Mitochondrial super complexes in the colorless chlorophycean alga Polytomella parva

Mitochondrial super complexes in the colorless chlorophycean alga Polytomella parva | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mitochondrial super complexes in the colorless chlorophycean alga Polytomella parva Marcos Ostolga-Chavarría, Anaiza Rico-Luna, Sergio Fuentes-Hernández, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7871941/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Chlorophycean algae of the genera Chlamydomonas and Polytomella share a common photosynthetic ancestor. However, members of the Polytomella lineage have adopted a heterotrophic lifestyle, having lost the photosynthetic apparatus and relying instead on acetate or ethanol as carbon sources, with energy production centered on oxidative phosphorylation (OXPHOS). In this study, we investigated the composition of the mitochondrial supercomplexes of the colorless alga Polytomella parva . OXPHOS complexes were solubilized using mild detergents such as glycol-diosgenin and digitonin, followed by separation of protein assemblies via Blue Native electrophoresis and Fast Protein Liquid Chromatography (FPLC). Additionally, complexome profiling of solubilized mitochondria resolved by Blue Native Gel Electrophoresis was carried out. The resulting data indicate that the OXPHOS supercomplexes of Polytomella closely resemble those observed in situ in the mitochondria of its green relative Chlamydomonas reinhardtii , as revealed by electron cryo-tomography and subtomogram averaging. Chlorophycean algae oxidative phosphorylation F1FO ATP synthase oligomeric complex V mitochondrial supercomplexes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Mitochondria are essential organelles in most eukaryotic cells, responsible for ATP production through oxidative phosphorylation (OXPHOS). Complexes I, III and IV oxidize substrates derived from the citric acid cycle, transferring electrons from NADH and succinate to oxygen while simultaneously generating a proton gradient across the inner mitochondrial membrane. This electrochemical gradient is then used by the F₁Fo-ATP synthase to make ATP (Vercellino and Sazanov, 2022 ). OXPHOS complexes often associate into higher-order oligomeric assemblies known as super complexes, which were first isolated by solubilization of mitochondria with mild detergents such as digitonin followed by Blue Native Electrophoresis (BN-PAGE) (Schägger, 2001 ). Across species, the catalytic cores of OXPHOS complexes share many conserved structural features, whereas variability is commonly found in peripheral regions, including N- or C-terminal extensions, subunit insertions, lineage-specific polypeptides, or even additional structural domains (He et al., 2024 ; Klusch et al., 2021 ; Maldonado et al., 2021 ; Mühleip et al., 2023 ; Parey et al., 2021 ). Consequently, the organization of OXPHOS supercomplexes exhibits substantial evolutionary diversity. To date, supercomplexes have been isolated and structurally characterized in representatives of four of the 13 eukaryotic supergroups (Guo et al., 2017 ; He et al., 2024 ; MacLean et al., 2025 ; Mühleip et al., 2023 ), while in situ characterization has been achieved for only two species (Waltz et al., 2025 ; Zheng et al., 2024 ). In all mitochondrial supercomplexes described so far, complex III serves as the central scaffold for the association of complexes I, II, or IV (Protasoni et al., 2020 ; Mühleip et al., 2023 ; Miranda-Astudillo and Rico-Luna, 2025 ). These supramolecular assemblies are thought to facilitate electron transfer between respiratory complexes, thereby reducing the production of reactive oxygen species (Berndtsson et al., 2020 ; Kohler et al., 2023 ; Chan et al., 2024 ). Moreover, they contribute to the efficient packing of OXPHOS components within the inner mitochondrial membrane and play a role in shaping cristae morphology—forming lamellar cristae in Opisthonkonta and Archaeplastida (Blum et al., 2019 ; Davies et al., 2011 ), discoid cristae in Discoba (Mühleip et al., 2017 ), tubular cristae in ciliates (Mühleip et al., 2016 ), and bulbous cristae in Apicomplexa (Mühleip et al., 2021 ). Previous work carried out with P. parva mitochondria solubilized with mild detergents, n-dodecyl-β-D-maltoside (here denoted as lauryl maltoside or LM) or digitonin, followed by separation on BN-PAGE, allowed the identification of three different associations, I-IV 6 , I-III 4 , and I-IV (Miranda-Astudillo et al., 2018 ). Here, we revisited the algal OXPHOS complexes using glyco-diosgenin (GDN)-solubilized mitochondria, BN-PAGE, chromatography and mass spectrometry-based complexome profiling. Materials and methods 1) Algal strain, growth conditions and mitochondria isolation. P. parva (strain number 198.80 from the Culture Collection of Algae at the University of Göttingen) was grown in liquid mineral Tris-phosphate medium supplemented with sodium acetate (30 mM), and vitamins (biotin 10%, B 12 vitamin 10%, and B 1 vitamin 2 × 10–5% (w/v)) (pH 7.0). Cells were harvested by centrifugation at 7,000 x g for 10 minutes and stored at -70°C until use. Sedimented cells were resuspended in SPT buffer (0.3 M sucrose, 4 mM potassium-EDTA, and 20 mM Tris-HCl pH 7.2) and disrupted with a Potter homogenizer. An enriched mitochondrial membrane fraction was obtained by a two-step differential centrifugation as earlier described (Miranda-Astudillo et al., 2018 ). 2) Blue Native-Polyacrylamide Gel Electrophoresis (BN-PAGE) Mitochondrial proteins were solubilized with 2.0 g lauryl maltoside (LM)/g protein (2.0%), or 4.0 g GDN/g protein (4.0%) in solubilization buffer (SB) containing 50 mM Tris-HCl, 1.5 mM MgSO 4 , 100 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 50 µ g/ml tosyl-lysyl-chloromethylketone (TLCK) (pH 8.4). The mixture was incubated at 4°C with gentle stirring for 30 min, and centrifuged at 30,000 × g for 30 min. The supernatants were subjected to BN-PAGE (Schägger, 2001 ) in 3%–10% acrylamide gradient gels. For 2D-BN-PAGE, a lane was excised and loaded to a second acrylamide gradient with 0.03% LM in the cathode buffer (Wittig and Schägger, 2005 ). 3) In-gel enzymatic activity staining. In-gel staining of NADH/NBT oxidoreductase, cytochrome c oxidase, and ATPase activities were performed as previously described (Miranda-Astudillo et al., 2018 ). 4) OXPHOS complexes separation by size exclusion chromatography. GDN-solubilized mitochondria were used in two independent approaches: anion-exchange chromatography and size exclusion chromatography. For anion-exchange chromatography, 200 mg of algal mitochondria were solubilized in SB and centrifuged at 35,000 x g for 30 min. The supernatant was diluted in SB without NaCl (1:3), loaded on a Source 15Q 10/100 column and eluted with a continuous NaCl gradient (from 0 to 500 mM). For size-exclusion chromatography, solubilized mitochondria were concentrated with an Amicon Ultra-15 Centrifugal Filter (EMD Millipore) to a final volume of 500 µL and injected to a couple of size exclusion Superose 6 10/300 columns connected in tandem (GE Healthcare Life Sciences) previously equilibrated with 50 mM Tris-HCl, 150 mM NaCl, 1mM MgSO 4 , 10% glycerol, 1 mM PMSF, 50 µ g/ml TLCK, and 0.01% GDN (pH 8.4). The elution was carried out at 0.25 mL/min. Fractions of 0.5 mL were collected and resolved by BN-PAGE. 5) Complexome profiling 5.1) In-gel digestion and peptide recovery Isolated mitochondria (100 µg) were solubilized with either lauryl-maltoside, digitonin or GDN at the following detergent/protein ratios (2 g of detergent per g of protein ) and then resolved by BN-PAGE on a 3–16% acrylamide gradient gel (Wittig et al., 2006 ). After electrophoresis, the gel was fixed overnight in 50% methanol, 10% acetic acid and 100 mM ammonium acetate. It was then stained for 45 min with 0.025% Coomassie G-250 in 10% acetic acid, destained in 10% acetic acid, and stored in deionized water allowing the gel to re-swell to its original dimensions. The gels were scanned, and a full real size image was used for the cutting procedure. Each lane was cut into 48 equal slices (upward from the bottom until the top of the gel), diced and transferred to 96-well filter plates (Millipore, MABVN1250) taped over waste collectors (Nunc MaxiSorp plates). Gel pieces were washed repeatedly in 50% methanol, 50 mM ammonium bicarbonate (ABC) until all blue dye had disappeared. Excess liquid was removed with brief centrifugations (1,000 x g , 20 s) between washes. Cysteines were reduced in 10 mM dithiothreitol, 50 mM ABC for 45 min, and further alkylated with 30 mM chloroacetamide, 50 mM ABC for 30 min in the dark. After a 15 min dehydration step in 50% methanol, 50 mM ABC and air-drying for 30–45 min at room temperature, 20 µl of a sequencing-grade trypsin solution at 5 ng µL⁻ 1 ) in 50 mM ABC, 1 mM CaCl 2 were added to each well. The gel pieces were incubated at 4°C for 20 min and covered with 50 µL of fresh ABC. The proteins were digested overnight at 37°C. The resultant peptides were collected into clean 96-well PCR microplates after centrifugation at 1,000 x g for 60 sec, followed by a 20 min incubation in 30% acetonitrile, 3% formic acid and finally eluted as in the previous step. The combined eluates were vacuum-dried (~ 3 h) in a Concentrator Plus (Eppendorf) and the peptides were resuspended in 20 µL of 5.0% acetonitrile, 0.5% formic acid. Samples were stored at − 20°C until used. 5.2) Nano LC–MS/MS analysis Thawed peptides were thoroughly agitated for ~ 20 min, and the plates were loaded onto an Ultimate 3000 UHPLC system coupled to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). Prior to electrospraying, 5 µL of peptides from each fraction were concentrated and desalted in a PepMap Neo Trap column (Thermo Fisher Scientific), followed by separation on an Elite Aurora column (1.7 µm C18, 15 cm × 75 µm ID (IonOpticks, Australia) maintained at 60°C. Peptide elution was performed over 55 min using a linear gradient of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) at a flow rate of 0.3 µL/min, with the gradient programmed as follows: sample loading 0–4 min (2% B); separation 4–5 min (2–5% B), 5–35 min (5–40%B), and 35–40 min (40–90% B). The column was washed for 8 min at 90% B, then adjusted back to 2% B in 1 min followed by a 6 min re-equilibration at 2% B. MS analysis was performed in positive mode. A NanoFlex source was used for electrospray ionization, applying 2.4 kV, with a source temperature of 275°C. MS data were acquired in data-dependent acquisition (DDA) mode. Full MS scans were recorded from 375 to 1500 m/z at a resolution of 120,000, with an RF lens setting of 30%. MS1 data were collected in profile mode. The 20 most abundant precursors (charge states 2–7) were selected for MS/MS analysis. Fragmentation was performed using collision-induced dissociation (CID) at 35% collision energy. All other instrument parameters (e.g., AGC, injection times, dynamic exclusion) were set to default. 5.3) Data processing and Profile Generation Raw spectra were processed with MaxQuant v2.6.5.0 against two databases: the transcriptome of P.parva SAG 63 − 3 (sample MMETSP0052 from the Marine Microbial Eukaryote Transcriptome Sequencing Project) (Mallet and Lee, 2006 ; Smith et al., 2010 ) and the mass spectrometry proteomic data obtained from Polytomella sp. SAG 198.80 (with identifier PXD035155 deposited at the ProteomeXchange Consortium) (Lacroux et al., 2022 ), with a false discovery rate (FDR) threshold of < 1%. The following modifications were specified: N-terminal acetylation (+ 42.01), deamidation of glutamine and asparagine (+ 0.98), and oxidation of methionine (+ 15.99) as variable modifications, while carbamidomethylation of cysteines (+ 57.02) was set as a fixed modification. were removed from the list. A total of 2,921 proteins were identified and annotated using BLAST in Phytozome, the Plant Comparative Genomics portal of the Department of Energy's Joint Genome Institute ( https://phytozome-next.jgi.doe.gov/ ) (Goodstein et al., 2012 ). Keratins, trypsin, and other common contaminants were not considered. Intensity-based absolute quantification (iBAQ) values were used to reflect protein abundances. Each profile was scaled to its own maximum, sorted by average-linkage hierarchical clustering with centered Pearson distance and displayed as black, yellow, and red heat maps in Microsoft Excel 2019. Further analysis was performed by manual inspection of the obtained profiles, and the iBAQ values were re-normalized within specific apparent molecular mass ranges. Bovine heart mitochondria (BHM) solubilized under the same conditions as P. parva mitochondria were used for mass calibration of the 3–16% BN-PAGE. 6) In silico modelling The 3D structural models of P. parva complexes III and IV were built using the SWISS-MODEL tool (Waterhouse, et al, 2018 ), based on the structures of C. reinhardtii complex III (PDB 9F5Z) and complex IV (PDB 9F60) respectively (Waltz, et al, 2025 ). The structure of P. parva complex I (PDB 7ARD) and the models of complex III and complex IV were assembled into the EMD 50210 electron density map and the PDB 9F62 structure of the C. reinhardtii respiratory supercomplexes (Waltz, et al, 2025 ) using ChimeraX 1.9 (Meng, et al, 2023 ). Results To investigate the supramolecular organization of the OXPHOS complexes in P. parva , isolated mitochondria solubilized with LM or GDN were subjected to BN-PAGE. The previously characterized pattern of algal OXPHOS complexes solubilized with LM and resolved in 3–10% acrylamide gradient gels stained with Coomassie blue exhibited three main bands corresponding to dimeric complex V (V 2 ), complex I (I), dimeric complex III (III 2 ). Four weaker bands were also noticed, two from oligomeric forms of complex V (V 4 and V 6 ) and two representing monomeric and dimeric complex IV (IV and IV 2 ) (Fig. 1 A lane 1). GDN solubilized mitochondria exhibited less V 2 but higher proportions of the oligomeric forms V 4 and V 6 . This was evident in both Coomassie Blue and ATPase activity staining, indicating that the milder detergent GDN preserves more complex-complex interactions than LM (Fig. 1 A, lanes 2 and 5). While both GDN and LM led to a similar band pattern of the complex V forms, the respiratory complexes displayed greater variation in migration. In-gel activity staining of complexes I and IV (Fig. 1 A, lanes 3 and 4) evidenced their presence in several supramolecular associations. Further resolution of GDN-solubilized samples by 2D-BN-PAGE in the presence of LM, revealed that V 6 dissociated into V 4 , V 2 , and V; V 4 was partially conserved, but also dissociated into V 2 and V; and lastly, V 2 fully dissociated to V. Complex I was found to be present in three larger super-complexes while complex IV appeared in four larger oligomeric forms. Furthermore, complex III 2 was found in three oligomeric states. In summary, these results indicate four main oligomeric associations of the respiratory complexes: III 2 -IV, I-III 2 , I-III 2 -IV x and I-III 2 -IV y (where x and y represent uncertain stoichiometries, difficult to define by this approach) (Fig. 1 B). Fast Protein Liquid Chromatography (FPLC) using either anion exchange or size-exclusion columns allowed us to identify different super complexes in GDN-solubilized Polytomella mitochondria. In the first case, protein complexes were eluted with a continuous gradient from 0 to 500 mM NaCl, giving rise to the elution profile shown in Fig. 2 A. Collected fractions were subjected to BN-PAGE and in-gel activity staining for both complexes I and IV. The ATP synthase oligomeric forms V 2 , V 4 and V 6 eluted earlier than respiratory complexes I, III and IV (Fig. 2 A) and their supramolecular associations, here identified as III 2 -IV, I-III 2 , and I-III 2 -IV x . In the second case, our tandem size exclusion chromatography setup allowed separation of proteins ranging 5–5000 kDa. The elution pattern shows the sequential separation of V 6 , V 4 and V 2 oligomers followed by the supramolecular associations of respiratory complexes I-III 2 and III 2 -IV (Fig. 2 B). To further explore the effect of the detergents GDN, digitonin, and LM on mitochondria solubilization and super-complex stability, composition, and abundance, complexome profiling (CP) was performed. This approach involves separating mitochondrial proteins by native electrophoresis, fractionating entire gel lanes, and identifying proteins in each individual fraction by tandem mass spectrometry followed by abundance pattern–based hierarchical clustering (Heide et al. 2012 ; Cabrera-Orefice et al., 2022 ). CP enables unbiased and comprehensive identification of potential protein–protein interactions. Complexome profiling analysis allowed us to identify around 2,900 proteins, among which we found most of the subunits that form part of the mitochondrial respiratory complexes. For complex I, 38 out of the 51 subunits were identified; for complex III, 7 out of 10; and for complex IV, 9 out of 12 (Fig. 3 , Supplementary Table S2). It should be noted that the subunits that cannot be identified, such as cytochrome b of complex III, or some subunits belonging to the P distal module of complex I, are highly hydrophobic membrane proteins, which makes them difficult to digest with trypsin and subsequently to be identified by MS. On the other hand, all the subunits of complex II and complex V could be identified (4 for complex II and 18 for complex V) (Supplementary Figure S1 ). In addition, this experimental approach allowed us to identify 5 isoforms of previously characterized proteins (Supplementary Table S1 and Supplementary Figure S2). For complex I, two isoforms were identified: one of NUOP4 belonging to the P distal module, and another for gamma carbonic anhydrase (CA3). In complex IV, the presence of an isoform of the Cox6b subunit was found (Fig. 3 , highlighted in red). In the case of complex II and complex V, which are not constituents of the supercomplexes, an isoform of subunit D and an isoform of subunit Asa9 were identified, respectively (Supplementary Figure S1 ). Under the growth conditions for the colorless alga, using acetate as carbon source, all these proteins are produced in different proportions. With GDN-solubilized mitochondria, the migration of complexes I, III and IV and its supramolecular associations were followed by mass spectrometry (Fig. 4 ). Normalization of the main peaks representing the supercomplexes allowed the identification of three different organizations: 1-IV (~ 1,188 kDa), I-III 4 -IV 2 (~ 2,320 kDa) and I 2 -III 4 -IV 2 (~ 3,130 kDa) (Fig. 4 , inset). In contrast, digitonin-solubilized mitochondria subjected to BN-PAGE in the same conditions seem to partially disrupt supramolecular associations, since higher levels of free complexes I, III and IV were present as compared with GDN-solubilized mitochondria (Fig. 5 ). In these conditions, the three following associations were identified: I-IV (~ 1,188 kDa), I-III 2 -IV (~ 1,445 kDa) and I 2 -III 2 -IV 2 (~ 2,532 kDa) (Fig. 5 , inset). Finally, and as expected, LM-solubilized mitochondria displayed a polypeptide pattern exhibiting mainly free respiratory complexes and negligible amounts of supercomplexes and (Fig. 6 ). Discussion Earlier studies recognized the mitochondrial ATP synthase of the colorless alga P. parva as a stable dimer resistant to dissociation by detergents such as LM (Vázquez-Acevedo et al., 2006 ; van Lis et al., 2007 ). The enzyme contains conserved subunits within its rotary and catalytic domains but also incorporates several atypical subunits known as ASA subunits, which form the peripheral arms of the complex and participate in the dimerization of the enzyme. In addition to its dimeric form, higher-order oligomers—including tetramers and hexamers—were observed by BN-PAGE analysis (Miranda-Astudillo et al., 2018 ). Low-resolution imaging further revealed the overall silhouette of the dimer, characterized by robust peripheral stalks (Dudkina et al., 2005 ). More recently, high-resolution 3D structures have provided detailed insights into subunit interactions and the mechanism of rotational catalysis (Allegretti et al., 2015 ; Murphy et al., 2019 ). Electron microscopy and cryo-electron tomography revealed the presence of densely packed cristae in Polytomella mitochondria (Dudkina et al., 2006 ). Using electron cryo-tomography and subtomogram averaging at resolutions of up to 4.2 Å, ATP synthase could be visualized in vivo following rapid freezing of actively growing algal cells. The 3D reconstruction of a mitochondrion showed series of ATP synthase dimers adopting a near-helical arrangement, with multiple parallel rows organized within the disk-shaped cristae (Dietrich et al., 2014). Additionally, individual ATP synthases were observed in proximity to the cristae junctions. As shown here, some of these higher V 2 oligomeric states can be preserved after mild extraction (Fig. 1 ), and these associations remain stable after size-exclusion or anion-exchange chromatography (Fig. 2 ). The existence of respiratory super complexes in Polytomella was suggested by the identification in LM solubilized mitochondria of I-IV 6 , I-III 4 , and I-IV associations by BN-PAGE and the in vitro reconstitution of supercomplexes III 2 -IV and I-III 2 -IV 2 by ion exchange chromatography (Miranda-Astudillo et al., 2018 ). Here, we demonstrate the presence of the super complex III 2 -IV in GDN-solubilized mitochondrial membranes (Fig. 1 A) as well as the existence of dimeric complex IV and of at least one respirasome. Recent advances in in situ cryo-electron microscopy have provided direct images of mitochondria, enabling the determination of the 3D structures of respiratory supercomplexes in their native states from both porcine (Zheng et al., 2024 )d reinhardtii mitochondria (Waltz et al., 2025 ). Therefore, there is no doubt about the existence of dynamical super-structures in the mitochondrial inner membrane that exhibit a huge diversity of associations and stoichiometries among different species (Eldeeb et al., 2024 ; Guan et al., 2022 ). In mammalian mitochondria, four distinct super complex organizations have been identified: I-III₂-IV (~ 1,980 kDa), I₂-III₂-IV₂ (~ 3,380 kDa), I-III₂-IV₂ (~ 2,128 kDa), and I₂-III₄-IV₂ (~ 3,906 kDa). Notably, the latter two arrangements had not previously been detected in vitro using mild non-ionic detergents for extraction (Vercellino and Sazanov, 2024 ), highlighting the unique ability of in situ cryo-electron microscopy to reveal otherwise elusive associations. In contrast, the C. reinhardtii respirasome displayed an in situ I₂-III₄-IV₆ arrangement (Waltz et al., 2025 ), which differs from supercomplexes reported in other organisms, including Sus scrofa [I-III₂-IV₂ (PDB 8UGI), I-III₂-IV₂ (PDB 8UGJ), I₂-III₂-IV₂ (PDB 8UGN), I₂-III₄-IV₂ (PDB 8UGR)] (Zheng et al., 2024 ); Mus musculus [I-III₂-IV (PDB 8PW7), I-III₂-IV₂ (PDB 8PW5)] (Vercellino and Sazanov, 2024 ); Euglena gracilis [I-III₂-IV] (He et al., 2024 ); and Tetrahymena thermophila [I₂-II₂-III₄-IV₂ (PDB 8GYM)] (Han et al., 2023 ). Among them, the T. thermophila super complex is the only respirasome characterized so far to include complex II, a feature attributed to lineage-specific subunits in both complexes II and IV, that promote an unusually large supramolecular assembly exceeding in size the I₂-III₄-IV₆ arrangement of the C. reinhardtii respirasome. These different supramolecular organizations may influence local membrane curvature, rendering it more convex or concave depending on their organization and stoichiometry (Zheng et al., 2024 ). Putative respiratory strings have been proposed to be present in mammalian, chlorophycean and plant mitochondria, where I 2 -III 2 -IV 2 and I 2 -III 4 -IV 2 associations should work as building blocks for larger circular or linear organizations, respectively (Bultema et al., 2009 ; Guo et al., 2018 ; Letts et al., 2016 ; Miranda-Astudillo et al., 2018 ). To date, no direct evidence of these large associations has been confirmed using in-situ studies (Davies et al., 2012 , 2011 ; Dietrich et al., 2024 ; Mühleip et al., 2016 , 2017 ; Zheng et al., 2024 ). Electron cryotomography of C. reinhardtii mitochondria further revealed a spatial segregation within cristae of respiratory complexes and rows of ATP synthase dimers, as has been reported in other systems. Biochemical isolation and characterization of the C. reinhardtii respirasome yielded a super complex with an I-III₂-IV₂ composition, whose structure was resolved at 2.8 Å using single-particle cryo-EM (Waltz et al., 2025 ). Apart from the absence of a dimeric complex IV, this isolated form appears to represent one half of the intact respirasome observed in situ . Multicellular chlorophycean algae such as Volvox diverged from their unicellular ancestors at least 200 million years ago (MYA) (Herron et al., 2009 ). It can be inferred that the Polytomella lineage separated from the Chlamydomonas less than 200 MYA. The loss of photosynthesis in Polytomella may have driven extensive changes in chloroplast composition and function, ultimately leading to the emergence of colorless plastids in this genus (Figueroa-Martínez et al., 2015 ). In contrast, mitochondrial evolution appears to have followed a more conservative trajectory, preserving many features of its green algal relatives. The stoichiometry of the P. parva respirasomes characterized in this study may reflect supramolecular associations derived from the dissociation of a larger, Chlamydomonas-like I 2 -III 4 -IV 6 respirasome. Nevertheless, we did not detect in vitro a complete respirasome comparable to the one observed in situ in the green algal mitochondria. This discrepancy may arise from several factors, including the disruptive effects of detergent extraction on complex organization or the loss of structural lipids such as cardiolipin (Walz et al., 2025). Alternatively, it is possible that the various supra complex species identified by complexome profiling assemble in a context-dependent manner, reflecting differential associations that adapt to the metabolic requirements of the cell. In the C. reinhardtii respirasome, four contact regions have been identified between complexes I and III, and two between complexes I and IV. It was proposed that the I/III super complex is the most stable, whereas complex IV is the least resistant to biochemical purification (Waltz et al., 2025 ). In the present work, we isolated an I 2 -III 4 -IV 2 super complex from P. parva , identifying each of the orthologous subunits that form the complexes taking as a reference the subunits that make up the same complexes in C. reinhardtii (Supplementary Table S3). However, it is likely that up to four complex IV units were lost during the isolation procedure. We propose that P. parva contains a higher-order I 2 -III 4 -IV 6 respirasome which, upon detergent solubilization of mitochondria, may dissociate into the various single complexes and supercomplexes detected in this study (Fig. 6 ). While differences between isolated supercomplexes and their counterparts observed in situ are well recognized, multiple supramolecular arrangements with varying stoichiometries can coexist within the same organism (Zheng et al., 2024 ). Accordingly, the I 2 -III 4 -IV 2 super complex characterized here may also represent a physiologically relevant organization. Declarations Acknowledgments Funding was provided with grants from Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT-DGAPA-UNAM) numbers IN204426 (to D.G-H.) and IA204524 (to H.V.M-A.). D.G.-H. also acknowledges financial support from grant CBF-2025-I-258 from Secretaría de Ciencia, Humanidades, Tecnología e Innovación, Mexico (SECIHTI). H.V.M.-A. also acknowledges financial support from the Instituto de Investigaciones Biomédicas under the Institutional Program [“Production of biomolecules of biomedical interest in microorganisms”]. The technical support of QBP Miriam Vázquez-Acevedo (IFC, UNAM) and of PhD Toshiko Takahashi Íñiguez (IIBO, UNAM) is also acknowledged. M. O-C. and S. F-H. are Ph.D. students of the Programa de Doctorado en Ciencias Bioquímicas de la Universidad Nacional Autónoma de México (UNAM) and have received fellowships from SECIHTI 710287 and 1146629 respectively. A. 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13:50:39","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":137387,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7871941/v1/6533860b95c6bda39755b6fd.html"},{"id":94985906,"identity":"c503c2e9-f203-4325-8db9-4b43f491d511","added_by":"auto","created_at":"2025-11-03 06:59:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":151625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATPase oligomers and respiratory supercomplexes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. parva \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emitochondria\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003eIsolated mitochondria were solubilized with the indicated detergent: lauryl maltoside(LM) at 2.0 g/g protein or glyco-diosgenin (GDN) at 4.0 g/g protein. (A) soluble fractions resolved by BN-PAGE in a 3–10% polyacrylamide gradient gel showing Coomassie Blue staining (lanes 1 and 2); \u003cem\u003ein-gel\u003c/em\u003e NADH-dehydrogenase activity staining (lane 3); \u003cem\u003ein-gel\u003c/em\u003e cytochrome \u003cem\u003ec\u003c/em\u003e oxidase (COX) activity staining (lane 4); and\u003cem\u003e in-gel\u003c/em\u003e ATPase activity staining (lane 5). (B) The GDN-solubilized samples resolved by BN-PAGE (Lanes 2 to 5 from Fig 1A) were incubated in the presence of LM and resolved in 2D-BN-PAGE gels, allowing the rupture of higher-order associations but maintaining intact the composition of individual complexes I, III and IV. The four 2D gels were subject to Coomassie Blue and activity staining, revealing the composition of the different supercomplexes.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7871941/v1/9dbcd9f4f2a63fc84984a005.png"},{"id":94865093,"identity":"50544f60-4d40-4b88-8822-8456ccfaa5c5","added_by":"auto","created_at":"2025-10-31 13:50:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":180485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOXPHOS supercomplexes are resolved by ion exchange chromatography and size exclusion chromatography.\u003c/strong\u003e Two hundred milligrams of mitochondria were solubilized with GDN and loaded into an anion exchange column, then eluted with continuous NaCl gradient. A) BN-PAGE of elution fractions from ion exchange chromatography, the upper gray triangle represents the NaCl gradient. B) BN-PAGE of fractions derived from size-exclusion chromatography. The gels were stained for complex I and IV activity.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7871941/v1/4cc6e81c0cf68e4b9f3768cd.png"},{"id":94865097,"identity":"756c7cd3-bfc4-4264-be37-f14c6e24922c","added_by":"auto","created_at":"2025-10-31 13:50:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":469459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComplexome profiling of mitochondria isolated from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. parva\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e solubilized with different detergents. \u003c/strong\u003e\u003cem\u003eP. parva\u003c/em\u003e mitochondria were solubilized with the detergents GDN, LM, and digitonin at a ratio of 2:1 mg of detergent per mg of protein. The samples were separated by BN-PAGE followed by quantitative mass spectrometry analysis. The amount (iBAQ) of each of the subunits that form the different complexes were represented by a heat map. For complex I, 42 out of 51 subunits that are known to form the complex were identified. For complex III, 8 out of 10, and for complex IV, 9 out of the 12.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7871941/v1/62046b7bf13cd68a7f6721c5.png"},{"id":94985928,"identity":"ba86b727-519d-4977-8986-c186b477b8ae","added_by":"auto","created_at":"2025-11-03 06:59:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":156320,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRespiratory complexes and supercomplexes in GDN-solubilized mitochondria.\u003c/strong\u003e Relative abundance graph and heat maps of the migration profiles of the averaged subunits of the algal respiratory complexes in their individual forms (OXPHOS-GDN): IV (232 kDa, green line), III\u003csub\u003e2\u003c/sub\u003e (468 kDa, orange line), and I (943 kDa, blue line). Inset: amplified region showing the migration zone of the supercomplexes (SC-GDN) (1,200-5,000 KDa), where the associations I/IV (1,200 kDa), I-III\u003csub\u003e4\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e (2,300 kDa) and I\u003csub\u003e2\u003c/sub\u003e-III\u003csub\u003e4\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e (3,100 kDa) could be identified.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7871941/v1/2587f2d497fe5325a6c1b915.png"},{"id":94865108,"identity":"fd0287bf-0b5e-4269-b0a0-81e5a34a6b99","added_by":"auto","created_at":"2025-10-31 13:50:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":172414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRespiratory complexes and supercomplexes in digitonin-solubilized mitochondria.\u003c/strong\u003e Relative abundance graph and heat maps of the migration profiles of the averaged subunits of the algal respiratory complexes in their individual forms (OXPHOS-digitonin): IV (192 kDa, green line), III\u003csub\u003e2\u003c/sub\u003e (421 kDa, orange line), and I (922 kDa, blue line). Inset: amplified region showing the migration zone of the supercomplexes (SC-digitonin) (1,200-5,000 KDa), where the associations I/IV (1,200 kDa), I-III\u003csub\u003e2\u003c/sub\u003e-IV\u003csub\u003e4\u003c/sub\u003e (1,500 kDa) and I\u003csub\u003e2\u003c/sub\u003e-III\u003csub\u003e2\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e (2,500 kDa) could be identified.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7871941/v1/00be2b46ec53f9d2cce8fc99.png"},{"id":94865112,"identity":"2a8311ab-f2d2-4b52-adc7-3aadc99ec535","added_by":"auto","created_at":"2025-10-31 13:50:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":174942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLM destabilizes supramolecular associations in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. parva\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mitochondria, preventing higher-order assemblies.\u003c/strong\u003e Relative abundance graph and heat maps of the migration profiles of the subunits of the complexes in their individual forms (OXPHOS-LM): IV (200 kDa, green line), III\u003csub\u003e2\u003c/sub\u003e (508 kDa, orange line), and I (1,086 kDa, blue line). Inset: amplified region of the migration zone of supercomplexes (SC-LM) showing negligible amounts of high-order associations (No SC´s).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7871941/v1/412ba0ae2af57fcf8f8dea44.png"},{"id":94986356,"identity":"4a76bd7d-e835-4a15-99cb-d49691f74d65","added_by":"auto","created_at":"2025-11-03 07:00:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":239427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe supramolecular associations of respiratory complexes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. parva.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eThe main, central figure shows a model for the putative \u003cem\u003eP. parva\u003c/em\u003e respirasome modelled upon the \u003cem\u003eC. reinhardtii\u003c/em\u003e mitochondrial respirasome I\u003csub\u003e2\u003c/sub\u003e-III\u003csub\u003e4\u003c/sub\u003e-IV\u003csub\u003e6\u003c/sub\u003e, and its fate after solubilization with each of the three detergents used in this work: LM, digitonin and GDN. Solubilization with the detergent LM caused the dissociation of the supercomplexes into their individual complexes I (blue), III (orange) and IV (green). Digitonin preserved three interactions: the supramolecular associations I-IV, I-III\u003csub\u003e2\u003c/sub\u003e-IV, and I\u003csub\u003e2\u003c/sub\u003e-III\u003csub\u003e2\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e. By contrast, solubilization GDN yielded six different supramolecular species, two of which were respirasomes with different stoichiometries: I-III\u003csub\u003e4\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e and I\u003csub\u003e2\u003c/sub\u003e-III\u003csub\u003e4\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e. \u003cem\u003eP. parva\u003c/em\u003e complexes III and IV were modeled upon the \u003cem\u003eC. reinhardtii\u003c/em\u003e PDB structures PF5Z and 9F60 (Walz et al., 2025) respectively, using Swiss-Model. For \u003cem\u003eP. parva\u003c/em\u003e Complex I the structure 7ARD from PDB was used (Klusch et al., 2021). The \u003cem\u003eP. parva\u003c/em\u003e respirasome was assembled in Chimera X using the density map EDM 50210 together with the PDB structure 9F62 (Walz et al., 2025).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7871941/v1/c9d9ba04926a6627513f676d.png"},{"id":94990522,"identity":"a3483c7d-5301-4a77-b884-6d8cee0a6178","added_by":"auto","created_at":"2025-11-03 07:17:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2481081,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7871941/v1/7ab47f60-6078-4e52-a17a-097fd77b70cf.pdf"},{"id":94986900,"identity":"f675e5e6-a38a-431c-b778-e389740b4306","added_by":"auto","created_at":"2025-11-03 07:00:57","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2531821,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7871941/v1/a75896a06576cc151d6ba263.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mitochondrial super complexes in the colorless chlorophycean alga Polytomella parva","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMitochondria are essential organelles in most eukaryotic cells, responsible for ATP production through oxidative phosphorylation (OXPHOS). Complexes I, III and IV oxidize substrates derived from the citric acid cycle, transferring electrons from NADH and succinate to oxygen while simultaneously generating a proton gradient across the inner mitochondrial membrane. This electrochemical gradient is then used by the F₁Fo-ATP synthase to make ATP (Vercellino and Sazanov, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). OXPHOS complexes often associate into higher-order oligomeric assemblies known as super complexes, which were first isolated by solubilization of mitochondria with mild detergents such as digitonin followed by Blue Native Electrophoresis (BN-PAGE) (Sch\u0026auml;gger, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Across species, the catalytic cores of OXPHOS complexes share many conserved structural features, whereas variability is commonly found in peripheral regions, including N- or C-terminal extensions, subunit insertions, lineage-specific polypeptides, or even additional structural domains (He et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Klusch et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Maldonado et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; M\u0026uuml;hleip et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Parey et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, the organization of OXPHOS supercomplexes exhibits substantial evolutionary diversity. To date, supercomplexes have been isolated and structurally characterized in representatives of four of the 13 eukaryotic supergroups (Guo et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; He et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; MacLean et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; M\u0026uuml;hleip et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), while \u003cem\u003ein situ\u003c/em\u003e characterization has been achieved for only two species (Waltz et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zheng et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In all mitochondrial supercomplexes described so far, complex III serves as the central scaffold for the association of complexes I, II, or IV (Protasoni et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; M\u0026uuml;hleip et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Miranda-Astudillo and Rico-Luna, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These supramolecular assemblies are thought to facilitate electron transfer between respiratory complexes, thereby reducing the production of reactive oxygen species (Berndtsson et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kohler et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Chan et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Moreover, they contribute to the efficient packing of OXPHOS components within the inner mitochondrial membrane and play a role in shaping cristae morphology\u0026mdash;forming lamellar cristae in Opisthonkonta and Archaeplastida (Blum et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Davies et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), discoid cristae in Discoba (M\u0026uuml;hleip et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), tubular cristae in ciliates (M\u0026uuml;hleip et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and bulbous cristae in Apicomplexa (M\u0026uuml;hleip et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePrevious work carried out with \u003cem\u003eP. parva\u003c/em\u003e mitochondria solubilized with mild detergents, n-dodecyl-β-D-maltoside (here denoted as lauryl maltoside or LM) or digitonin, followed by separation on BN-PAGE, allowed the identification of three different associations, I-IV\u003csub\u003e6\u003c/sub\u003e, I-III\u003csub\u003e4\u003c/sub\u003e, and I-IV (Miranda-Astudillo et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Here, we revisited the algal OXPHOS complexes using glyco-diosgenin (GDN)-solubilized mitochondria, BN-PAGE, chromatography and mass spectrometry-based complexome profiling.\u003c/p\u003e"},{"header":"Materials and methods","content":"\n\u003ch3\u003e1) Algal strain, growth conditions and mitochondria isolation.\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eP. parva\u003c/em\u003e (strain number 198.80 from the Culture Collection of Algae at the University of G\u0026ouml;ttingen) was grown in liquid mineral Tris-phosphate medium supplemented with sodium acetate (30 mM), and vitamins (biotin 10%, B\u003csub\u003e12\u003c/sub\u003e vitamin 10%, and B\u003csub\u003e1\u003c/sub\u003e vitamin 2 \u0026times; 10\u0026ndash;5% (w/v)) (pH 7.0). Cells were harvested by centrifugation at 7,000 x \u003cem\u003eg\u003c/em\u003e for 10 minutes and stored at -70\u0026deg;C until use. Sedimented cells were resuspended in SPT buffer (0.3 M sucrose, 4 mM potassium-EDTA, and 20 mM Tris-HCl pH 7.2) and disrupted with a Potter homogenizer. An enriched mitochondrial membrane fraction was obtained by a two-step differential centrifugation as earlier described (Miranda-Astudillo et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003e2) Blue Native-Polyacrylamide Gel Electrophoresis (BN-PAGE)\u003c/h3\u003e\n\u003cp\u003eMitochondrial proteins were solubilized with 2.0 g lauryl maltoside (LM)/g protein (2.0%), or 4.0 g GDN/g protein (4.0%) in solubilization buffer (SB) containing 50 mM Tris-HCl, 1.5 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 100 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 50 \u003cem\u003e\u0026micro;\u003c/em\u003eg/ml tosyl-lysyl-chloromethylketone (TLCK) (pH 8.4). The mixture was incubated at 4\u0026deg;C with gentle stirring for 30 min, and centrifuged at 30,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 30 min. The supernatants were subjected to BN-PAGE (Sch\u0026auml;gger, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) in 3%\u0026ndash;10% acrylamide gradient gels. For 2D-BN-PAGE, a lane was excised and loaded to a second acrylamide gradient with 0.03% LM in the cathode buffer (Wittig and Sch\u0026auml;gger, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003e3) In-gel enzymatic activity staining.\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eIn-gel\u003c/em\u003e staining of NADH/NBT oxidoreductase, cytochrome \u003cem\u003ec\u003c/em\u003e oxidase, and ATPase activities were performed as previously described (Miranda-Astudillo et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003e4) OXPHOS complexes separation by size exclusion chromatography.\u003c/h3\u003e\n\u003cp\u003eGDN-solubilized mitochondria were used in two independent approaches: anion-exchange chromatography and size exclusion chromatography.\u003c/p\u003e\u003cp\u003eFor anion-exchange chromatography, 200 mg of algal mitochondria were solubilized in SB and centrifuged at 35,000 x \u003cem\u003eg\u003c/em\u003e for 30 min. The supernatant was diluted in SB without NaCl (1:3), loaded on a Source 15Q 10/100 column and eluted with a continuous NaCl gradient (from 0 to 500 mM).\u003c/p\u003e\u003cp\u003eFor size-exclusion chromatography, solubilized mitochondria were concentrated with an Amicon Ultra-15 Centrifugal Filter (EMD Millipore) to a final volume of 500 \u0026micro;L and injected to a couple of size exclusion Superose 6 10/300 columns connected in tandem (GE Healthcare Life Sciences) previously equilibrated with 50 mM Tris-HCl, 150 mM NaCl, 1mM MgSO\u003csub\u003e4\u003c/sub\u003e, 10% glycerol, 1 mM PMSF, 50 \u003cem\u003e\u0026micro;\u003c/em\u003eg/ml TLCK, and 0.01% GDN (pH 8.4). The elution was carried out at 0.25 mL/min. Fractions of 0.5 mL were collected and resolved by BN-PAGE.\u003c/p\u003e\n\u003ch3\u003e5) Complexome profiling\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e5.1) In-gel digestion and peptide recovery\u003c/h2\u003e\u003cp\u003eIsolated mitochondria (100 \u0026micro;g) were solubilized with either lauryl-maltoside, digitonin or GDN at the following detergent/protein ratios (2 g of detergent per g of protein ) and then resolved by BN-PAGE on a 3\u0026ndash;16% acrylamide gradient gel (Wittig et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). After electrophoresis, the gel was fixed overnight in 50% methanol, 10% acetic acid and 100 mM ammonium acetate. It was then stained for 45 min with 0.025% Coomassie G-250 in 10% acetic acid, destained in 10% acetic acid, and stored in deionized water allowing the gel to re-swell to its original dimensions. The gels were scanned, and a full real size image was used for the cutting procedure.\u003c/p\u003e\u003cp\u003eEach lane was cut into 48 equal slices (upward from the bottom until the top of the gel), diced and transferred to 96-well filter plates (Millipore, MABVN1250) taped over waste collectors (Nunc MaxiSorp plates). Gel pieces were washed repeatedly in 50% methanol, 50 mM ammonium bicarbonate (ABC) until all blue dye had disappeared. Excess liquid was removed with brief centrifugations (1,000 x \u003cem\u003eg\u003c/em\u003e, 20 s) between washes. Cysteines were reduced in 10 mM dithiothreitol, 50 mM ABC for 45 min, and further alkylated with 30 mM chloroacetamide, 50 mM ABC for 30 min in the dark. After a 15 min dehydration step in 50% methanol, 50 mM ABC and air-drying for 30\u0026ndash;45 min at room temperature, 20 \u0026micro;l of a sequencing-grade trypsin solution at 5 ng \u0026micro;L⁻\u003csup\u003e1\u003c/sup\u003e) in 50 mM ABC, 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e were added to each well. The gel pieces were incubated at 4\u0026deg;C for 20 min and covered with 50 \u0026micro;L of fresh ABC. The proteins were digested overnight at 37\u0026deg;C. The resultant peptides were collected into clean 96-well PCR microplates after centrifugation at 1,000 x \u003cem\u003eg\u003c/em\u003e for 60 sec, followed by a 20 min incubation in 30% acetonitrile, 3% formic acid and finally eluted as in the previous step. The combined eluates were vacuum-dried (~\u0026thinsp;3 h) in a Concentrator Plus (Eppendorf) and the peptides were resuspended in 20 \u0026micro;L of 5.0% acetonitrile, 0.5% formic acid. Samples were stored at \u0026minus;\u0026thinsp;20\u0026deg;C until used.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e5.2) Nano LC\u0026ndash;MS/MS analysis\u003c/h2\u003e\u003cp\u003eThawed peptides were thoroughly agitated for ~\u0026thinsp;20 min, and the plates were loaded onto an Ultimate 3000 UHPLC system coupled to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). Prior to electrospraying, 5 \u0026micro;L of peptides from each fraction were concentrated and desalted in a PepMap Neo Trap column (Thermo Fisher Scientific), followed by separation on an Elite Aurora column (1.7 \u0026micro;m C18, 15 cm \u0026times; 75 \u0026micro;m ID (IonOpticks, Australia) maintained at 60\u0026deg;C. Peptide elution was performed over 55 min using a linear gradient of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) at a flow rate of 0.3 \u0026micro;L/min, with the gradient programmed as follows: sample loading 0\u0026ndash;4 min (2% B); separation 4\u0026ndash;5 min (2\u0026ndash;5% B), 5\u0026ndash;35 min (5\u0026ndash;40%B), and 35\u0026ndash;40 min (40\u0026ndash;90% B). The column was washed for 8 min at 90% B, then adjusted back to 2% B in 1 min followed by a 6 min re-equilibration at 2% B.\u003c/p\u003e\u003cp\u003eMS analysis was performed in positive mode. A NanoFlex source was used for electrospray ionization, applying 2.4 kV, with a source temperature of 275\u0026deg;C. MS data were acquired in data-dependent acquisition (DDA) mode. Full MS scans were recorded from 375 to 1500 m/z at a resolution of 120,000, with an RF lens setting of 30%. MS1 data were collected in profile mode. The 20 most abundant precursors (charge states 2\u0026ndash;7) were selected for MS/MS analysis. Fragmentation was performed using collision-induced dissociation (CID) at 35% collision energy. All other instrument parameters (e.g., AGC, injection times, dynamic exclusion) were set to default.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e5.3) Data processing and Profile Generation\u003c/h2\u003e\u003cp\u003eRaw spectra were processed with MaxQuant v2.6.5.0 against two databases: the transcriptome of \u003cem\u003eP.parva\u003c/em\u003e SAG 63\u0026thinsp;\u0026minus;\u0026thinsp;3 (sample MMETSP0052 from the Marine Microbial Eukaryote Transcriptome Sequencing Project) (Mallet and Lee, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Smith et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and the mass spectrometry proteomic data obtained from \u003cem\u003ePolytomella\u003c/em\u003e sp. SAG 198.80 (with identifier PXD035155 deposited at the ProteomeXchange Consortium) (Lacroux et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), with a false discovery rate (FDR) threshold of \u0026lt;\u0026thinsp;1%. The following modifications were specified: N-terminal acetylation (+\u0026thinsp;42.01), deamidation of glutamine and asparagine (+\u0026thinsp;0.98), and oxidation of methionine (+\u0026thinsp;15.99) as variable modifications, while carbamidomethylation of cysteines (+\u0026thinsp;57.02) was set as a fixed modification. were removed from the list. A total of 2,921 proteins were identified and annotated using BLAST in Phytozome, the Plant Comparative Genomics portal of the Department of Energy's Joint Genome Institute (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Goodstein et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Keratins, trypsin, and other common contaminants were not considered. Intensity-based absolute quantification (iBAQ) values were used to reflect protein abundances. Each profile was scaled to its own maximum, sorted by average-linkage hierarchical clustering with centered Pearson distance and displayed as black, yellow, and red heat maps in Microsoft Excel 2019. Further analysis was performed by manual inspection of the obtained profiles, and the iBAQ values were re-normalized within specific apparent molecular mass ranges. Bovine heart mitochondria (BHM) solubilized under the same conditions as \u003cem\u003eP. parva\u003c/em\u003e mitochondria were used for mass calibration of the 3\u0026ndash;16% BN-PAGE.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e6) In silico modelling\u003c/h3\u003e\n\u003cp\u003eThe 3D structural models of \u003cem\u003eP. parva\u003c/em\u003e complexes III and IV were built using the SWISS-MODEL tool (Waterhouse, et al, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), based on the structures of \u003cem\u003eC. reinhardtii\u003c/em\u003e complex III (PDB 9F5Z) and complex IV (PDB 9F60) respectively (Waltz, et al, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The structure of \u003cem\u003eP. parva\u003c/em\u003e complex I (PDB 7ARD) and the models of complex III and complex IV were assembled into the EMD 50210 electron density map and the PDB 9F62 structure of the \u003cem\u003eC. reinhardtii\u003c/em\u003e respiratory supercomplexes (Waltz, et al, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) using ChimeraX 1.9 (Meng, et al, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTo investigate the supramolecular organization of the OXPHOS complexes in \u003cem\u003eP. parva\u003c/em\u003e, isolated mitochondria solubilized with LM or GDN were subjected to BN-PAGE. The previously characterized pattern of algal OXPHOS complexes solubilized with LM and resolved in 3\u0026ndash;10% acrylamide gradient gels stained with Coomassie blue exhibited three main bands corresponding to dimeric complex V (V\u003csub\u003e2\u003c/sub\u003e), complex I (I), dimeric complex III (III\u003csub\u003e2\u003c/sub\u003e). Four weaker bands were also noticed, two from oligomeric forms of complex V (V\u003csub\u003e4\u003c/sub\u003e and V\u003csub\u003e6\u003c/sub\u003e) and two representing monomeric and dimeric complex IV (IV and IV\u003csub\u003e2\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA lane 1). GDN solubilized mitochondria exhibited less V\u003csub\u003e2\u003c/sub\u003e but higher proportions of the oligomeric forms V\u003csub\u003e4\u003c/sub\u003e and V\u003csub\u003e6\u003c/sub\u003e. This was evident in both Coomassie Blue and ATPase activity staining, indicating that the milder detergent GDN preserves more complex-complex interactions than LM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, lanes 2 and 5). While both GDN and LM led to a similar band pattern of the complex V forms, the respiratory complexes displayed greater variation in migration. \u003cem\u003eIn-gel\u003c/em\u003e activity staining of complexes I and IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, lanes 3 and 4) evidenced their presence in several supramolecular associations. Further resolution of GDN-solubilized samples by 2D-BN-PAGE in the presence of LM, revealed that V\u003csub\u003e6\u003c/sub\u003e dissociated into V\u003csub\u003e4\u003c/sub\u003e, V\u003csub\u003e2\u003c/sub\u003e, and V; V\u003csub\u003e4\u003c/sub\u003e was partially conserved, but also dissociated into V\u003csub\u003e2\u003c/sub\u003e and V; and lastly, V\u003csub\u003e2\u003c/sub\u003e fully dissociated to V. Complex I was found to be present in three larger super-complexes while complex IV appeared in four larger oligomeric forms. Furthermore, complex III\u003csub\u003e2\u003c/sub\u003e was found in three oligomeric states. In summary, these results indicate four main oligomeric associations of the respiratory complexes: III\u003csub\u003e2\u003c/sub\u003e-IV, I-III\u003csub\u003e2\u003c/sub\u003e, I-III\u003csub\u003e2\u003c/sub\u003e-IV\u003csub\u003ex\u003c/sub\u003e and I-III\u003csub\u003e2\u003c/sub\u003e-IV\u003csub\u003ey\u003c/sub\u003e (where x and y represent uncertain stoichiometries, difficult to define by this approach) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFast Protein Liquid Chromatography (FPLC) using either anion exchange or size-exclusion columns allowed us to identify different super complexes in GDN-solubilized Polytomella mitochondria. In the first case, protein complexes were eluted with a continuous gradient from 0 to 500 mM NaCl, giving rise to the elution profile shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. Collected fractions were subjected to BN-PAGE and \u003cem\u003ein-gel\u003c/em\u003e activity staining for both complexes I and IV. The ATP synthase oligomeric forms V\u003csub\u003e2\u003c/sub\u003e, V\u003csub\u003e4\u003c/sub\u003e and V\u003csub\u003e6\u003c/sub\u003e eluted earlier than respiratory complexes I, III and IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and their supramolecular associations, here identified as III\u003csub\u003e2\u003c/sub\u003e-IV, I-III\u003csub\u003e2\u003c/sub\u003e, and I-III\u003csub\u003e2\u003c/sub\u003e-IV\u003csub\u003ex\u003c/sub\u003e. In the second case, our tandem size exclusion chromatography setup allowed separation of proteins ranging 5\u0026ndash;5000 kDa. The elution pattern shows the sequential separation of V\u003csub\u003e6\u003c/sub\u003e, V\u003csub\u003e4\u003c/sub\u003e and V\u003csub\u003e2\u003c/sub\u003e oligomers followed by the supramolecular associations of respiratory complexes I-III\u003csub\u003e2\u003c/sub\u003e and III\u003csub\u003e2\u003c/sub\u003e-IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further explore the effect of the detergents GDN, digitonin, and LM on mitochondria solubilization and super-complex stability, composition, and abundance, complexome profiling (CP) was performed. This approach involves separating mitochondrial proteins by native electrophoresis, fractionating entire gel lanes, and identifying proteins in each individual fraction by tandem mass spectrometry followed by abundance pattern\u0026ndash;based hierarchical clustering (Heide et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Cabrera-Orefice et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). CP enables unbiased and comprehensive identification of potential protein\u0026ndash;protein interactions.\u003c/p\u003e\u003cp\u003eComplexome profiling analysis allowed us to identify around 2,900 proteins, among which we found most of the subunits that form part of the mitochondrial respiratory complexes. For complex I, 38 out of the 51 subunits were identified; for complex III, 7 out of 10; and for complex IV, 9 out of 12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary Table S2). It should be noted that the subunits that cannot be identified, such as cytochrome \u003cem\u003eb\u003c/em\u003e of complex III, or some subunits belonging to the P distal module of complex I, are highly hydrophobic membrane proteins, which makes them difficult to digest with trypsin and subsequently to be identified by MS. On the other hand, all the subunits of complex II and complex V could be identified (4 for complex II and 18 for complex V) (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In addition, this experimental approach allowed us to identify 5 isoforms of previously characterized proteins (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Supplementary Figure S2). For complex I, two isoforms were identified: one of NUOP4 belonging to the P distal module, and another for gamma carbonic anhydrase (CA3). In complex IV, the presence of an isoform of the Cox6b subunit was found (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, highlighted in red). In the case of complex II and complex V, which are not constituents of the supercomplexes, an isoform of subunit D and an isoform of subunit Asa9 were identified, respectively (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Under the growth conditions for the colorless alga, using acetate as carbon source, all these proteins are produced in different proportions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWith GDN-solubilized mitochondria, the migration of complexes I, III and IV and its supramolecular associations were followed by mass spectrometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Normalization of the main peaks representing the supercomplexes allowed the identification of three different organizations: 1-IV (~\u0026thinsp;1,188 kDa), I-III\u003csub\u003e4\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e (~\u0026thinsp;2,320 kDa) and I\u003csub\u003e2\u003c/sub\u003e-III\u003csub\u003e4\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e (~\u0026thinsp;3,130 kDa) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, inset).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast, digitonin-solubilized mitochondria subjected to BN-PAGE in the same conditions seem to partially disrupt supramolecular associations, since higher levels of free complexes I, III and IV were present as compared with GDN-solubilized mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In these conditions, the three following associations were identified: I-IV (~\u0026thinsp;1,188 kDa), I-III\u003csub\u003e2\u003c/sub\u003e-IV (~\u0026thinsp;1,445 kDa) and I\u003csub\u003e2\u003c/sub\u003e-III\u003csub\u003e2\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e (~\u0026thinsp;2,532 kDa) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, inset).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFinally, and as expected, LM-solubilized mitochondria displayed a polypeptide pattern exhibiting mainly free respiratory complexes and negligible amounts of supercomplexes and (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEarlier studies recognized the mitochondrial ATP synthase of the colorless alga \u003cem\u003eP. parva\u003c/em\u003e as a stable dimer resistant to dissociation by detergents such as LM (V\u0026aacute;zquez-Acevedo et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; van Lis et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The enzyme contains conserved subunits within its rotary and catalytic domains but also incorporates several atypical subunits known as ASA subunits, which form the peripheral arms of the complex and participate in the dimerization of the enzyme. In addition to its dimeric form, higher-order oligomers\u0026mdash;including tetramers and hexamers\u0026mdash;were observed by BN-PAGE analysis (Miranda-Astudillo et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Low-resolution imaging further revealed the overall silhouette of the dimer, characterized by robust peripheral stalks (Dudkina et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). More recently, high-resolution 3D structures have provided detailed insights into subunit interactions and the mechanism of rotational catalysis (Allegretti et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Murphy et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eElectron microscopy and cryo-electron tomography revealed the presence of densely packed cristae in Polytomella mitochondria (Dudkina et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Using electron cryo-tomography and subtomogram averaging at resolutions of up to 4.2 \u0026Aring;, ATP synthase could be visualized \u003cem\u003ein vivo\u003c/em\u003e following rapid freezing of actively growing algal cells. The 3D reconstruction of a mitochondrion showed series of ATP synthase dimers adopting a near-helical arrangement, with multiple parallel rows organized within the disk-shaped cristae (Dietrich et al., 2014). Additionally, individual ATP synthases were observed in proximity to the cristae junctions. As shown here, some of these higher V\u003csub\u003e2\u003c/sub\u003e oligomeric states can be preserved after mild extraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and these associations remain stable after size-exclusion or anion-exchange chromatography (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe existence of respiratory super complexes in Polytomella was suggested by the identification in LM solubilized mitochondria of I-IV\u003csub\u003e6\u003c/sub\u003e, I-III\u003csub\u003e4\u003c/sub\u003e, and I-IV associations by BN-PAGE and the \u003cem\u003ein vitro\u003c/em\u003e reconstitution of supercomplexes III\u003csub\u003e2\u003c/sub\u003e-IV and I-III\u003csub\u003e2\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e by ion exchange chromatography (Miranda-Astudillo et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Here, we demonstrate the presence of the super complex III\u003csub\u003e2\u003c/sub\u003e-IV in GDN-solubilized mitochondrial membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) as well as the existence of dimeric complex IV and of at least one respirasome.\u003c/p\u003e\u003cp\u003eRecent advances in \u003cem\u003ein situ\u003c/em\u003e cryo-electron microscopy have provided direct images of mitochondria, enabling the determination of the 3D structures of respiratory supercomplexes in their native states from both porcine (Zheng et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)d \u003cem\u003ereinhardtii\u003c/em\u003e mitochondria (Waltz et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Therefore, there is no doubt about the existence of dynamical super-structures in the mitochondrial inner membrane that exhibit a huge diversity of associations and stoichiometries among different species (Eldeeb et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Guan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In mammalian mitochondria, four distinct super complex organizations have been identified: I-III₂-IV (~\u0026thinsp;1,980 kDa), I₂-III₂-IV₂ (~\u0026thinsp;3,380 kDa), I-III₂-IV₂ (~\u0026thinsp;2,128 kDa), and I₂-III₄-IV₂ (~\u0026thinsp;3,906 kDa). Notably, the latter two arrangements had not previously been detected \u003cem\u003ein vitro\u003c/em\u003e using mild non-ionic detergents for extraction (Vercellino and Sazanov, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), highlighting the unique ability of \u003cem\u003ein situ\u003c/em\u003e cryo-electron microscopy to reveal otherwise elusive associations. In contrast, the \u003cem\u003eC. reinhardtii\u003c/em\u003e respirasome displayed an \u003cem\u003ein situ\u003c/em\u003e I₂-III₄-IV₆ arrangement (Waltz et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), which differs from supercomplexes reported in other organisms, including \u003cem\u003eSus scrofa\u003c/em\u003e [I-III₂-IV₂ (PDB 8UGI), I-III₂-IV₂ (PDB 8UGJ), I₂-III₂-IV₂ (PDB 8UGN), I₂-III₄-IV₂ (PDB 8UGR)] (Zheng et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2024\u003c/span\u003e); \u003cem\u003eMus musculus\u003c/em\u003e [I-III₂-IV (PDB 8PW7), I-III₂-IV₂ (PDB 8PW5)] (Vercellino and Sazanov, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2024\u003c/span\u003e); \u003cem\u003eEuglena gracilis\u003c/em\u003e [I-III₂-IV] (He et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e); and \u003cem\u003eTetrahymena thermophila\u003c/em\u003e [I₂-II₂-III₄-IV₂ (PDB 8GYM)] (Han et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among them, the \u003cem\u003eT. thermophila\u003c/em\u003e super complex is the only respirasome characterized so far to include complex II, a feature attributed to lineage-specific subunits in both complexes II and IV, that promote an unusually large supramolecular assembly exceeding in size the I₂-III₄-IV₆ arrangement of the \u003cem\u003eC. reinhardtii\u003c/em\u003e respirasome.\u003c/p\u003e\u003cp\u003eThese different supramolecular organizations may influence local membrane curvature, rendering it more convex or concave depending on their organization and stoichiometry (Zheng et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Putative respiratory strings have been proposed to be present in mammalian, chlorophycean and plant mitochondria, where I\u003csub\u003e2\u003c/sub\u003e-III\u003csub\u003e2\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e and I\u003csub\u003e2\u003c/sub\u003e-III\u003csub\u003e4\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e associations should work as building blocks for larger circular or linear organizations, respectively (Bultema et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Letts et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Miranda-Astudillo et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To date, no direct evidence of these large associations has been confirmed using \u003cem\u003ein-situ\u003c/em\u003e studies (Davies et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Dietrich et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; M\u0026uuml;hleip et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zheng et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Electron cryotomography of \u003cem\u003eC. reinhardtii\u003c/em\u003e mitochondria further revealed a spatial segregation within cristae of respiratory complexes and rows of ATP synthase dimers, as has been reported in other systems. Biochemical isolation and characterization of the \u003cem\u003eC. reinhardtii\u003c/em\u003e respirasome yielded a super complex with an I-III₂-IV₂ composition, whose structure was resolved at 2.8 \u0026Aring; using single-particle cryo-EM (Waltz et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Apart from the absence of a dimeric complex IV, this isolated form appears to represent one half of the intact respirasome observed \u003cem\u003ein situ\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eMulticellular chlorophycean algae such as Volvox diverged from their unicellular ancestors at least 200\u0026nbsp;million years ago (MYA) (Herron et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). It can be inferred that the Polytomella lineage separated from the Chlamydomonas less than 200 MYA. The loss of photosynthesis in Polytomella may have driven extensive changes in chloroplast composition and function, ultimately leading to the emergence of colorless plastids in this genus (Figueroa-Mart\u0026iacute;nez et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In contrast, mitochondrial evolution appears to have followed a more conservative trajectory, preserving many features of its green algal relatives. The stoichiometry of the \u003cem\u003eP. parva\u003c/em\u003e respirasomes characterized in this study may reflect supramolecular associations derived from the dissociation of a larger, Chlamydomonas-like I\u003csub\u003e2\u003c/sub\u003e-III\u003csub\u003e4\u003c/sub\u003e-IV\u003csub\u003e6\u003c/sub\u003e respirasome. Nevertheless, we did not detect \u003cem\u003ein vitro\u003c/em\u003e a complete respirasome comparable to the one observed \u003cem\u003ein situ\u003c/em\u003e in the green algal mitochondria. This discrepancy may arise from several factors, including the disruptive effects of detergent extraction on complex organization or the loss of structural lipids such as cardiolipin (Walz et al., 2025). Alternatively, it is possible that the various supra complex species identified by complexome profiling assemble in a context-dependent manner, reflecting differential associations that adapt to the metabolic requirements of the cell.\u003c/p\u003e\u003cp\u003eIn the \u003cem\u003eC. reinhardtii\u003c/em\u003e respirasome, four contact regions have been identified between complexes I and III, and two between complexes I and IV. It was proposed that the I/III super complex is the most stable, whereas complex IV is the least resistant to biochemical purification (Waltz et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In the present work, we isolated an I\u003csub\u003e2\u003c/sub\u003e-III\u003csub\u003e4\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e super complex from \u003cem\u003eP. parva\u003c/em\u003e, identifying each of the orthologous subunits that form the complexes taking as a reference the subunits that make up the same complexes in \u003cem\u003eC. reinhardtii\u003c/em\u003e (Supplementary Table S3). However, it is likely that up to four complex IV units were lost during the isolation procedure. We propose that \u003cem\u003eP. parva\u003c/em\u003e contains a higher-order I\u003csub\u003e2\u003c/sub\u003e-III\u003csub\u003e4\u003c/sub\u003e-IV\u003csub\u003e6\u003c/sub\u003e respirasome which, upon detergent solubilization of mitochondria, may dissociate into the various single complexes and supercomplexes detected in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). While differences between isolated supercomplexes and their counterparts observed \u003cem\u003ein situ\u003c/em\u003e are well recognized, multiple supramolecular arrangements with varying stoichiometries can coexist within the same organism (Zheng et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Accordingly, the I\u003csub\u003e2\u003c/sub\u003e-III\u003csub\u003e4\u003c/sub\u003e-IV\u003csub\u003e2\u003c/sub\u003e super complex characterized here may also represent a physiologically relevant organization.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding was provided with grants from Programa de Apoyo a Proyectos de Investigaci\u0026oacute;n e Innovaci\u0026oacute;n Tecnol\u0026oacute;gica (PAPIIT-DGAPA-UNAM) numbers IN204426 (to D.G-H.) and IA204524 (to H.V.M-A.). D.G.-H. also acknowledges financial support from grant CBF-2025-I-258 from Secretar\u0026iacute;a de Ciencia, Humanidades, Tecnolog\u0026iacute;a e Innovaci\u0026oacute;n, Mexico (SECIHTI). H.V.M.-A. also acknowledges financial support from the Instituto de Investigaciones Biom\u0026eacute;dicas under the Institutional Program [\u0026ldquo;Production of biomolecules of biomedical interest in microorganisms\u0026rdquo;]. The technical support of QBP Miriam V\u0026aacute;zquez-Acevedo (IFC, UNAM) and of PhD Toshiko Takahashi \u0026Iacute;\u0026ntilde;iguez (IIBO, UNAM) is also acknowledged. M. O-C. and S. F-H. are Ph.D. students of the Programa de Doctorado en Ciencias Bioqu\u0026iacute;micas de la Universidad Nacional Aut\u0026oacute;noma de M\u0026eacute;xico (UNAM) and have received fellowships from SECIHTI 710287 and 1146629 respectively. A. R-L is a doctoral student from the Programa de Doctorado en Ciencias Biom\u0026eacute;dicas, UNAM and has received a SECIHTI fellowship 927622.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAllegretti M, Klusch N, Mills DJ, Vonck J, Kühlbrandt W, Davies KM (2015) Horizontal membrane-intrinsic α-helices in the stator a-subunit of an F-type ATP synthase. Nature 521:237-240. doi: 10.1038/nature14185. \u003c/li\u003e\n \u003cli\u003eBerndtsson J, Kohler A, Rathore S, Marin-Buera L, Dawitz H, Diessl J, Kohler V, Barrientos A, Büttner S, Fontanesi F, Ott M (2020) Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance. EMBO Rep 21:e51015. doi: 10.15252/embr.202051015. \u003c/li\u003e\n \u003cli\u003eBlum TB, Hahn A, Meier T, Davies KM, Kühlbrandt W (2019) Dimers of mitochondrial ATP synthase induce membrane curvature and self-assemble into rows. 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Nature 631: 232–239. https://doi.org/10.1038/s41586-024-07488-9\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-bioenergetics-and-biomembranes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jobb","sideBox":"Learn more about [Journal of Bioenergetics and Biomembranes](http://link.springer.com/journal/10863)","snPcode":"10863","submissionUrl":"https://submission.nature.com/new-submission/10863/3","title":"Journal of Bioenergetics and Biomembranes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chlorophycean algae, oxidative phosphorylation, F1FO ATP synthase, oligomeric complex V, mitochondrial supercomplexes","lastPublishedDoi":"10.21203/rs.3.rs-7871941/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7871941/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChlorophycean algae of the genera Chlamydomonas and Polytomella share a common photosynthetic ancestor. However, members of the Polytomella lineage have adopted a heterotrophic lifestyle, having lost the photosynthetic apparatus and relying instead on acetate or ethanol as carbon sources, with energy production centered on oxidative phosphorylation (OXPHOS). In this study, we investigated the composition of the mitochondrial supercomplexes of the colorless alga \u003cem\u003ePolytomella parva\u003c/em\u003e. OXPHOS complexes were solubilized using mild detergents such as glycol-diosgenin and digitonin, followed by separation of protein assemblies via Blue Native electrophoresis and Fast Protein Liquid Chromatography (FPLC). Additionally, complexome profiling of solubilized mitochondria resolved by Blue Native Gel Electrophoresis was carried out. The resulting data indicate that the OXPHOS supercomplexes of Polytomella closely resemble those observed \u003cem\u003ein situ\u003c/em\u003e in the mitochondria of its green relative \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e, as revealed by electron cryo-tomography and subtomogram averaging.\u003c/p\u003e","manuscriptTitle":"Mitochondrial super complexes in the colorless chlorophycean alga Polytomella parva","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 13:50:34","doi":"10.21203/rs.3.rs-7871941/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-12T18:56:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-12T17:34:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"304161889811966580622029802337585636824","date":"2025-11-09T08:22:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-06T14:17:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171496367585254872326988025907721227128","date":"2025-11-03T14:17:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"269964377160217935413814192605594716131","date":"2025-10-27T09:04:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-21T15:23:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-21T11:00:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-21T10:59:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Bioenergetics and Biomembranes","date":"2025-10-15T23:11:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-bioenergetics-and-biomembranes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jobb","sideBox":"Learn more about [Journal of Bioenergetics and Biomembranes](http://link.springer.com/journal/10863)","snPcode":"10863","submissionUrl":"https://submission.nature.com/new-submission/10863/3","title":"Journal of Bioenergetics and Biomembranes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5ccb1024-34bd-4c24-9f98-6cf9607c2fac","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-18T19:53:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-31 13:50:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7871941","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7871941","identity":"rs-7871941","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}