Cytochrome c oxidase is required for the assembly/stability of respiratory complex I in mouse fibroblasts

Abstract

Cytochrome c oxidase (COX) biogenesis requires COX10, which encodes a protoheme:heme O farnesyl transferase that participates in the biosynthesis of heme a. We created COX10 knockout mouse cells that lacked cytochrome aa3, were respiratory deficient, had no detectable complex IV activity, and were unable to assemble COX. Unexpectedly, the levels of respiratory complex I were markedly reduced in COX10 knockout clones. Pharmacological inhibition of COX did not affect the levels of complex I, and transduction of knockout cells with lentivirus expressing wild-type or mutant COX10 (retaining residual activity) restored complex I to normal levels. Pulse-chase experiments could not detect newly assembled complex I, suggesting that either COX is required for assembly of complex I or the latter is quickly degraded. These results suggest that in rapidly dividing cells, complex IV is required for complex I assembly or stability.

FIG. 1.

Generation of COX10 knockout mouse cells. (A) loxP sites were introduced into COX10 by homologous recombination of a plasmid containing a floxed exon 6 (box marked “6”) in embryonic stem cells. The diagram shows the position of the loxP sites in the gene (black triangles). A primary culture from skin fibroblasts was established from a homozygous mouse with the floxed COX10 and transfected with the pCre-Hygro plasmid to generate KO cells. To generate KO cells, several hygromycin-resistant clones were obtained and characterized. (B) Genetic characterization of the COX10 locus. The parental fibroblast line (C) and five hygromycin-resistant clones were analyzed by Southern blotting to detect the deletion of COX10 exon 6. The deleted allele was detected in the K clones (K11, K18, and K19); the wild-type floxed allele was detected in two C clones (C12 and C16). Clone C12 showed the presence of both the floxed and the deletion allele, indicating that it was heterozygous for the deletion. DNA was digested with BamHI, and the location of the probe is shown as a bar (P) in panel A.

FIG. 2.

Function of mitochondrial enzymes in COX10 KO cells. (A) Total cell respiration of COX10 KO cells was determined by the rate of oxygen consumption on a Clark O₂ electrode. Knockout clones (K11, K18, and K19) did not respire, either with endogenous substrates or with ascorbate-TMPD, which donates electrons directly to complex IV. (B) Respiratory complex activities of COX10 KO cells. Enzymatic activities were measured spectrophotometrically in isolated mitochondria. Specific activities, initially obtained as micromoles/minute/milligram, are expressed as ratios to citrate synthase activity. Error bars represent standard deviations of at least six independent measurements. (C) Cytochrome spectra of COX10 KO cells. The differential oxidized spectra minus reduced spectra for C12 and K11 mitochondrial cytochromes are shown. The COX10 KO clone K11 lacked the cytochrome a and a₃ peak at 603 nm. cyt, cytochrome; Ctrl, control.

FIG. 3.

Synthesis and steady-state levels of OXPHOS proteins in COX10 KO cells. (A) We analyzed the steady-state levels of several subunits of cytochrome oxidase by Western blotting, including Cox1, Cox4, Cox5b, Cox6b, and cytochrome c. The lower part of the panel shows the steady-state levels of subunits of complex I (Ndufa9), complex II [SDH(Fp)], complex III (Uqcrc2 and Uqcrfs1), and complex V (ATPase-β). Protein loading was analyzed using an antibody against VDAC1. (B) [³⁵S]methionine-labeled mitochondrial proteins (60 μg) were separated on SDS-15% PAGE. Fluorographic bands were assigned as described previously (11). LMTK ρ°, mouse cell line devoid of mtDNA. (C) Southern analyses of total DNA digested with SacI or with NheI. The membrane was hybridized with a ³²P-labeled mitochondrial probe (mouse mtDNA nucleotide positions 5556 to 6268). Ctrl, control; cyt, cytochrome.

FIG. 4.

Mitochondrial morphological abnormalities in COX10 KO cells. Cell lines were immunostained for Cox1 and MitoTracker red. Cox1 colocalized with MitoTracker in control cells but was absent in KO clones. Control cells displayed a typical mitochondrial filamentous network, whereas COX10 KO cells contained round enlarged mitochondria.

FIG. 5.

Steady-state levels of respiratory holocomplexes in COX10 KO cells. A Western blot of one-dimension BN-PAGE was probed with monoclonal antibodies against (A) the Ndufa9 subunit of complex I (C I) and ATPase-β of complex V, (B) Uqcrc2 of complex III, (C) and Cox1 of complex IV. The same samples were subjected to SDS-PAGE, and VDAC was immunodetected as a loading control (D). Ctrl, control.

FIG. 6.

Complex I is not destabilized by decreased COX enzyme activity. (A) In-gel activity for complexes I, IV, and V showed a severe defect in complex I in the K clones. F1 is the dissociated F1 component of complex V. (B) The concentration of KCN required for the complete inhibition of COX activity in a control cell was determined to be 250 to 300 μM. Cells were grown in the presence of 400 μM KCN for different times (1 or 3 days), and enzymatic activities of complex I were determined after BN-PAGE. Panel C shows complex I in-gel activity stain (upper) of control and C12 cells at 0, 1, and 3 days of KCN treatment. The lower part of panel C shows a Western blot demonstrating that although the activity of COX was inhibited by KCN, complex IV was still present. Ctrl, control.

FIG. 7.

Levels of complexes IV and I are restored by transduction of COX10 KO fibroblasts with recombinant COX10. The COX-deficient clone K19 was infected with recombinant lentivirus carrying the wild-type and two mutant forms of COX10. The mutations corresponded to alterations in the human COX10 gene observed in patients with mitochondrial disorders. (A) Infection with wild-type COX10 restored complex IV (CIV) activity to 65% of control levels, whereas the mutants had a less potent effect. (B) Similar results were observed by BN in-gel COX activity and Western blotting for COX subunits. (C) The steady-state levels of complexes I, III, IV, and V are shown by BN-PAGE Western blots. (D) The mtDNA levels in the different COX10-transduced lines are shown. Ctrl, control; GFP, green fluorescent protein; wt, wild type.

FIG. 8.

Lack of COX does not prevent complex I and III association. (A) Western one-dimension BN-PAGE showed that COX10 KO cells (clone K19 or K19 transduced with lentivirus-green fluorescent protein [GFP]) had decreased levels of both complex I and I/III supercomplex. Clone K19 transduced with a lentivirus expressing the wild-type COX10 cDNA had most of complexes I and III as a stable I/III supercomplex. (B) Two-dimension (2ndD) BN Western blots were performed sequentially in the same membrane. Pseudocolors identifying the different complexes were added for clarity. This experiment showed the presence of I/III/IV and I/III supercomplexes in control cells (B). Because Cox1 migrates close to Ndufa9, we added an isolated exposure of a Cox1 Western blot to the bottom of panel B. (C) Using different antibodies on 2D gels showed that the I/III/IV supercomplex was absent in KO clones and the I/III supercomplex was markedly reduced. wt, wild type; Ctrl, control; CI, complex I.

FIG. 9.

Cardiolipin levels are not markedly altered by COX deficiency. (A) One-dimension TLC comparing the phospholipids composition of two COX-competent cell lines (control [Ctrl] and K19/wild type [wt]) and two COX-deficient lines (K19 and K19/green fluorescent protein [GFP]). (B) Separation of cardiolipin by two-dimension TLC using a different solvent system (see Materials and Methods). The identity of cardiolipin was established not only by analyzing standards on two dimensions but also by the analysis of a yeast mutant in cardiolipin synthesis (data not shown). PI, phosphatidylinositol; PA, phosphatidic acid; PS, phosphatidylserine; PG, phosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; CL, cardiolipin. PDME (phosphatidyldimethylethanolamine) was used as a two-dimension standard, but this phospholipid was not detected in the cells. Std, standard; D, dimension.

FIG. 10.

Newly assembled complex I is undetectable in cells with COX deficiency. A COX-deficient cell line (K19) and two controls were pulse labeled for 2 h with Trans³⁵S label and chased for the indicated times as described in Materials and Methods. (A) Autoradiogram of a BN gel (4 to 10% acrylamide gel). (B) The same samples analyzed by Western blotting using antibodies to Ndufa9 and ATPase-β. S³⁵-Met, [³⁵S]methionine; C I, complex I; Ctrl, control.