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• W4387570511 abstract "Full text Figures and data Side by side Abstract eLife assessment Introduction Results Discussion Materials and methods Appendix 1 Data availability References Peer review Author response Article and author information Abstract Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages compared with the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from Drosophila melanogaster tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. Here, we show that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, we conclude that SC formation is necessary to stabilize the complexes in suboptimal biogenesis conditions, but not for the enhancement of respiratory chain catalysis. eLife assessment This study presents valuable findings on the organization of respiratory chain complexes in mitochondria. It provides solid evidence that respiratory supercomplex formation in the fruit fly does not impact respiratory function, suggesting the role of these complexes is structural, rather than catalytic. However, whether the conclusions extend to other species requires further evidence. This manuscript will be of broad interest to the field of mitochondrial bioenergetics. https://doi.org/10.7554/eLife.88084.3.sa0 About eLife assessments Introduction Mitochondria are the organelles providing most of the cellular energy in form of adenosine triphosphate (ATP) in aerobic eukaryotes. The molecular machinery responsible for energy transformation is the oxidative phosphorylation (OXPHOS) system, which is canonically composed of five multiprotein complexes embedded in the inner mitochondrial membrane. OXPHOS consists of two tightly regulated processes: electron transport and ATP synthesis. Electron transport takes place between complexes I-IV and two mobile electron carriers (coenzyme Q and cytochrome c). During electron transport, complexes I, III, and IV pump protons from the mitochondrial matrix to the intermembrane space, generating a proton gradient that provides the protonmotive force exploited by complex V to synthesize ATP. In mammalian mitochondria, mitochondrial respiratory chain (MRC) complexes I, III, and IV can interact with each other forming supramolecular structures described generally by the term ‘supercomplexes’ (Schägger and Pfeiffer, 2000; Schägger and Pfeiffer, 2001). MRC supercomplexes (SCs) can have different stoichiometries and compositions, ranging from the binding of only two complexes, such as the I1III2 and III2IV1 SCs (Letts et al., 2019; Vercellino and Sazanov, 2021), to higher order associations between complexes I, III and IV, with the SC of I1III2IV1 stoichiometry known as the ‘respirasome’ (Schägger and Pfeiffer, 2000; Letts et al., 2016; Gu et al., 2016; Sousa et al., 2016; Wu et al., 2016; Guo et al., 2017). Now that the association of individual MRC complexes into supramolecular structures is well-established, with structures of several SC species being resolved, the debate is centered on what the functional significance of these structures might be. Several possible roles have been proposed for SCs. First, it was suggested that the association between complex I (CI) and the obligate dimer of complex III (CIII2) would allow substrate channeling, sequestering a dedicated coenzyme Q (CoQ) pool and allowing a more efficient electron transfer between the two complexes, while separating this electronic route from those of FADH2-linked dehydrogenases (e.g. complex II) to the CIII2 not bound to CI (Schägger and Pfeiffer, 2000; Bianchi et al., 2004; Lenaz and Genova, 2007; Lenaz and Genova, 2009; Lapuente-Brun et al., 2013; Calvo et al., 2020; García-Poyatos et al., 2020). This increased efficiency would in turn decrease electron leak and, as a consequence, produce less reactive oxygen species (ROS) than the individual free complexes (Maranzana et al., 2013; Lopez-Fabuel et al., 2016). However, the available high-resolution respirasome structures show that the distance between the CoQ binding sites in CI and CIII2 are far apart and exposed to the membrane, thus not supporting the notion of substrate channeling within the SC structure (Vercellino and Sazanov, 2021; Hirst, 2018). In addition, exogenously added CoQ was necessary to sustain CI activity in the purified I1III2 SC (Letts et al., 2019), arguing against a tightly bound and segregated CoQ pool as a functional component of the SC. A large body of work from the late 1960s to the 1980s, resulted in the ‘random collision model’ to explain electron transfer between the respiratory chain complexes, and in the evidence that CoQ is present as an undifferentiated pool (Green and Tzagoloff, 1966; Kröger and Klingenberg, 1973a; Kröger and Klingenberg, 1973b; Hackenbrock et al., 1986; Chazotte and Hackenbrock, 1988). More recently, additional proof of the non-compartmentalized electronic routes from CI to CIII2 and from complex II (CII) to CIII2, came from kinetic measurements in sub-mitochondrial particles. In these systems, MRC organization in SCs was conserved but b- and c-type cytochromes in CIII2 were equally accessible to CI-linked and CII-linked substrates (Blaza et al., 2014), and CoQ reduced by CI in the respirasomes was able to reach and readily reduce external enzymes to the SCs (Fedor and Hirst, 2018). In addition, growing evidence supports the notion that different MRC organizations exist in vivo, where varying proportions of SC vs. free complexes do not result in separate and distinct CI-linked and CII-linked respiratory activities (Mourier et al., 2014; Lobo-Jarne et al., 2018; Bundgaard et al., 2020; Fernández-Vizarra et al., 2022). This is in contrast with the idea that segregation into different types of SCs and in individual complexes is necessary for the functional interplay of the MRC, leading to the adaptation of the respiratory activity to different metabolic settings (Lapuente-Brun et al., 2013). The physical proximity of CIII2 and CIV has also been suggested to promote faster electron transfer kinetics via cytochrome c (Vercellino and Sazanov, 2021; Berndtsson et al., 2020; Stuchebrukhov et al., 2020), although this is a matter of debate as well (Trouillard et al., 2011; Nesci and Lenaz, 2021). The second main explanation to justify the existence of SCs is that they play a structural function, stabilizing the individual complexes (Acín-Pérez et al., 2004; Diaz et al., 2006) and/or serving as a platform for the efficient assembly of the complexes, with a special relevance for the biogenesis of mammalian CI (Moreno-Lastres et al., 2012; Protasoni et al., 2020; Fernández-Vizarra and Ugalde, 2022). Notably, the MRC structural organization, especially the stoichiometry, arrangements and stability of the SCs, may not be conserved in all eukaryotic species (Maldonado et al., 2021; Zhou et al., 2022; Maldonado et al., 2023; Klusch et al., 2023). This is the case even within mammals, as human cells and tissues barely contain free CI, which is rather contained in SC I1+III2 and the respirasome (Fernández-Vizarra et al., 2022; Protasoni et al., 2020). In contrast, other mammalian mitochondria (bovine, ovine, rat or mouse) contain larger amounts of CI in its free form, even if the majority is still in the form of SCs (Schägger and Pfeiffer, 2001; Lopez-Fabuel et al., 2016; Bundgaard et al., 2020; Acín-Pérez et al., 2008; Letts and Sazanov, 2017; Davies et al., 2018). The distribution of the MRC complexes between free complexes and SCs seems to differ even more in non-mammalian animal species. Several reptile species contain a very stable SC I+III2 that lacks CIV (Bundgaard et al., 2020), and in Drosophila melanogaster practically all of CI is free, with SCs being almost completely absent (Garcia et al., 2017; Shimada et al., 2018). However, comparative studies of MRC function in diverse animal species suggest that higher amounts and stability of the SCs do not correlate with increased respiratory activity/efficiency and/or reduced ROS production (Bundgaard et al., 2020; Shimada et al., 2018). Here we show that SCs can be stably formed in D. melanogaster mitochondria upon mild perturbations of individual CIV, CIII2 and CI biogenesis. This finding enabled us to test whether increased SC formation translated into enhanced respiration proficiency. However, MRC performance of fruit fly mitochondria did not change regardless of the presence or absence of SCs. These observations have led us to conclude that: (1) the efficiency in the assembly of the individual complexes is likely to be the main determinant of SC formation and (2) these supramolecular complexes play a more relevant role in maintaining the stability and/or supporting the biogenesis of the MRC than in promoting catalysis. Results D. melanogaster MRC organization does rely on SC formation under physiological conditions To obtain a detailed characterization of MRC organization in D. melanogaster, we isolated mitochondria from wild-type adults and, after solubilization with digitonin, we performed Blue-Native Gel Electrophoresis (BNGE) followed by mass spectrometry analysis of the gel lanes, using ‘Complexome Profiling (CP)’ (Cabrera-Orefice et al., 2021) and thus obtaining a profile of peptide intensities from most OXPHOS subunits along the electrophoresis lane (Figure 1A). This allowed us to unequivocally determine the identity of the main bands that can be visualized by Coomassie staining of BNGE gels (Figure 1B). The identity of the bands corresponding to complex I (CI) and complex IV (CIV) was also confirmed using specific in-gel activity (IGA) assays (Figure 1B). These analyses verified that D. melanogaster mitochondria contain extremely low amounts of high-molecular weight CI-containing SCs (Garcia et al., 2017; Shimada et al., 2018), using the same solubilization and electrophoresis conditions in which the SCs are readily detectable in mammalian cells and tissues (Fernández-Vizarra et al., 2022; Acín-Pérez et al., 2008; Wittig et al., 2006). As previously described (Shimada et al., 2018), dimeric complex V (CV2) is easily visualized by BNGE and present in similar amounts as monomeric CV in D. melanogaster mitochondrial membranes solubilized with digitonin (Figure 1A and B). This CV2 species is not a strongly bound dimer, as it disappears when the samples are solubilized using a harsher detergent such as dodecylmaltoside (DDM) (Shimada et al., 2018; Figure 1C). Conversely, CI is mainly found as a free complex in the native gels irrespective of whether the mitochondria had been solubilized with digitonin or DDM (Figure 1A, B and C). The other minor CI-containing band corresponding to the fraction associated with CIII2, accounts for about 3% of the total amounts of CI and CIII2, according to label-free mass spectrometry quantifications in the CP analysis (Figure 1A). CIV activity is absent in this band both in digitonin- and DDM- solubilized samples (Figure 1B and C), whereas it is present in the bands that correspond to individual CIV and dimeric CIV (CIV2) detected both in digitonin- and DDM-treated samples, as well as in SC III2IV1, which is present only in the digitonin-solubilized samples. This is different from mammalian mitochondria in which SC III2IV1 is present also in DDM-solubilized mitochondria, probably due to the tight binding of CIII2 to CIV through COX7A2L/SCAF1 (Vercellino and Sazanov, 2021; Fernández-Vizarra et al., 2022; Perales-Clemente et al., 2010). The latter does not have a homolog in D. melanogaster even though this species has three different COX7A isoforms (named COX7A, COX7AL, and COX7AL2) that exhibit a tissue-specific expression pattern, according to FlyBase (https://www.flybase.org/). CP analysis of Drosophila mitochondria only detected COX7A (mammalian COX7A1 homolog) and COX7AL2 (mammalian COX7A2 homolog), whereas COX7AL, that is solely expressed in testis, was not found. Therefore, SC I1III2 can be considered the only stable SC species in physiological conditions in D. melanogaster, yet containing a minute fraction of the total CI and CIII2. Figure 1 Download asset Open asset 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), 96th 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). Perturbations of CIV assembly result in increased formation of SC I1III2 COA8 is a CIV assembly factor the defects of which cause isolated mitochondrial CIV deficiency in human and mouse (Melchionda et al., 2014; Signes et al., 2019), as well as in Drosophila melanogaster (Brischigliaro et al., 2019; Brischigliaro et al., 2022a). Consistent with the role of Coa8 in CIV biogenesis, CP analysis of mitochondria from Coa8 knockout (Coa8KO) flies showed a clear decrease in fully assembled CIV and in all the CIV-containing species (Figure 2A and B) when compared to the corresponding wild-type (WT) individuals (Figure 1A and Figure 2B). Curiously, CP also showed that the amounts of SC I1III2 were noticeably increased in the Coa8KO mitochondria (Figure 2A and B). In these samples, complexes I and III2 build a stable SC species containing ~16% of the total amount of CI, as visualized by CI-IGA, as well as by western blot (WB) and immunodetection of specific CI and CIII2 subunits after BNGE in DDM-solubilized mitochondria (Figure 2C and D, Figure 2—figure supplement 1). We initially speculated that the ~fivefold increase of SC I1III2 formation could be linked to the release of III2 from SC III2IV1 induced by the strong reduction in CIV amounts when Coa8 is absent. Figure 2 with 1 supplement see all Download asset Open asset 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 (Coa8KO) flies. (D) BN-PAGE, western blot immunodetection of MRC complexes from a pool of three control wild-type (w1118) and three Coa8 KO (Coa8KO) 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). To test this hypothesis, we modulated Coa8 expression via the UAS/GAL4 system using RNAi driven by a ‘mild’ ubiquitous GAL4 driver (da-gal4). With this system, the Coa8 mRNA levels were reduced to ~60% of the control (Figure 3A). However, these flies showed comparable levels of fully assembled CIV (Figure 3B, Figure 3—figure supplement 1). Interestingly, in this case there was also an increased formation of SC I1III2 from ~3% in the control to ~10% in the mild Coa8RNAi (Figure 3B and C, Figure 3—figure supplement 1). Therefore, both strong and weak perturbations of CIV assembly produce an increased formation of CI-containing SCs in D. melanogaster, irrespective of whether they result in CIV deficiency or not. Figure 3 with 1 supplement see all Download asset Open asset 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 respiratory rates SC formation was proposed to serve as a means to favor electron transfer between the complexes and therefore increase the efficiency of CI-fueled respiration (Schägger and Pfeiffer, 2000). With this in mind, the complete Coa8KO and mild Coa8RNAi fly mitochondria, which show increased amounts of SC I1III2 compared to the WT controls, provide an excellent opportunity to test this possibility. Oxygen consumption activities of fly homogenates in the presence of different substrates and inhibitors were analyzed by high-resolution respirometry (Figure 4A and B). The significant decrease in CIV enzymatic activity in the Coa8KO (Figure 4C) was not reflected by reduced oxygen consumption (Figure 4A). This could be explained as a result of a high CIV excess in fly mitochondria, in which the observed 60% reduction in CIV enzyme activity is still above the threshold at which the CIV defect determines lower respiratory rates (Villani et al., 1998; Villani and Attardi, 2000). In contrast, the mild reduction in Coa8 mRNA levels did not result in CIV enzymatic deficiency but produced a slight elevation in CI activity (Figure 4D), which is most likely due to the increased SC I1III2 formation (Figure 3B and C, Figure 3—figure supplement 1). However, the Coa8-KD mitochondria did not show any differences in respiration with either CI-linked or CII-linked substrates. Also, the increased and stable interactions between complexes I and III2 in the Coa8 deficient models did not produce a preferential utilization of electrons coming from CI, which would be the prediction if SC formation increased electron transfer efficiency (Lapuente-Brun et al., 2013). Figure 4 Download asset Open asset 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 also enhances SC formation in D. melanogaster To determine whether increased SC I1III2 formation was specific for CIV deficient flies, we targeted CIII2 by knocking down the expression of Bcs1. BCS1L, the human homolog, is fundamental for a correct CIII2 biogenesis, being responsible for the incorporation of the catalytic subunit UQCRFS1 in the last steps of CIII2 maturation (Fernandez-Vizarra et al., 2007; Fernandez-Vizarra and Zeviani, 2018). To obtain a severe CIII2 defect in D. melanogaster, we crossed a ‘strong’ ubiquitous GAL4 driver (act5c-gal4) line with a UAS-Bcs1 RNAi responder line (Brischigliaro et al., 2021). The knockdown efficiency was high, with a~75% decrease in Bcs1 expression at the mRNA level (Figure 5A). In this model, D. melanogaster development was severely impaired causing an arrest at the larval stage (Brischigliaro et al., 2021). The strong Bcs1RNAi caused also a significant decrease in fully assembled CIII2 levels (Figure 5B and C, Figure 5—figure supplement 1) and in CIII2 enzymatic activity of about 50% (Figure 5D). Consistent with the observed CIII2 deficiency, both the CI- and CII- linked respiration rates were significantly decreased by around 40% (Figure 5E). Figure 5 with 1 supplement see all Download asset Open asset 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). In contrast, less pronounced decreases in Bcs1 expression (Figure 5F) by using the mild da-gal4 driver instead, did not produce a noticeable CIII2 enzymatic defect (Figure 5I). However, the mild Bcs1-KD mitochondria showed a very different pattern of CI distribution than the controls (Figure 5G and H, Figure 5—figure supplement 1), with the formation of a ‘respirasome-like’ SC I1III2IV1, and also of a higher molecular weight supercomplex (HMW-SC), of unknown stoichiometry, containing also complexes I, III2 and IV, as revealed by WB and immunodetection analyses (Figure 5G and H). At the functional level, this was associated with a~1.5-fold increase in CI enzymatic activity (Figure 5I), which is proportional to the increase in total CI amounts (Figure 5—figure supplement 1), and in higher CI-linked respiration rates but only by ~1.2-fold (Figure 5J). CII-linked respiration was the same in the mild Bcs1RNAi samples as in the controls. Therefore, the formation of respirasome-like SCs in these mitochondria did neither increase the efficiency of electron transfer from CI nor determine a diversion of the electronic routes giving preference to the SC-bound CI. Mild perturbation of CI biogenesis also leads to increased SC assembly To understand the effect of the strong and mild perturbations in CI biogenesis on MRC organization in D. melanogaster, we employed a similar strategy as that for CIII (see above). Crossing the strong ubiquitous act5c-gal4 driver fly line with the UAS-Ndufs4 RNAi responder line, produced a decrease in Ndufs4 mRNA expression of ~90% (Figure 6A). Defects in NDUFS4 are a major cause of CI deficiency-associated mitochondrial disease in humans (Ortigoza-Escobar et al., 2016) and the mouse and D. melanogaster animal models display CI deficiency and pathological phenotypes (Kruse et al., 2008; Foriel et al., 2018). Accordingly, the strong reduction in Ndufs4 expression observed in our models resulted in developmental arrest and a significant decrease in fully assembled CI levels by ~40% (Figure 6B and C, Figure 6—figure supplement 1) and in a proportional decrease in NADH:CoQ oxidoreductase enzyme activity in the larvae (Figure 6D). BNGE analysis of the strong Ndufs4RNAi D. melanogaster mitochondria, revealed the presence of a CI subassembly, containing the core Ndufs3 subunit (Figure 6C, Figure 6—figure supplement 1) but lacking NADH-dehydrogenase activity (Figure 6B). This is similar to what is observed in NDUFS4-deficient human and mouse, which accumulate the so-called ~830 kDa intermediate lacking the N-module and stabilized by the NDUFAF2 assembly factor (Vogel et al., 2007; Assouline et al., 2012; Calvaruso et al., 2012). In contrast, CIV levels and enzyme activity were significantly increased by 1.5-fold in the strong Ndufs4-KD (Figure 6C and D, Figure 6—figure supplement 1). CI-linked respiration measured in isolated mitochondria from these flies was significantly lower than in the controls, whereas the CII-linked respiration was comparable to the control (Figure 6E). These observations are compatible with the isolated CI defect displayed by the strong Ndufs4-KD flies. Figure 6 with 1 supplement see all Download asset Open asset 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" @default.
• W4387570511 created "2023-10-13" @default.
• W4387570511 creator A5048731424 @default.
• W4387570511 date "2023-10-12" @default.
• W4387570511 modified "2023-10-13" @default.
• W4387570511 title "eLife assessment: Structural rather than catalytic role for mitochondrial respiratory chain supercomplexes" @default.
• W4387570511 doi "https://doi.org/10.7554/elife.88084.3.sa0" @default.
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