Role of nuclear-encoded subunit Vb in the assembly and stability of cytochrome c oxidase complex: implications in mitochondrial dysfunction and ROS production

Abstract

CcO (cytochrome c oxidase) is a multisubunit bigenomic protein complex which catalyses the last step of the mitochondrial electron transport chain. The nuclear-encoded subunits are thought to have roles either in regulation or in the structural stability of the enzyme. Subunit Vb is a peripheral nuclear-encoded subunit of mammalian CcO that is dramatically reduced under hypoxia. Although it has been shown to contain different ligand-binding sites and undergo modifications, its precise function is not known. In the present study we generated a cell line from RAW 264.7 murine macrophages that has a more than 80% reduced level of Vb. Functional analysis of these cells showed a loss of CcO activity, membrane potential and less ability to generate ATP. Resolution of complexes on blue native gel and two-dimensional electrophoretic analysis showed an accumulation of subcomplexes of CcO and also reduced association with supercomplexes of the electron transfer chain. Furthermore, the mitochondria from CcO Vb knock-down cells generated increased ROS (reactive oxygen species), and the cells were unable to grow on galactose-containing medium. Pulse-chase experiments suggest the role of the CcO Vb subunit in the assembly of the complex. We show for the first time the role of a peripheral, non-transmembrane subunit in the formation as well as function of the terminal CcO complex.

Figure 1. siRNA mediated silencing of CcO Vb mRNA in RAW 264.7 macrophages

A. 50 μg of mitochondrial protein from clones 1, 3 or 8 stably expressing siRNA against CcO Vb mRNA, or the scrambled siRNA (control), were subjected to immunoblot analysis using monoclonal antibodies to CcO Vb or TOM 20 proteins. The protein level was quantitated using Bio-Rad Versa Doc imaging software by normalizing the intensity of the CcO Vb band to that of TOM 20. B. The level of CcO Vb mRNA from clone exhibiting the lowest protein level (VbKD1) was assayed using Real Time-PCR. The CcO Vb level was normalized to the β-actin mRNA level and is represented as the amount relative to that of control. C, D. CcO activity in Vb KD1 cells was determined by measuring the rate of oxygen consumption (C) and the rate of cytochrome c oxidation (D). The relative activities were calculated based on 2.1 μmole cytochrome C oxidized/min/mg protein in C and 118 μmole of O₂ consumed/min/μg protein in D, considered to be 1. For both assays, the slope of the linear portion of triplicate experiments was averaged and the value of the control experiments was arbitrarily set to one.

Figure 2. Silencing of subunit Vb leads to loss of CcO

A, 100 μg of mitochondrial extract was separated using 6-13% BN-PAGE and stained bands were visualized, B, 150 μg of mitochondrial protein was resolved on BN-PAGE as in A and transferred to PVDF membrane and probed with monoclonal antibodies for CcO I, CcO II, CcO IV, CcO Va, CcO Vb proteins. Complex II was used as loading control. C. 800 μg of mitochondrial protein solubilized with 2% lauryl maltoside was used for spectral analysis. The reduced-minus oxidized difference spectra was recorded and analyzed for the a/a3 specific peak at 605 nm and the cytochrome c/c1 peak at 548 nm. D. X-band EPR of the normal and VbKD cells were measured at 10 K using 5 mW microwave power as described in Materials and Methods. D show the Fe/S cluster and E shows the heme a, and heme a3/CuB binuclear center of CcO respectively.

Figure 3. CcO Vb silencing caused a reduction in the steady state levels of other CcO subunits

A,B. 50 μg (mitochondrial extract) or 80 μg (whole cell extract) protein from control and VbKD1 cells were subjected to immunoblot analysis using monoclonal antibodies for CcO subunits I, II, IV, Va, and Vb, and either TOM 20 for mitochondrial samples or β-actin for whole cell extract as loading controls. C,D. The protein levels for various subunits were quantitated as described in Figure 1A. E. The mRNA levels for indicated CcO subunits were determined using Real Time-PCR. The mRNA levels were normalized to β-actin and the level of each subunit present in control was arbitrarily set at one.

Figure 4. Accumulation of subcomplexes of CcO in VbKD cells

A. Two dimensional BN PAGE/SDS analysis of mitochondrial samples from metabolically labeled cells. Cells were labeled with 20 μCi S³⁵-Methionine in presence of 30 μg/ml cycloheximide for 2h and chased for 3h with excess unlabeled Met. CcO subcomplexes (S1, S2 and S3) are indicated. Autoradiogram of SDS gel has been presented. B. Quantitation of the bands from A. The sum of the band intensities of the holoenzyme and subcomplexes was taken as 100% for calculating the % distribution. C. Two dimensional BN PAGE/SDS analysis of subcomplexes and supercomplexes. 150 μg of mitochondrial protein was used in each case and CcO I antibody was used for probing the blot. An SDS gel pattern is presented. D. Quantitation of the bands from C.

Figure 5. Levels of other electron transfer chain complexes and loss of supercomplexes in VbKD cells

A. Resolution of complexes I, III and V from control and VbKD cells by blue native gel analysis. Details were as in Figure 2 and the Materials and Methods section. B. Relative activities of complexes I, II + III and CcO in control and Vb KD cells. 50 μg protein was used in each case as described in the Materials and Methods section. The relative activities were calculated based on 2.1 μmole cytochrome C oxidized/min/mg protein for CcO, 265 nmoles NADH oxidized/min/mg protein for complex I and 218 nmoles cytochrome C reduced/min/mg protein for complex II/III, considered as 1. C. Immunoblots showing supercomplexes in control and VbKD mitochondria separated by two dimensional Blue Native gel analysis. The blots were probed with CcO subunit I antibody.

Figure 6. Impaired mitochondrial function and growth defects in VbKD cells

A. Relative cellular ATP levels in control and VbKD cells. ATP was measured in 10⁶ cells using Somatic cell assay kit, as described in Materials and Methods. The relative ATP level was calculated based on 2.9 nmoles of ATP/ 10⁶ cells considered as 1. B. Spectrofluorometric analysis of mitochondrial membrane potential (ΔΨm) following the uptake of mitotracker orange (50 nM), as described in Materials and Methods. Excitation at 525 nm and emission at 575 nm were followed. C, D. Growth rates of control and VbKD cells in normal medium (C) and galactose medium (D).

Figure 7. Increased mitochondrial ROS formation in VbKD cells

A. Formation of ROS in isolated mitochondria from control and VbKD cells measured by DCFDA oxidation. Azide (Az, 1mM), N-Acetyl Cysteine (NAC, 10mM) were added at 0 time. B. EPR spectrum obtained from isolated mitochondria following spin trapping with mito-DEPMPO or DEPMPO. Mitochondria (200 μg) were incubated with Mito-DEPMPO (50 mM) in phosphate buffer at pH 7.3 for 20 min at 37 °C. C. Structure of the adduct of superoxide and mito-DEPMPO