates of wt, 6B1KO c.1, 6B1KO c.1 + AOX, 6B1KO c.2, 6B1KO c.2 + AOX, and 6B1KO + 6B1. FLAG-tagged COX6B1 and endogenous COX6B1 proteins are marked with 6B1FLAG and 6B1endo (in italics), respectively. See also quantification in Figure S3A. B, differential content of cIV subunits and assembly factors (AFs) between 6B1KO and 6B1KO + AOX cells. Volcano plots represent LFQ–MS analysis (6B1KO c.2: n = 3, 6B1KO + AOX: n = 3) of all analyzed proteins (gray) and subunits of cIV modules (initial: orange, MT-CO1: magenta, MT-CO2: green, MT-CO3: yellow, and NDUFA4: blue) on the left, cIV assembly factors (MT-CO1 metalation: triangle in magenta, MT-CO1 maturation: circle in magenta, MT-CO2 metalation: triangle in green, MT-CO2 maturation: circle in green, MT-CO1 + MT-CO2 metalation: triangle in yellow, MT-CO1 + MT-CO2 association: circle in orange, and other: circle in blue) on the right. COX6B1 protein missing in 6B1KO was visualized; thanks to the imputation step (missing values replaced from normal distribution) performed during the Perseus analysis of the LFQ–MS data. C, 2D (BN/SDS)-PAGE–WB detection of cIV (MT-CO1, MT-CO2, COX4I1, COX5A, and COX6C antibodies), cIII (UQCRC2 antibody), and cI (NDUFC2 antibody) in 6B1KO c.2 mitochondrial fraction. Antibody against cII (SDHA) was used as a loading control. For 6B1KO c.1, see also Figure S3B. D, 2D (BN/SDS)-PAGE/WB detection of cIV (MT-CO1, MT-CO2, COX4I1, COX5A, and COX6C antibodies), cIII (UQCRC2 antibody), and cI (NDUFC2 antibody) in 6B1KO + AOX mitochondrial fraction. Antibody against cII (SDHA) was used as a loading control. For 6B1KO c.1 + AOX, see also Figure S3B. E, respiratory rates representing maximal capacities of OXPHOS, ETS, and COX measured in digitonin-permeabilized cells (0.4–1.5 mg of protein) are plotted as the mean ± SD value for wt (n = 3), 6B1KO (n = 3), 6B1KO + 6B1 (n = 4) and (n = 3). One-way ANOVA (∗∗∗p < 0.001) was performed. Plotted data of wt, 6B1KO, and 6B1KO + 6B1 originate from Figure S1G. Representative traces of oxygen consumption in 6B1KO + AOX in the presence of the AOX inhibitor SHAM or the cIV inhibitor KCN are shown in Figure S3, C and D, respectively. F, respiratory capacity in COX state plotted as the mean ± SD value of 6B1KO (n = 3) and 6B1KO + AOX (n = 3). Unpaired, two-tailed t test (##p < 0.01) was performed. The graph represents y-axis zoomed-in representation of 6B1KO and 6B1KO + AOX rates displayed in Figure S4E. cI/III/IV, complexes I/III/IV; COX, cytochrome c oxidase; ETS, electron transporting system; KCN, potassium cyanide; LFQ–MS, label-free quantification–mass spectrometry; OXPHOS, oxidative phosphorylation; SHAM, salicylhydroxamic acid.
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