Arrangement of the Respiratory Chain Complexes in Saccharomyces cerevisiae Supercomplex III₂IV₂ Revealed by Single Particle Cryo-Electron Microscopy^*

Yeast Strain and Growth

S. cerevisiae strain USY00b (Mat α, ade2-1, his3-11-15, leu2-3-112, trp1-1, ura3-52, can1R-100, atp2::LEU2, TRP1::ATP2-His₆) was supplied by Dr. David Mueller (Rosalind Franklin University of Medicine and Science, North Chicago, IL) and expresses the F_oF₁-ATPase β-subunit with a His₆ extension (27). The strain was cultivated in YP-lactate growth medium (pH 5.6) containing 1% yeast extract, 2% peptone, and 3.7% of lactic acid. The cells were harvested (A₆₀₀ of 2.0) by centrifugation and washed with cold TBS buffer (50 mm Tris-HCl, pH 7.4 containing 150 mm NaCl).

Isolation of Mitochondria and Mitoplasts

Mitochondria were isolated from spheroplasts of yeast cells as previously described (10) except that 3 mg of Zymolase-20T per g of cells were used, and incubation time was 90 min. Isolated mitochondria were further purified by centrifugation at 30,000 rpm (SW41 Ti) at 4 °C for 90 min through a density gradient containing 60, 32, 23, and 15% layers of sucrose in 15 mm Tris-HCl buffer, pH 7.4 and 20 mm KCl. Mitochondria were either used immediately or flash-frozen with liquid N₂ and stored at −80 °C. Mitoplasts (mitochondria without the outer mitochondrial membrane) for phospholipid analysis were prepared from purified mitochondria according to the protocol described in (10).

Purification of Mitochondrial Supercomplex III₂IV₂

Isolated mitochondria (8 mg of protein) were suspended in the 1 ml of lysis buffer containing 2% (w/v) digitonin (Invitrogen), 50 mm potassium acetate, 10% glycerol, 1:50 volume Protease Inhibitor mixture set III (Calbiochem), 1.5 mm phenylmethylsulfonyl fluoride, and 30 mm HEPES-KOH (pH 7.4) for 1 h at 4 °C with gentle shaking. After incubation the lysate was centrifuged at 4 °C for 20 min at 45,000 rpm (TLA55 rotor), and the supernatant was incubated with 1 μl of TBS-equilibrated magnetized Cobalt Beads (Dynabeads TALON catalogue 101.02D, Invitrogen) per 2.2 μl of lysate to remove F_oF₁-ATPase. Incubation of 1 ml of lysate with magnetic beads was performed for 45 min at 4 °C with constant shaking. After incubation the beads were removed using a magnetic separator. The supernatant (1 ml) was immediately transferred to an 8 ml sucrose gradient (0.75 m to 1.5 m sucrose in 15 mm Tris-HCl (pH 7.2), 20 mm KCl, and 0.4% digitonin) and centrifuged at 4 °C for 20 h at 30,000 rpm (TLS-55 swinging bucket rotor). Fractions (80–100 μl) from the gradient were analyzed by Blue Native polyacrylamide gel electrophoresis (BN-PAGE) and Western blotting.

Analysis of the Supercomplex

Aliquots of fractions obtained after sucrose gradient centrifugation were subjected to BN-PAGE in a 3–12% acrylamide gradient gel in Bis-Tris buffer (Invitrogen) according to manufacturer's instructions. After electrophoresis gels were either stained with Bio-Safe Coomassie G-250 (Bio-Rad) or used for Western blotting analysis as we described earlier (28) with primary monoclonal antibody (generous gift of Dr. Bernard L. Trumpower, Dartmouth University, Hanover, NH) to the Qcr7 subunit of complex III and secondary anti-mouse antibody (Stabilized Peroxidase Conjugate, Goat anti-mouse, Thermo Fisher). SuperSignal Western Femto Sensitivity kit was used to develop the signal, which was captured using a Fluro-S Max multi imager (29). For Western blotting with primary antibody against yeast cytochrome c (generous gift of Dr. Alexander Tzagoloff, Columbia University, New York, NY) followed by goat anti-rabbit secondary antibody (Stabilized Peroxidase Conjugate, goat anti-rabbit, Thermo Fisher), the purified supercomplex was separated by NuPAGE in a 4–12% acrylamide gradient gel in Bis-Tris buffer with NuPAGE MES SDS Running buffer (Invitrogen) according to the manufacturer's instructions. Protein concentrations were determined using the BCA protein assay kit (Thermo Scientific) according to manufacturer's instructions.

Measurements of Supercomplex Enzymatic Activity

Respiratory activity of the purified supercomplex III₂IV₂ was measured using a Clark oxygen electrode as KCN-sensitive oxygen consumption with NaBH₄-reduced decylubiquinone (Sigma), QH₂, as substrate at 30 °C. Rates were determined from the slope of a plot of O₂ concentration versus time. Supercomplex purified in the sucrose gradient (about 55 μg of protein) was incubated in 50 mm Tris-HCl buffer, pH 7.0, 100 mm KCl, 0.13 m sucrose, 0.05% digitonin in a final volume of 3 ml. QH₂ (40 μm) was added to start the reaction, and oxygen consumption was stopped by addition of 50 μm KCN.

Phospholipid Extraction from Supercomplex for Mass Spectrometry

Phospholipids were extracted from the supercomplex and mitoplasts (∼20 μg of protein) with a mixture of methanol:chloroform:0.1 n HCl (containing 11 mm ammonium acetate) in a proportion of 1:1:0.9. Samples were vortexed for 1 min and centrifuged at 3000 × g. The organic layer was transferred to a glass tube and dried under a stream of N₂. Dried phospholipid samples were resuspended in 100 μl of hexane-isopropanol (30:40. v/v) (30). For quantification of CL using mass spectrometry, 0.8 nmol of 1,1′, 2,2′-tetramyristoyl cardiolipin (CL (14:0)₄) (Avanti Polar Lipids, Alabaster, AL) was added as an internal standard prior to extraction of lipids.

ESI-MS Analysis of CL

ESI-MS was performed as previously described (30). Samples for CL quantitation were injected into a normal-phase HPLC column (Luna 5 μm silica 100 Å, 2.0 × 150 mm column; Phenomenex) coupled to an API 3200 mass spectrometer running in negative ion mode. Negative ion ESI was carried out at −4,000 V, with declustering potential of −100 V, focusing potential of −350 V, and entrance potential of −10 V. The solvent system consisted of hexane:isopropanol:20 mm ammonium acetate in water, pH 5.5, in a ratio of 30:40:7 (v/v/v) for solvent A and hexane:isopropanol (30:40, v/v) for solvent B. In separate runs 0.34, 0.69 or 1.37 nmol of CL containing four oleic acids (CL (18:1)₄) (Avanti Polar Lipids, Alabaster, AL) as a reference and 0.8 nmol of CL ((14:1)₄) as an internal standard were used to establish the standard curve. There was a linear relationship between the ratio of the negative molecular ion ([M-H]⁻) current from CL (18:1)₄ (reference standard), and the [M-H]⁻ from the internal standard, CL (14:0)₄, as a function of the concentration of CL (18:1)₄. The nmol of individual molecular species of CL in analyzed samples of the supercomplex or mitoplasts were calculated by determining the ratio of the peak area of the CL molecular species of interest to the internal standard. This ratio, along with the slope and intercept of the standard curve made with reference standard CL (18:1)₄ was used to calculate nmol of CL as described in (30). Total CL was determined as the sum of the most prevalent CL species. The value in nmol per mg of protein subjected to extraction was calculated. This number represents the number of CL molecules per mol of the supercomplex since 1 nmol of the supercomplex III₂IV₂ is about 1 mg.

Cryo-EM

Selected fractions containing the purified supercomplex III₂IV₂, were combined and used for cryo-EM. Samples were diluted 1:9 in 15 mm Tris-HCl, pH 7.2, and 20 mm KCl buffer to reduce digitonin and sucrose concentrations and deposited on freshly glow discharged (2 min) Quantifoil holey carbon grids (West Chester, PA). Prior to glow discharge, the holey carbon grids were coated with a continuous thin carbon film that was prepared by evaporating carbon on a freshly cleaved mica sheet from a graphite rod using a Denton Vacuum DU-502A Evaporator. Grids were rapidly frozen in liquid ethane using a home made gravity-driven plunger apparatus. Cryo-frozen samples were imaged at liquid nitrogen temperature using a Polara 300 kV electron microscope (FEI Company) using a 4K × 4K CCD camera (TVIPS GmbH, Gauting, Germany). Images were recorded at low dose conditions at 3 to 7 μm underfocus and a ×39,000 nominal magnification.

Data Processing, Map Visualization, and Analysis

All image processing steps were done using the SPARX software package (31). From 200 micrographs a total of 23,715 particles were manually selected and windowed using e2boxer.py with a pixel size of 4.58 Å on the specimen scale. Class averages were calculated using the Iterative Stable Alignment and Clustering (ISAC) program, from which 252 averages were produced (32). By visual inspection, we selected from this set a subset of 230 plausible views of the supercomplex III₂IV₂.

An initial model was calculated with the three-dimensional projection matching method using the selected class averages and starting from a template structure constructed using the electron density map obtained from the atomic model of S. cerevisiae complex III (PDB accession number 1KYO) embedded in an oblate spheroid (lens-shape) representing the undefined position of two complexes IV in the supercomplex (see Fig. 1). Prior to conversion to an electron density map, the PDB file of the yeast cytochrome bc₁ complex (complex III) was modified by removing chains j, u, k, v, and w. Subsequently, individual projection image-based three-dimensional projection alignment was carried out with enforcement of C2 symmetry of the structure and starting from the model obtained from class averages. Finally, images whose projection directions (as determined by the refinement) were over-represented were removed to obtain a subset of 7,824 images with approximately evenly distributed projection directions and the refinement was continued until we obtained a stable three-dimensional structure at a resolution of 24 Å, as determined by the FSC @ 0.5 test (33). During the refinement the power spectrum of the structure was adjusted to match that of the electron density map calculated from the atomic model of yeast cytochrome bc₁ complex and low-pass filtered with frequency cut-off calculated from the FSC curve. The atomic models of S. cerevisiae complex III (PDB accession numbers 1KYO or 3CX5, both giving the same results) and bovine complex IV (PDB accession number 1OCC) were used for modeling the supercomplex structure. For the former case the PDB file was modified by removing the antibody fragment chains and, unless otherwise indicated, the cytochrome c (chain w) structure; subunit Qcr10p is not present in the PDB file since it was lost prior to structural determination. Only a monomer of the dimeric bovine complex IV was used while chains K and M not present in yeast were removed. The initial approximate placement of each x-ray structure in the supercomplex EM density map was done manually based on distinct structural features and subsequently refined using the UCSF Chimera program by maximization of the local correlation coefficient between the two maps (34).

Purification of the III₂IV₂ Supercomplex

The approach used previously (35) was modified for purification of the active supercomplex III₂IV₂. Extraction of the supercomplex by digitonin and purification using sucrose gradient centrifugation resulted in contamination of the supercomplex with the F_oF₁-ATPase dimer, which has a similar molecular mass. To decrease this contamination, the ratio of digitonin to the protein previously used was 6:1 (wt/wt) to induce dissociation of the F_oF₁-ATPase dimer to monomers. However, these conditions also increased the level of the III₂IV supercomplex trimer, which could not be completely separated from the intact supercomplex in the sucrose gradient (35). Several modifications of the published method were made to eliminate the above contaminants thus improving the quality of material subjected to cryo-EM. Strain USY00b expresses a His₆-tagged β-subunit of F_oF₁-ATPase, which was easily removed by incubation of the digitonin extract with cobalt affinity beads. The strain was grown on lactate-containing media with intense aeration, which resulted in high levels of the III₂IV₂ supercomplex and greatly reduced levels of the III₂IV supercomplex trimer due to higher level of expression of complex IV (24). For extraction of the supercomplex, a ratio of digitonin to protein of 2.5:1 (wt/wt) was used, which also reduced the latter contaminant. Purification of the digitonin extract of mitochondria by sucrose gradient centrifugation after removing F_oF₁-ATPase with cobalt beads resulted in samples containing highly purified III₂IV₂ supercomplex (Fig. 2A).

It was previously demonstrated that the purified mammalian respirasome contained bound CoQ and cytochrome c (20, 36) and can perform NADH oxidation without addition of any external cytochrome c (36). According to negative stain EM and single particle analysis, some percentage of the yeast supercomplex population of the sample purified using a sucrose gradient appears to contain attached cytochrome c (26). SDS-PAGE followed by Western blot analysis with antibody to cytochrome c confirmed the presence of cytochrome c in our purified supercomplex samples (8 different purifications). Since III₂IV₂ represents a part of respirasome lacking dehydrogenases, QH₂ was used to donate electrons to complex III (Complex III contains bound CoQ). Oxygen consumption upon oxidation of QH₂ by the purified supercomplex (Fig. 2B) was recorded without any addition of the external cytochrome c. The process was completely inhibited by 50 μm of KCN, an inhibitor of the complex IV. Activity of the supercomplex sample calculated from the data presented in Fig. 2B was about 240 nmol of O₂ min⁻¹ per mg of protein. Taking into account that the concentration of the supercomplex used in this experiment was about 18 μg per ml (or 18 nm) and that cytochrome c was at sub-saturating levels with respect to the supercomplex, we concluded that the recorded reaction was supported by the channeling of cytochrome c between complexes III and IV in the supercomplex and represents activity of the fraction of supercomplex containing bound cytochrome c. Activity of the supercomplex upon addition of 50 μm of cytochrome c was about 1 μmol of O₂ min⁻¹ per mg of protein, which is comparable with published results (24).

EM and Image Processing

A total of 23,715 particle images of the purified active III₂IV₂ supercomplex were manually selected from 200 micrographs and a total of 252 class averages were calculated from which 230 averages were selected; see Fig. 2 for a typical micrograph. An initial model for projection image-based three-dimensional refinement was determined using an approach based on calculating a three-dimensional map from the 230 selected averages, for which the three-dimensional projection-matching strategy with enforcement of C2 symmetry was employed (37). The reference structure for the procedure was constructed to avoid introducing any bias in the orientation between complexes III and IV in the reconstructed map. Toward this end, we obtained the reference structure by combining the electron density map of complex III converted from the atomic model of yeast cytochrome bc₁ complex and an oblate spheroid as described in “Materials and Methods” (Fig. 1). Furthermore, to determine the self-consistency and reproducibility of the three-dimensional maps computed by this approach, we made ten noise-corrupted models of the oblate spheroid model. Each of the ten noisy models was used as the initial template for three-dimensional projection alignment of the set of 230 selected averages. The resulting ten three-dimensional maps were similar demonstrating that orientation between complexes III and IV was reliably established using this approach. Using the initial model as a starting structure and a subset of projection images (see “Materials and Methods” for details), a three-dimensional map (Fig. 3, A–D) with a resolution of 24 Å was obtained.

A pseudo-atomic model of the three-dimensional structure of the supercomplex was constructed by superimposing the x-ray structures of complex III (S. cerevisiae, PDB 3CX5) and complex IV (bovine, PDB 1OCC, monomer) (Fig. 4). The protruding densities at the top center of the supercomplex correspond to the position of Cor1 and Cor2 subunits in complex III (Figs. 4A and 5A). Our three-dimensional map (Fig. 4) and the derived pseudo-atomic model (Fig. 5) confirm the previously suggested overall organization of the S. cerevisiae supercomplex, which was based on an arrangement inferred from two-dimensional class averages obtained by processing of negative stain data (26). The main finding was that each of the two individual complexes IV interacts with complex III through convex surface of IV, which is opposite to the interaction surface between the cytochrome c oxidase monomers in the dimer of complex IV present in the crystal structure (38). However, the three-dimensional structure obtained by cryo-EM suggests some differences in orientation of IV relative to III (Fig. 5) compared with conclusions based on the two-dimensional work (26). The distance between cytochrome c binding sites on cytochrome c₁ subunit of complex III and subunit II of complex IV is about 6 nm (Fig. 5) versus 4.0 nm as previously reported. This larger distance would still enable channeling of cytochrome c between these sites in the supercomplex. Complexes III and IV are closer to each other at the aqueous-membrane interface exposed to the mitochondrial inter-membrane space, while larger spacing between III and IV that is seen in the hydrophobic lipid bilayer is consistent with what is observed in the bovine supercomplex (20, 22). This “gap” (spacing) is sufficiently spacious for insertion of lipids between the individual complexes in the supercomplex.

Determination and Quantification of CL in the Supercomplex

The phospholipid CL is required for formation of the stable III₂IV₂ supercomplex (10, 14, 15) and therefore should be associated with the supercomplex. We determined the number of CL molecules and their fatty acid species distribution in the supercomplex. CL associated with the purified supercomplex showed the same proportion between the five major CL species (with values of singly ionized CL of m/z 1,344, 1,372, 1,400, 1,428, and 1,456) as CL found in mitoplasts (representing the inner mitochondrial membrane) (Fig. 6). These numbers correspond to CL species with acyl chains (16:1)₄, (16:1)₃(18:1)₁, (16:1)₂ (18:1)₂, and (18:1)₄, respectively. Thus the major CL species contain only monounsaturated 16:1 and 18:1 acyl chains, which corresponds to published results for yeast mitochondria (39). Total CL was determined as the sum of these five prevalent species. By using CL (14:0)₄ as an internal standard and CL (18:1)₄ as a reference standard (see “Materials and Methods”) 40–60 mol of CL per mol of the yeast supercomplex were determined in two independent experiments.