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, 39 (3), e102817

Respiratory Supercomplexes Act as a Platform for Complex III-mediated Maturation of Human Mitochondrial Complexes I and IV

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Respiratory Supercomplexes Act as a Platform for Complex III-mediated Maturation of Human Mitochondrial Complexes I and IV

Margherita Protasoni et al. EMBO J.

Abstract

Mitochondrial respiratory chain (MRC) enzymes associate in supercomplexes (SCs) that are structurally interdependent. This may explain why defects in a single component often produce combined enzyme deficiencies in patients. A case in point is the alleged destabilization of complex I in the absence of complex III. To clarify the structural and functional relationships between complexes, we have used comprehensive proteomic, functional, and biogenetical approaches to analyze a MT-CYB-deficient human cell line. We show that the absence of complex III blocks complex I biogenesis by preventing the incorporation of the NADH module rather than decreasing its stability. In addition, complex IV subunits appeared sequestered within complex III subassemblies, leading to defective complex IV assembly as well. Therefore, we propose that complex III is central for MRC maturation and SC formation. Our results challenge the notion that SC biogenesis requires the pre-formation of fully assembled individual complexes. In contrast, they support a cooperative-assembly model in which the main role of complex III in SCs is to provide a structural and functional platform for the completion of overall MRC biogenesis.

Keywords: complex I; complex III; cytochrome b mutation; mitochondrial respiratory chain assembly; supercomplexes.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Complex I and IV enzymatic deficiencies in Δ4‐CYB cells

The activities (mUnits/g of protein) of the MRC enzymes were determined by spectrophotometric kinetic measurements in WT and Δ4‐CYB cells and normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n = 4–6 biological replicates). Unpaired Student's t‐test **P = 0.0100; ***P = 0.0002; ****P < 0.0001.

Complex I in‐gel activity assays (IGA) after blue‐native gel electrophoresis (BNGE) of WT and Δ4‐CYB samples solubilized with either 1.6 mg DDM/mg protein or 4 mg digitonin/mg protein. The gels were incubated in the reaction mixture for 1.5 h (lighter signals in DDM gels) or were left to continue the reaction for 24 h to obtain darker signals (DDM and Digitonin gels).

BNGE, Western blot, and immunodetection, with anti‐NDUFS1 (cI), anti‐ATP5A (cV), and anti‐SDHB (cII) antibodies, of samples from the WT cybrids and from Δ4‐CYB clones E (#17.3E) and B (#17.3B). Clone E was the cell line of choice for the analysis shown in panels (A and B), and all the figures hereafter.

Source data are available online for this figure.
Figure 2
Figure 2. Reduced steady‐state levels of structural MRC subunits in Δ4‐CYB cells

Scatter plot generated from the peptide content analyzed by mass spectrometry in each of the 64 slices excised from BNGE and after quantifying the heavy‐to‐light (H/L) and light‐to‐heavy (L/H) ratios in both reciprocal labeling experiments performed with mitochondria isolated from WT and Δ4‐CYB cells (see also Fig EV1). The logarithmic ratios were calculated using MaxQuant (Cox & Mann, 2008), and the statistical significance of the differences for the enrichment or depletion of the proteins was determined with Perseus (Cox & Mann, 2011; Tyanova et al, 2016).

Labeling of the thirteen mtDNA‐encoded MRC structural subunits. Cells were incubated with [35S]‐L‐Met for 1 h in the presence of emetine 100 μg/ml to inhibit cytoplasmic translation.

Immunodetection of complex III structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and Δ4‐CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β‐Actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). Two‐way ANOVA with Sidak's multiple comparisons test ****P < 0.0001; ***P = 0.0007.

Immunodetection of complex I structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and Δ4‐CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β‐actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). Two‐way ANOVA with Sidak's multiple comparisons test ****P < 0.0001; **P = 0.0024 (NDUFS3); **P = 0.0061 (NDUFB8).

Immunodetection of complex IV structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and Δ4‐CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β‐Actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). Two‐way ANOVA with Sidak's multiple comparisons test **P = 0.0011.

Immunodetection of complex II structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and Δ4‐CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β‐actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). Two‐way ANOVA with Sidak's multiple comparisons test ****P < 0.0001.

Immunodetection of complex V structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and Δ4‐CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β‐actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). There were no differences in the steady‐state levels of the tested subunits (2‐way ANOVA with Sidak's multiple comparisons test).

Source data are available online for this figure.
Figure EV1
Figure EV1. Complexome profiling of cIII2‐containing structures in samples from in WT and Δ4‐CYB cells solubilized with DDM (related to Figs 2, 3, 4)

Heatmaps of cIII2 structural subunits and assembly factors derived from the DDM‐solubilized samples in the experiment where Δ4‐CYB cells were labeled with the heavy (H) amino acids. Black = 0; yellow = 0.5; red = 1 relative peptide intensities of the most frequent peptide found in each of the samples individually.

Complexome profiles of the cIII2 structural subunits found in both cell lines in the two reciprocal labeling experiments. The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1.0 versus the molecular mass, calculated using the individual complexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines were determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and Δ4‐CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments.

Heatmaps of cIII2 structural subunits and assembly factors derived from the digitonin‐solubilized samples in the experiment where WT cells were labeled with the heavy (H) amino acids. Black = 0; yellow = 0.5; red = 1 relative peptide intensities of the most frequent peptide found in each of the samples individually.

Profiles of the subunits only found in the WT samples in the two reciprocal labeling experiments. The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1 versus the molecular mass, calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The represented values are the mean ± SEM of the two reciprocal labeling experiments.

Figure 3
Figure 3. Blue‐native gel electrophoresis (BNGE) mass spectrometry and immunodetection analysis of cIII2‐related proteins

Complexome profiles of cIII2 structural subunits generated by analyzing the peptide content in each of the 64 slices in which the gel lanes were excised (see also Figs EV1 and EV2). The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1.0, versus the molecular mass calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines were determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and Δ4‐CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments.

Second‐dimension BNGE of digitonin‐solubilized samples from WT and Δ4‐CYB cells, Western blot and immunodetection of the indicated cIII2 structural subunits with specific antibodies. The immunodetection patterns were equivalent to the complexome profiles.

Quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated cIII2 subunits. The x‐axis values were the slice number (1‐64), and the y‐axis values were the relative peptide intensity. The graph shows the mean ± SD (n = 2). Two‐way ANOVA with Sidak's multiple comparisons test **P = 0.0083 (UQCRC2); **P = 0.0033 (UQCRFS1); *P = 0.0224; n.s. = non‐significant.

Complexome profiles of two cIII2 assembly factors (BCS1L and LYRM7 or MZM1L) generated in the same way as in (A). The represented values are the mean ± SEM of the two reciprocal labeling experiments.

Source data are available online for this figure.
Figure EV2
Figure EV2. Complexome profiling and area quantification of internal control proteins (related to Fig 3)
The complexome profiles of the three chosen proteins, citrate synthase (CS), and two members of the translocase of the outer membrane family (TOM20 and TOM22) were generated as in the main Fig 3. The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1 versus the molecular mass, calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines were determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and Δ4‐CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments. The bar graph represents the quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated proteins. The x‐axis values were the slice number (1–64), and the y‐axis values were the relative peptide intensity. The plotted values are mean ± SD (n = 2). The differences were not significant according to 2‐way ANOVA with Sidak's multiple comparisons test.
Figure 4
Figure 4. Proteomic analyses of UQCR10 and CYC1‐containing protein associations in Δ4‐CYB cells. See also Fig EV3

SDS–PAGE, Western blot, and immunodetection, with the indicated specific antibodies, of Δ4‐CYB and WT cells expressing HA‐tagged versions of UQCRQ and UQCR10 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty).

BNGE, Western blot, and immunodetection, with an anti‐HA tag antibody, of samples from the same cell lines as in (A) solubilized either with digitonin or DDM.

BNGE, Western blot, and immunodetection, with the monoclonal (M) anti‐UQCRQ antibody (Abcam ab110255), of non‐transduced Δ4‐CYB and WT cells. The mitoplast samples were solubilized with DDM (See also Fig EV1).

Scatter plot generated from the analysis of the logarithmic heavy (H)‐to‐light (L) ratios in the x‐axis and the reverse in the y‐axis, in the two reciprocal labeling SILAC experiments (1 and 2) and anti‐HA immunopurification of Δ4‐CYB and WT cells expressing UQCR10HA.

Complexome profiles, generated as in Fig 3, for the proteins found specifically enriched in Δ4‐CYB UQCR10HA, according to the SILAC immunopurification experiments shown in (D). The represented values are the mean ± SEM of the two reciprocal labeling experiments.

SDS–PAGE, Western blot, and immunodetection, with the indicated specific antibodies, of Δ4‐CYB and WT cells expressing an HA‐tagged version of CYC1 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty).

BNGE, Western blot, and immunodetection, with an anti‐HA tag antibody, of samples from the same cell lines as in (F) solubilized either with digitonin.

Scatter plot generated from the analysis of the logarithmic heavy (H)‐to‐light (L) ratios in the x‐axis and the reverse in the y‐axis, in the two reciprocal labeling SILAC experiments (A and B) of anti‐HA immunopurification of Δ4‐CYB and WT cells expressing CYC1HA.

Source data are available online for this figure.
Figure EV3
Figure EV3. Knock‐down of either GHITM or CHCHD3 does not produce cIII2 functional nor assembly defects (related to Fig 4)

Oxygen consumption rates measured in WT cells transduced with lentiviral vectors encoding two different shRNAs specific for GHITM mRNA (shRNA GHITM 1 and shRNA GHITM 2) and with pLKO.1 without any shRNA insert (empty vector, EV). Respiration was measured in whole cells in the basal state (Routine), in the presence of oligomycin (Leak) and uncoupled with CCCP (ETS capacity), using a O2K high‐resolution respirometer (Oroboros instruments). The plotted values are the mean ± SD (n = 4 for WT EV and WT shRNA GHITM 1 and n = 3 for WT shRNA GHITM 2).

MRC enzyme activities normalized to the activity of citrate synthase (CS) measured in the same cell lines shown in (A). The plotted values are the mean ± SD (n = 2 biological replicates). Two‐way ANOVA Tukey's multiple comparisons test *P = 0.0462 (CIV).

SDS–PAGE, Western blot, and immunodetection analysis with the indicated specific antibodies.

1D BNGE, Western blot, and immunodetection analysis of digitonin‐solubilized samples from the same three cell lines shown in (A).

Oxygen consumption rates measured in 143B WT cells transduced with lentiviral vectors encoding two different shRNAs specific for CHCHD3 mRNA (shRNA CHCHD3 1 and shRNA CHCHD3 2) and with pLKO.1 without any shRNA insert (empty vector, EV). Respiration was measured in whole cells in the basal state (Routine), in the presence of oligomycin (Leak) and uncoupled with CCCP (ETS capacity), using a O2K high‐resolution respirometer (Oroboros instruments). The plotted values are the mean ± SD (n = 4). Two‐way ANOVA Tukey's multiple comparisons test *P = 0.0126 (Routine shRNA 2); *P = 0.0386 (ETS capacity shRNA 1); ***P = 0.0002 (ETS capacity shRNA 2).

MRC enzyme activities normalized to the activity of citrate synthase (CS) measured in the same cell lines shown in (E). The plotted values are the mean ± SD (n = 4 biological replicates).

SDS–PAGE, Western blot, and immunodetection analysis with the indicated specific antibodies.

1D BNGE, Western blot, and immunodetection analysis of digitonin‐solubilized samples from the same three cell lines shown in (E).

Source data are available online for this figure.
Figure 5
Figure 5. Complex I alterations in Δ4‐CYB cells

Complexome profiles of the different cI structural modules. The graphs were generated as in Fig 3, but in this case, the peptide intensity values for the individual subunits belonging to each module (Stroud et al, 2016) were averaged to simplify the analysis (see also Figs EV4 and EV5). The represented values are the mean ± SEM of the two reciprocal labeling experiments.

Complexome profiles of the cI assembly factor NDUFAF2 (see main text for details) in WT (blue) and Δ4‐CYB (red) mitochondria (see also Figs EV4 and EV5). The represented values are the mean ± SEM of the two reciprocal labeling experiments.

Quantification of the total area under the curve (calculated as in Fig 3C) in the profiles corresponding to each cI module (top graph). The area of the NDUFA9 subunit was calculated separately as its profile did not correspond to any of the other structural subunit modules. The plotted values are the mean ± SD (n = 2). Two‐way ANOVA with Sidak's multiple comparisons test ***P = 0.0006; **P = 0.0012 (N‐module); **P = 0.0016 (ND1‐module); **P = 0.0032 (ND2‐module); **P = 0.0057 (ND4‐module); **P = 0.0020 (ND5‐module); *P = 0.0140.

Quantification of the area of the N‐module peak corresponding to the molecular mass of the fully assembled cI: 1,172 kDa in Δ4‐CYB samples and the respirasome (cI+cIII2+cIV) SCs in #4.1 WT samples (2,110 kDa). The plotted values are the mean ± SD (n = 2). Unpaired Student's t‐test ****P < 0.0001.

Figure EV4
Figure EV4. Complexome profiling of cI‐containing structures in WT and Δ4‐CYB cells (related to Fig 5)
Heatmaps of the individual cI structural subunits and the NDUFAF2 assembly factor derived from the digitonin‐solubilized samples in the experiment where WT cells were labeled with the heavy (H) amino acids. Black = 0; yellow = 0.5; red = 1 relative peptide intensities of the most frequent peptide found in each of the samples individually.
Figure EV5
Figure EV5. CI assembly in WT and Δ4‐CYB cells solubilized with DDM and in modulated NDUFAF2 levels (related to Fig 5)

Complexome profiles of the cI structural modules found in both cell lines in the two reciprocal labeling experiments. The graphs plot the average of relative peptide peak intensities along the lane corresponding to the subunits of each module, setting the maximum to 1 versus the molecular mass, calculated using the individual complexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines were determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and Δ4‐CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments. The bar graph represents the quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated cI modules. The x‐axis values were the slice number (1‐64), and the y‐axis values were the relative peptide intensity. The plotted values are mean ± SD (n = 2). Two‐way ANOVA with Sidak's multiple comparisons test **P = 0.0072 (Q‐module); **P = 0.0029 (N‐module); *P = 0.0102.

A Myc‐DDK (FLAG) tagged version of NDUFAF2 (AF2Myc‐DDK) was stably expressed in both WT and Δ4‐CYB cybrids by transfection of the cloned cDNA in the pCMV6‐Entry mammalian expression vector (Origene Cat#: RC207387). As negative controls, the two cell lines were also transfected with the empty pCMV6‐Entry vector (EV), providing the resistance to neomycin but no expression of other relevant proteins.

Complex I‐IGA assay performed in the NDUAF2‐overexpressing (AF2Myc‐DDK) and negative control (EV) cell lines after solubilizing the samples with digitonin and separating them on BNGE.

Complex I enzymatic activity assay performed in the NDUAF2‐overexpressing (AF2Myc‐DDK) and negative control (EV) cell lines. Results are expressed as mean ± SD (n = 3 biological replicates). No significant differences (ns) in cI activity were found between the NDUFAF2‐overexpressing WT or Δ4‐CYB cells and their corresponding negative control (one‐way ANOVA with Tukey's multiple comparisons test).

NDUFAF2 expression was knocked down both WT and Δ4‐CYB cybrids by transfection of two different siRNAs targeting the NDUFAF2 transcript (AF2 siRNA1 and AF2 siRNA2, Sigma‐Aldrich). Sigma's siRNA Universal Negative Control #1 was used in the experiments as well.

Complex I‐IGA assay performed in the cells transfected with both siRNAs and the negative control (CT(‐) siRNA), after solubilizing the samples with digitonin and separating them on BNGE.

Complex I enzymatic activity assay performed in the cells transfected with both siRNAs and the negative control cell lines. Results are expressed as mean ± SD (n = 3 biological replicates). No significant differences (ns) in cI activity were found between the silenced WT or Δ4‐CYB cells and their corresponding negative control (one‐way ANOVA with Tukey's multiple comparisons test).

Source data are available online for this figure.
Figure 6
Figure 6. Complex I assembly kinetics in Δ4‐CYB cells

SDS–PAGE resolving the radioactively labeled mitochondrial translation products after a 2‐h pulse (P). The 35S‐Met and the cycloheximide were removed from the medium, and cells were collected at the indicated chase times (2, 5, and 24 h).

First‐dimension (1D) BNGE analysis of the same cells as in (A), prepared with digitonin.

Denaturing second‐dimension (2D) BNGE analysis of the same samples allowed following the incorporation of the individual labeled subunits inside their corresponding complex and supercomplex species.

Complex I‐IGA (cI‐IGA) analysis of digitonin‐solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days (0 h). After removing the drug and restoring synthesis of the mtDNA‐encoded subunits, the cells were collected at the indicated times to follow the appearance of cI reactivity with time. The gels were incubated in the reaction mixture for 24 h. SS = steady state. The asterisk indicates the presence of a high‐molecular‐weight cI‐containing band of unknown nature (see main text).

2D BNGE, Western blot, and immunodetection analysis of WT and Δ4‐CYB mitochondria from cells collected at the same times after doxycycline treatment to follow the incorporation kinetics of the indicated cI nuclear‐encoded subunits, belonging to different structural modules. The blots shown were either exposed for 16 s. (low exposures) or 160 s. (high exposures) in order to visualize the qualitative signals in the Δ4‐CYB samples.

Source data are available online for this figure.
Figure 7
Figure 7. Alternative oxidase (AOX) expression and function in WT and Δ4‐CYB cells

SDS–PAGE, Western blot, and immunodetection of AOXHA expression in whole‐cell lysates from WT and Δ4‐CYB cells transduced with the AOXHA/pWPXLd‐ires‐HygroR lentiviral vector. The transduction and selection controls were the same cell lines transfected with an empty pWPXLd‐ires‐HygroR vector (EV).

Growth curves of the AOXHA expressing cell lines and their corresponding EV controls. Cell growth was monitored every 6 h after substituting the medium in two replicate 24‐well plates, one plate with medium without uridine (Uridine), and the second plate with medium supplemented with 50 μg/ml uridine (Uridine+). The graphs show the average confluence ± SD at each time point (n = 6 wells per cell line).

Immunodetection of cI structural subunits and NDUFAF2 in the same samples as in panel (A).

1D BNGE, Western blot, and immunodetection analyses of digitonin‐solubilized mitochondria from WT and Δ4‐CYB cybrids expressing AOXHA and their corresponding EV controls.

Complex I in‐gel activity assays (IGA) after BNGE as in panel (D). The gels were incubated in the IGA reaction mixture for 5 h. The asterisk indicates the presence of a high‐molecular‐weight cI‐containing band of unknown nature (see main text).

Spectrophotometric kinetic measurements of cI activity in WT and Δ4‐CYB AOXHA and EV samples normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n = 5 biological replicates). Two‐way ANOVA with Tukey's multiple comparisons test **P = 0.0077 (WT AOXHA versus Δ4‐CYB AOXHA); **P = 0.0044 (Δ4‐CYB EV versus Δ4‐CYB AOXHA); ***P = 0.0003; ****P < 0.0001.

High‐resolution respirometry analyses performed in intact cells in an Oroboros instrument. ROUTINE: cellular basal oxygen consumption rate (in pmol O2/sec) per million cells in DMEM medium. LEAK is the non‐phosphorylating respiration in the presence of the ATP synthase inhibitor oligomycin. ETS: maximal respiration rate in the presence of the uncoupler CCCP. AOX: oxygen consumption in the presence of antimycin A, inhibiting cIII2 activity but not that of AOX. ROTENONE: oxygen consumption in the presence of the cI inhibitor rotenone. In all cases, this was equal to the background. Results are expressed as mean ± SD (n = 2 biological replicates). Two‐way ANOVA with Tukey's multiple comparisons test ****P < 0.0001.

Respirometry analyses, performed as in panel (G), in WT EV controls and AOXHA‐expressing cells untreated or treated with 2.5 μM antimycin A for 7 days (+AA). Results are expressed as mean ± SD (n = 4 biological replicates). Two‐way ANOVA with Tukey's multiple comparisons test ****P < 0.0001.

1D BNGE followed by cI‐IGA (right) or Western blot and immunodetection of cIII2 subunit CYC1 (left) in digitonin‐solubilized samples from WT EV controls and AOXHA‐expressing cells untreated (−) or treated (+) with 2.5 μM antimycin A for 7 days.

Source data are available online for this figure.
Figure 8
Figure 8. Assembly state of cV, cII, and cIV in Δ4‐CYB cells

Complexome profiles of the two cV structural and assembly modules. The graphs were generated as in Fig 3, but in this case, the peptide intensity values for the individual subunits belonging to each module (He et al, 2018) were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments.

Complexome profiles of the three detected cII subunits. The graphs were generated as in Fig 3. The represented values are the mean ± SEM of the two reciprocal labeling experiments.

Complexome profiles of the different cIV assembly modules and of the last subunit to be incorporated (NDUFA4). The graphs were generated as in Fig 3, but in this case, the peptide intensity values for the individual subunits belonging to each assembly module (Vidoni et al, 2017) were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments. The bar graph represents the quantification of the total area under the curve (calculated as in Fig 3C) in the profiles corresponding to each cIV module. The plotted values are mean ± SD (n = 2). Two‐way ANOVA with Sidak's multiple comparisons test **P = 0.0088 (Early); **P = 0.0063 (MT‐CO2); **P = 0.0040 (NDUFA4); *P = 0.0264.

1D BNGE, Western blot, and immunodetection of two cIV subunits (MT‐CO1 and COX7B) in samples from WT and Δ4‐CYB cells solubilized with DDM and digitonin (Dig).

Source data are available online for this figure.

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