Biochemistry and Chemical Biology

Structural rather than catalytic role for mitochondrial respiratory chain supercomplexes

Michele Brischigliaro author has email address
Alfredo Cabrera-Orefice
Susanne Arnold
Carlo Viscomi
Massimo Zeviani
Erika Fernández-Vizarra author has email address

Department of Biomedical Sciences, University of Padova, Padova, Italy
Veneto Institute of Molecular Medicine, Padova, Italy
Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands
Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
Department of Neurosciences, University of Padova, Padova, Italy

https://doi.org/10.7554/eLife.88084.2

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Figures and data

D. melanogaster mitochondrial respiratory chain does not rely on SC formation under physiological conditions.
(A) Complexome profiling of wild-type D. melanogaster mitochondria. Heatmaps show relative abundance of MRC subunits belonging to complex I (CI), complex II (CII), complex III₂ (CIII) and complex IV (CIV). Color scale of normalized peptide intensities are 0 (black), 96^th percentile (yellow) and 1 (red). (B) BN-PAGE separation of mitochondria from wild-type D. melanogaster solubilized with digitonin. Native gels were either stained with Coomassie R250 or analyzed by in-gel activity (IGA) for complex I (CI) and complex IV (CIV). (C) BN-PAGE separation of mitochondria from wild-type D. melanogaster solubilized with dodecylmaltoside (DDM). Native gels were either stained with Coomassie R250 or analyzed by in-gel activity assay (IGA) for complex I (CI) and complex IV (CIV).

Severely perturbed CIV assembly results in increased formation of SC I₁III₂.
(A) Complexome profiling of Coa8 KO D. melanogaster mitochondria. Heatmaps show relative abundance of MRC subunits belonging to complex I (CI), complex II (CII), complex III₂ (CIII) and complex IV (CIV). Color scale of normalized peptide intensities are 0 (black), 96° percentile (yellow) and 1 (red). (B) Average MS profiles depicted as relative abundance of MRC enzymes in natively separated complexes from wild-type (top) and Coa8 KO (bottom) fly mitochondria. Profiles of complexes I, III₂ and V (CI, CIII and CV) are plotted as average peptide intensity of the specific subunits identified by MS for each complex vs. apparent molecular weight. The increase in the relative abundance of SC I₁III₂ in Coa8 KO mitochondria is indicated by a black arrow. (C) In gel-activity assays for MRC complex I (CI), complex II (CII) and complex IV (CIV) in DDM-solubilized mitochondria from wild-type (w¹¹¹⁸) and Coa8 KO (Coa8^KO) flies. (D) BN-PAGE, western blot immunodetection of MRC complexes from a pool of three control wild-type (w¹¹¹⁸) and and three Coa8 KO (Coa8^KO) fly mitochondria preparations, using antibodies against specific subunits: anti-UQCRC2 (complex III), anti-NDUFS3 (complex I), anti-COX4 (complex IV) and anti-SDHA (complex II).

Mildly perturbed CIV assembly results in increased formation of SC I₁III₂.
(A) Relative quantification (RQ) of Coa8 mRNA expression in control (da-gal4>+) and Coa8 KD (da-gal4>Coa8^RNAi) flies measured by qPCR. Data are plotted as mean[±[SD (n[=[3 biological replicates, Student’s t test *p[≤[0.05). (B) In gel-activity assays for MRC complex I (CI), complex II (CII) and complex IV (CIV) in DDM-solubilized mitochondria from control (da-gal4>+) and Coa8 KD (da-gal4>Coa8^RNAi) flies. (C) BN-PAGE, western blot immunodetection of MRC complexes from a pool of three control (da-gal4>+) and three Coa8 KD (da-gal4>Coa8^RNAi) fly mitochondria samples, using antibodies against specific subunits: anti-UQCRC2 (complex III), anti-NDUFS3 (complex I), anti COX4 (complex IV) and anti-SDHA (complex II).

Enhanced formation of SC I₁III₂ does not result in increased respiration.
(A-B) High-resolution respirometry (HRR) analyses of whole-fly homogenates. Respiration is represented by oxygen flux (JO₂) measured by oxygen consumption rates (OCR – pmol/s*fly). OCR have been measured via substrate-driven respiration under saturating concentrations of substrates inducing either complex I (CI) or complex II (CII) -linked respiration. Rotenone was used to block CI-linked respiration before measuring CII-linked respiration. HRR was performed on (A) Coa8 KO and (B) Coa8 KD fly homogenates compared to relative controls. Data are plotted as mean[±[SD (n[=[4 biological replicates). (C-D) Kinetic enzyme activity of individual MRC complexes in (C) Coa8 KO and (D) Coa8 KD compared with the relative control individuals, normalized by citrate synthase (CS) activity. Data are plotted as mean[±[SD (n[=[3 biological replicates, pairwise comparisons by unpaired Student’s t test *p[≤[0.05, **p[≤[0.01, ****p[≤[0.0001).

Mild perturbation of CIII₂ biogenesis enhances SC formation in D. melanogaster.
(A) Relative quantification (RQ) of Bcs1 mRNA expression in control (act5c-gal4>+) and Bcs1 KD (act5c-gal4>Bcs1^RNAi) larvae measured by qPCR. Data are plotted as mean[±[SD (n[=[3 biological replicates, Student’s t test ***p[≤[0.001). (B) In gel-activity assays for MRC complex I (CI), complex II (CII) and complex IV (CIV) in DDM-solubilized mitochondria from control (act5c-gal4>+) and Bcs1 KD (act5c-gal4>Bcs1^RNAi) larvae. (C) BN-PAGE, western blot immunodetection of MRC complexes from a pool of three control (act5c-gal4>+) and three Bcs1 KD (act5c-gal4>Bcs1^RNAi) larvae mitochondria samples, using antibodies against specific subunits: anti-UQCRC2 (complex III), anti-NDUFS3 (complex I), anti-COX4 (complex IV) and anti-SDHA (complex II). (D) Kinetic enzyme activity of individual MRC complexes in control (act5c-gal4>+) and Bcs1 KD (act5c-gal4>Bcs1^RNAi) larvae mitochondria normalized by citrate synthase (CS) activity. Data are plotted as mean[±[SD (n[=[3 biological replicates, pairwise comparisons by unpaired Student’s t test, *p[≤[0.05, ****p[≤[0.0001). (E) High resolution respirometry (HRR) analyses of whole-fly homogenates. Respiration is represented by oxygen flux (JO₂) measured by oxygen consumption rates (OCR – pmol/s*fly). OCR have been measured via substrate-driven respiration under saturating concentrations of substrates inducing either complex I (CI) or complex II (CII) -linked respiration. Rotenone was used to block CI-linked respiration before measuring CII-linked respiration. HRR was performed on control (act5c-gal4>+) and Bcs1 KD (act5c-gal4>Bcs1^RNAi) homogenates compared to relative controls. Data are plotted as mean[±[SD (n[=[3 biological replicates, two-way ANOVA with Sidak’s multiple comparisons, ****p[≤[0.0001). (F) Relative quantification (RQ) of Bcs1 mRNA expression in control (da-gal4>+) and Bcs1 KD (da-gal4>Bcs1^RNAi) larvae measured by qPCR. Data are plotted as mean[±[SD (n[=[3 biological replicates, Student’s t test ***p[≤[0.001). (G) In gel-activity assays for MRC complex I (CI), complex II (CII) and complex IV (CIV) in DDM-solubilized mitochondria from control (da-gal4>+) and Bcs1 KD (da-gal4>Bcs1^RNAi) larvae. (H) BN-PAGE, western blot immunodetection of MRC complexes from a pool of three control (da-gal4>+) and three Bcs1 KD (da-gal4>Bcs1^RNAi) larvae mitochondria samples using antibodies against specific subunits: anti-UQCRC2 (complex III), anti NDUFS3 (complex I), anti-COX4 (complex IV) and anti-SDHA (complex II). HWM-SC: high molecular weight supercomplex. (I) Kinetic enzyme activity of individual MRC complexes in control (da-gal4>+) and Bcs1 KD (da-gal4>Bcs1^RNAi) larvae mitochondria normalized by citrate synthase (CS) activity. Data are plotted as mean[±[SD (n[=[3 biological replicates, pairwise comparisons by unpaired Student’s t test, *p[≤[0.05). (J) High resolution respirometric (HRR) analyses of whole-fly homogenates. Respiration is represented by oxygen flux (JO₂) measured by oxygen consumption rates (OCR - pmol/s*fly). OCR have been measured via substrate-driven respiration under saturating concentrations of substrates inducing either complex I (CI) or complex II (CII) -linked respiration. Rotenone was used to block CI-linked respiration before measuring CII-linked respiration. HRR was performed on control (act5c-gal4>+) and Bcs1 KD (act5c-gal4>Bcs1^RNAi) homogenates compared to relative controls. Data are plotted as mean[±[SD (n[=[3 biological replicates, two-way ANOVA with Sidak’s multiple comparisons, ****p[≤[0.0001).

Mild perturbation of CI biogenesis enhances SC formation in D. melanogaster.
(A) Relative quantification (RQ) of Ndufs4 mRNA expression in control (act5c-gal4>+) and Ndufs4 KD (act5c-gal4>Ndufs4^RNAi) larvae measured by qPCR. Data are plotted as mean[±[SD (n[=[3 biological replicates, Student’s t test **p[≤[0.01). (B) In gel-activity assays for MRC complex I (CI), complex II (CII) and complex IV (CIV) in DDM-solubilized mitochondria from control (act5c-gal4>+) and Ndufs4 KD (act5c-gal4>Ndufs4^RNAi) larvae. (C) BN-PAGE, western blot immunodetection of MRC complexes from a pool of three control (act5c-gal4>+) and three Ndufs4 KD (act5c-gal4>Ndufs4^RNAi) larvae mitochondria samples, using antibodies against specific subunits: anti-UQCRC2 (complex III), anti-NDUFS3 (complex I), anti-COX4 (complex IV) and anti-SDHA (complex II). (D) Kinetic enzyme activity of individual MRC complexes in control (act5c-gal4>+) and Ndufs4 KD (act5c-gal4>^RNAi) larvae mitochondria normalized by citrate synthase (CS) activity. Data are plotted as mean[±[SD (n[=[3 biological replicates, pairwise comparisons by unpaired Student’s t test, **p[≤[0.01). (E) High-resolution respirometry (HRR) analyses of whole-fly homogenates. Respiration is represented by oxygen flux (JO₂) measured by oxygen consumption rates (OCR – pmol/s*fly). OCR have been measured via substrate-driven respiration under saturating concentrations of substrates inducing either complex I (CI) or complex II (CII) -linked respiration. Rotenone was used to block CI-linked respiration before measuring CII-linked respiration. HRR was performed on control (act5c-gal4>+) and Ndufs4 KD (act5c-gal4>Ndufs4^RNAi) homogenates compared to relative controls. Data are plotted as mean[±[SD (n[=[3 biological replicates, two-way ANOVA with Sidak’s multiple comparisons, ****p[≤[0.0001). (F) Relative quantification (RQ) of Ndufs4 mRNA expression in control (da-gal4>+) and Ndufs4 KD (da-gal4>Ndufs4^RNAi) larvae measured by qPCR. Data are plotted as mean[±[SD (n[=[3 biological replicates, Student’s t test ***p[≤[0.001). (G) In gel-activity assays for MRC complex I (CI), complex II (CII) and complex IV (CIV) in DDM-solubilized mitochondria from control (da-gal4>+) and Ndufs4 KD (da-gal4>Ndufs4^RNAi) larvae. (H) BN-PAGE, western blot immunodetection of MRC complexes from a pool of three control (da-gal4>+) and three Ndufs4 KD (da-gal4>Ndufs4^RNAi) larvae mitochondria samples, using antibodies against specific subunits: anti-UQCRC2 (complex III), anti-NDUFS3 (complex I), anti COX4 (complex IV) and anti-SDHA (complex II). (I) Kinetic enzyme activity of individual MRC complexes in control (da-gal4>+) and Ndufs4 KD (da-gal4>Ndufs4^RNAi) larvae mitochondria normalized by citrate synthase (CS) activity. Data are plotted as mean[±[SD (n[=[3 biological replicates, pairwise comparisons by unpaired Student’s t test, *p[≤[0.05). (J) High-resolution respirometry (HRR) analyses of whole-fly homogenates. Respiration is represented by oxygen flux (JO₂) measured by oxygen consumption rates (OCR - pmol/s*fly). OCR have been measured via substrate-driven respiration under saturating concentrations of substrates inducing either complex I (CI) or complex II (CII) -linked respiration. Rotenone was used to block CI-linked respiration before measuring CII-linked respiration. HRR was performed on control (act5c-gal4>+) and Ndufs4 KD (act5c-gal4>^RNAi) homogenates compared to relative controls. Data are plotted as mean[±[SD (n[=[4 biological replicates, two-way ANOVA with Sidak’s multiple comparisons).

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