Lack of cytochrome c in mouse fibroblasts disrupts assembly/stability of respiratory complexes I and IV

Abstract

Cytochrome c (cyt c) is a heme-containing protein that participates in electron transport in the respiratory chain and as a signaling molecule in the apoptotic cascade. Here we addressed the effect of removing mammalian cyt c on the integrity of the respiratory complexes in mammalian cells. Mitochondria from cyt c knockout mouse cells lacked fully assembled complexes I and IV and had reduced levels of complex III. A redox-deficient mutant of cyt c was unable to rescue the levels of complexes I and IV. We found that cyt c is associated with both complex IV and respiratory supercomplexes, providing a potential mechanism for the requirement for cyt c in the assembly/stability of complex IV.

FIGURE 1.

Expression and localization of cyt c in fibroblasts. Immunostaining of control (LF), dKO (L3), WT cyt c reintroduced (CL1) and W60S mutant cyt c reintroduced (CL18) cells with cyt c antibodies. Cells were co-stained with MitoTracker red to confirm the mitochondrial localization.

FIGURE 2.

Mouse fibroblasts with disrupted cyt c (somatic and testis) alleles are OXPHOS-deficient. A, control (LF), cyt c dKO (L3, L4, and L7), wild-type cyt c cDNA reintroduced (CL1 and CL15), and mutant cyt c cDNA reintroduced (CL18) intact cells were analyzed for KCN-sensitive oxygen consumption. All cell lines were also analyzed for the ratio of the ascorbate-TMPD respiration (where electrons are donated directly to cyt c) to endogenous respiration (starting at complexes I and II). cyt c dKO cells and those reintroduced with the mutant cyt c cDNA did not respire. B and D, respiratory complexes (III and IV) activities measured by spectrophotometric assays. Citrate synthase activity was measured in all the samples (C) as a measure of mitochondrial levels. cyt c dKO cells and those reintroduced with the W60S mutant cyt c cDNA (CL18 and CL25) lacked complex IV and had reduced complex III activities.

FIGURE 3.

cyt c-deficient cells lack OXPHOS complexes I and IV. A, Western analysis of mitochondria isolated from the different cell lines using antibodies raised against cyt c, Ndufa9, COX1, SDH, Core2, and ATPase-β. VDAC1 antibody was used as a loading control. cyt c dKO cells (L3 and L4) lacked COX1. B, BN-PAGE was performed on a 4–13% gel. The proteins were transferred onto a PVDF membrane and probed with antibodies raised against the different complex subunits, namely, Ndufa9 (complex I), Core2 (complex III), COX1 (complex IV), and ATPase-β (complex V). CI, CIII, CIV, and CV are complexes I, III, IV, and V, respectively. cyt c dKO cells (L3) and those reintroduced with the W60S mutant cyt c cDNA (CL18) lacked complexes I and IV. Complex I was also very reduced in CL1 and CL15. C, two-dimensional BN-PAGE of control (LF) mitochondria followed by Western analysis with antibodies against cyt c and complexes I (NDUFA9), III (core 2), and IV (COX 1).

FIGURE 4.

Complexes I and IV are not synthesized or are extremely unstable in cyt c dKO cells. Cells were labeled with [³⁵S]methionine and cysteine, followed by a chase for 2, 4, and 24 h, respectively. Lysates from the different cell lines were resolved on an SDS-PAGE (A). Lysates of LF and L3 (dKO) were also analyzed by BN-PAGE (B) and subjected to autoradiography. In C, the same samples shown in B were transferred onto a membrane and probed with antibodies raised against the different complex subunits, namely, Ndufa9 (complex I), COXI (complex IV), and ATPase-β (complex V). VDAC1 and CII(Fp) antibodies were used as loading controls.

FIGURE 5.

Mutant cyt c does not localize normally in the mitochondria. A, mitochondria and mitoplasts from LF, CL1, and CL18 were treated with proteinase K and subjected to Western analysis using antibodies against cyt c, COX1 (inner membrane protein), COXVb (inner membrane protein), and HSP60 (matrix protein). B, 250 μg of mitochondria from LF, L3 (dKO), CL1, and CL18 were resolved on a lithium dodecyl sulfate-PAGE, blotted, and subjected to heme staining to detect mitochondrial c-type cytochromes. Horse cyt c (200 pmol, Sigma) was loaded as a positive control.

FIGURE 6.

Lipid composition of mitochondria lacking cyt c. Quantitation of the total cardiolipin (CL) and triacylglycerol (TAG) content in the different cell lines by electrospray ionization-mass spectrometry was performed as shown in supplemental Figs. S5A and S5B. The results are expressed as a ratio to the respective lipid in control cells (LF).