Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects

Abstract

Mitochondrial protein SURF1 is a specific assembly factor of cytochrome c oxidase (COX), but its function is poorly understood. SURF1 gene mutations cause a severe COX deficiency manifesting as the Leigh syndrome in humans, whereas in mice SURF1(-/-) knockout leads only to a mild COX defect. We used SURF1(-/-) mouse model for detailed analysis of disturbed COX assembly and COX ability to incorporate into respiratory supercomplexes (SCs) in different tissues and fibroblasts. Furthermore, we compared fibroblasts from SURF1(-/-) mouse and SURF1 patients to reveal interspecies differences in kinetics of COX biogenesis using 2D electrophoresis, immunodetection, arrest of mitochondrial proteosynthesis and pulse-chase metabolic labeling. The crucial differences observed are an accumulation of abundant COX1 assembly intermediates, low content of COX monomer and preferential recruitment of COX into I-III2-IVn SCs in SURF1 patient fibroblasts, whereas SURF1(-/-) mouse fibroblasts were characterized by low content of COX1 assembly intermediates and milder decrease in COX monomer, which appeared more stable. This pattern was even less pronounced in SURF1(-/-) mouse liver and brain. Both the control and SURF1(-/-) mice revealed only negligible formation of the I-III2-IVn SCs and marked tissue differences in the contents of COX dimer and III2-IV SCs, also less noticeable in liver and brain than in heart and muscle. Our studies support the view that COX assembly is much more dependent on SURF1 in humans than in mice. We also demonstrate markedly lower ability of mouse COX to form I-III2-IVn supercomplexes, pointing to tissue-specific and species-specific differences in COX biogenesis.

Keywords: Cytochrome c oxidase; Doxycycline; Leigh syndrome; Pulse-chase; Respiratory supercomplexes; SURF1(−/−) mouse knockout.

Fig. 1

Two-dimensional electrophoretic analysis of different COX forms present in SURF1^−/− mouse tissues and fibroblasts and SURF1 patient fibroblasts. Respiratory complexes and supercomplexes were solubilized using 8 g digitonin/g protein of isolated mitochondria, separated by BNE in the first dimension and SDS PAGE in the second dimension and detected by Western blots using specific antibodies to COX1 (cIV), CORE1 (cIII), NDUFB6 (cI) and NDUFS3 (cI). For analysis, heart (A), muscle (B), liver (C), brain (D) and fibroblast (E) of SURF1^+/+ and SURF1^−/− mice as well as fibroblasts (F) of human control and SURF1 patient were used. COX1 assembly intermediates (COX1 AI), COX monomer (M), COX dimer (D), III₂–IV SC (III₂–IV), I–III₂ SC (I–III₂), I–III₂–IV_n SCs (I–III₂–IV_n), complex III dimer (cIII₂), complex I (cI).

Fig. 2

Distribution profiles of the COX1 signal in different COX forms resolved by BNE/SDS PAGE analysis. COX1 signals from two-dimensional electrophoretic analysis in Fig. 1 were expressed as quantitative distribution profiles. SURF1^+/+ and SURF1^−/− mice heart (A), muscle (B), liver (C), brain (D) and fibroblasts (E) and human control and SURF1 patient fibroblasts (F). Individual COX forms are indicated: COX1 assembly intermediates (COX1 AI), COX monomer (M), COX dimer (D), III₂–IV SC (III₂–IV), I–III₂–IV_n SCs (I–III₂–IV_n).

Fig. 3

Analysis of COX assembly in SURF1-deficient mouse and human fibroblasts following the release of doxycycline-arrested mitochondrial protein translation. BNE/SDS PAGE representative Western blot analysis using antibody to COX1 subunit performed in doxycycline treated (A) human control and (B) SURF1 patient fibroblasts and (C) SURF1^+/+ and (D) SURF1^−/− mouse fibroblasts. For BNE analysis, cell membranes were isolated and solubilized by digitonin (4 g dig/g protein). COX1 assembly intermediates (AI), COX monomer (M), COX supercomplexes (SC) are marked. Control cells without DOX (C_-DOX), times t0–t96 represent time points in hours after DOX removal, when the cells were harvested. Two independent DOX experiments for each cell line were performed to generate 2D maps showing distribution of COX1 forms along the DOX experiments at time points t0–t96 h (0 h–96 h) in (E) human control and (F) SURF1 patient fibroblasts and (G) SURF1^+/+ and (H) SURF1^−/− mouse fibroblasts. Relative quantities of individual COX forms (assembly intermediates, monomer, dimer, supercomplexes) were used to divide the respective COX1 signal for given time point from 1D SDS PAGE (see Fig. 4 E–H in the Data in Brief appendix), normalized to SDHA signal. The resulting datasets from each experiment representing individual COX forms in human and mice cells were rescaled (minimum = 0, maximum = 100) and averaged to plot in comparative 2D maps. COX assembly intermediates (AI), COX monomer (M), COX supercomplexes (SC).

Fig. 4

Pulse-chase metabolic labeling of mitochondrially synthesized proteins in SURF1-deficient mouse and human fibroblasts. (A, B) Human control and SURF1 patient fibroblasts, (C, D) SURF1^+/+ and SURF1^−/− mouse fibroblasts. Mitochondrial translation products of mouse and human fibroblasts were labeled with [³⁵S] methionine + cysteine for 2 h in the presence of cycloheximide. After indicated time of chase (0.5 h, 6 h, 16 h, 24 h) with cold methionine and cysteine, cell membranes were isolated, solubilized by digitonin (4 g/g protein) and analyzed by BNE/SDS PAGE. Radioactivity was detected in stained dried gels. On the right side of each gel, individual mtDNA coded subunits are marked and mtDNA coded COX subunits COX1, COX2, COX3 are highlighted in frames. COX assembly intermediates (AI), COX monomer (M) are marked by dotted lines; COX SCs (SC).

Fig. 5

2D maps of pulse-chase metabolic labeling of mitochondrially synthesized COX1 subunit. (A) Human control and (B) SURF1 patient fibroblasts, (C) SURF1^+/+ and (D) SURF1^−/− mouse fibroblasts. 2D maps show biogenesis of COX1 subunit along the pulse-chase experiment at chase times t0.5, t6, t16 and t24 h (0.5 h–24 h). Relative quantities of individual COX forms (assembly intermediates, monomer, supercomplexes) were used to divide the respective COX1 signal for given time chase from 1D SDS PAGE, normalized to overall radioactive signal in each time chase. The resulting datasets from each experiment representing individual COX forms in human and mouse cells were rescaled (minimum = 0, maximum = 100) and plotted in comparative 2D maps. COX assembly intermediates (AI), COX monomer (M), COX supercomplexes (SC).