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First published online April 2, 2004; 10.1104/pp.103.038018 Plant Physiology 134:1450-1459 (2004) © 2004 American Society of Plant Biologists Identification and Characterization of Respirasomes in Potato Mitochondria1Institut für Angewandte Genetik, Universität Hannover, D–30419 Hannover, Germany
Plant mitochondria were previously shown to comprise respiratory supercomplexes containing cytochrome c reductase (complex III) and NADH dehydrogenase (complex I) of I1III2 and I2III4 composition. Here we report the discovery of additional supercomplexes in potato (Solanum tuberosum) mitochondria, which are of lower abundance and include cytochrome c oxidase (complex IV). Highly active mitochondria were isolated from potato tubers and stems, solubilized by digitonin, and subsequently analyzed by Blue-native (BN) polyacrylamide gel electrophoresis (PAGE). Visualization of supercomplexes by in-gel activity stains for complex IV revealed five novel supercomplexes of 850, 1,200, 1,850, 2,200, and 3,000 kD in potato tuber mitochondria. These supercomplexes have III2IV1, III2IV2, I1III2IV1, I1III2IV2, and I1III2IV4 compositions as shown by two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE and BN/BN-PAGE in combination with activity stains for cytochrome c oxidase. Potato stem mitochondria include similar supercomplexes, but complex IV is partially present in a smaller version that lacks the Cox6b protein and possibly other subunits. However, in mitochondria from potato tubers and stems, about 90% of complex IV was present in monomeric form. It was suggested that the I1III2IV4 supercomplex represents a basic unit for respiration in mammalian mitochondria termed respirasome. Respirasomes also occur in potato mitochondria but were of low concentrations under all conditions applied. We speculate that respirasomes are more abundant under in vivo conditions.
Prerequisite for oxidative phosphorylation (OXPHOS) in mitochondria are five protein complexes termed NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and ATP synthase (complex V). These protein complexes can be separated by biochemical procedures and are well characterized for several organisms. However, there is mounting evidence that in vivo these protein complexes specifically interact forming supermolecular structures called supercomplexes: (1) purification protocols for individual OXPHOS complexes sometimes lead to the isolation of stoichiometric assemblies of two or more complexes which are functionally active (Hatefi et al., 1961  ; Hatefi and Rieske, 1967); (2) stable and enzymatically active supercomplexes can be reconstituted upon mixture of complexes I and III (Fowler and Hatefi, 1961; Fowler and Richardson, 1963; Hatefi, 1978; Ragan and Heron, 1978); (3) respiratory protein complexes from several bacteria were found to form specific supermolecular structures (Berry and Trumpower, 1985; Sone et al., 1987; Iwasaki et al., 1995; Niebisch and Bott, 2003); (4) inhibitor titration experiments reveal that the respiratory chain of yeast (Saccharomyces cerevisiae) behaves like a single functional unit (Boumans et al., 1998); and (5) flux control experiments indicate specific interactions of respiratory protein complexes (Genova et al., 2003). Several physiological roles were proposed for these respiratory supercomplexes, like substrate channeling, catalytic enhancement, protection of reactive reaction intermediates, and stabilization of individual protein complexes (Schägger and Pfeiffer, 2000; Genova et al., 2003).
Recently, characterization of mitochondrial supercomplexes was very much facilitated by the introduction of a novel experimental strategy which is based on protein solubilizations using mild nonionic detergents and separation of the solubilized protein complexes by Blue-native (BN) gel electrophoresis or gel chromatography (Arnold et al., 1998
In yeast, dimeric complex III (this protein complex always is dimeric for functional reasons) forms supercomplexes with one or two copies of complex IV. Furthermore, complex V was shown to partially occur in a dimeric state, which includes some dimer-specific subunits. In contrast, complex II from yeast does not form part of supermolecular structures under all experimental conditions applied. In beef, the complexes III2 and I form a supercomplex. Additionally, this supercomplex can include one to four copies of complex IV. The resulting large structures are called respirasomes, because they can autonomously carry out respiration in the presence of cytochrome c and ubiquinone (Schägger and Pfeiffer, 2000
Meanwhile, protein solubilizations using nonionic detergents and separations of solubilized protein complexes by BN-PAGE were used to systematically investigate the structure of the OXPHOS system of plants (Eubel et al., 2003 Here we report a continuation of our efforts to carefully characterize the supermolecular structure of the OXPHOS system of plant mitochondria. Using highly active mitochondria isolated from freshly harvested potato tubers, five additional supercomplexes of about 850, 1,150, 1,850, 2,200, and 3,000 kD are visible on BN gels. All five protein complexes include complex IVa as shown by one-dimensional (1D) BN-PAGE, two-dimensional (2D) BN/SDS-PAGE, and 2D BN/BN-PAGE in combination with in-gel activity measurements for cytochrome c oxidase. The novel supercomplexes are of comparatively low abundance and have III2IV1, III2IV2, I1III2IV1, I1III2IV2, and I1III2IV4 compositions. Slightly smaller versions of these protein complexes occur in potato stem mitochondria, which include complex IVb instead of complex IVa. Hence, the OXPHOS complexes of plant mitochondria partially form respirasomes, which most likely have important physiological and/or regulatory functions.
Identification of Novel Supercomplexes in Potato Mitochondrial Fractions
Previous investigations of digitonin-solubilized mitochondrial fractions from Arabidopsis, potato, bean, and barley by BN-PAGE led to the identification of I1III2 and I2III4 supercomplexes and dimeric ATP synthase (Eubel et al., 2003
All molecular masses of protein complexes given in this publication represent apparent molecular masses as deduced from separations on BN gels. These values should be considered with caution, because protein separations on BN gels do not exactly reflect calculated molecular masses. Some values for apparent molecular masses in this publication were corrected in comparison to the values given in Eubel et al. (2003) : 600 kD for complex V (previously 550 kD), 350 kD for complex IVa (previously 300 kD), and 270 kD for complex IVb (previously 220 kD). As expected, all known protein complexes of the OXPHOS system are visible on our gels (Fig. 1): complex I (approximately 1,000 kD), complex V (approximately 600 kD), and dimeric complex III (approximately 500 kD). Complex IVa (approximately 350 kD), complex IVb (approximately 270 kD), and complex II result in diffuse bands on the 1D gels but were clearly identified upon resolution of their subunits on second gel dimensions, which were carried out in the presence of SDS (data not shown). Finally, the I1III2 and I2III4 supercomplexes are visible. However, the occurrence of the I2III4 supercomplex and complex IVb was restricted to potato stem mitochondria. Dimeric ATP synthase could not be detected in both fractions, most likely because digitonin concentrations were too high. Besides the known mitochondrial protein complexes and supercomplexes, additional complexes of low abundance showed up on our gels at approximately 850 kD and above 1,500 kD in both mitochondrial fractions (Fig. 1). To test if these protein supercomplexes include complex IV, in-gel activity measurements for cytochrome c oxidase were carried out. Indeed, five novel bands of approximately 850, 1,200, 1,850, 2,200, and 3,000 kD specifically were labeled in the potato tuber mitochondrial fraction (Fig. 1A). The 850- and 1,850-kD bands also are present in potato stem mitochondria and additionally two bands at approximately 770 and approximately 1,770 kD (Fig. 1). Identities of the newly discovered protein complexes were analyzed by 2D gel electrophoresis systems and are given below.
Oxygen uptake measurements were carried out using a Clark-type oxygen electrode to ensure that mitochondria used for the characterization of the novel supercomplexes are intact and physiologically active (Fig. 2 ). Organelles prepared from freshly harvested potato tubers exhibited high oxygen consumption rates (on average 155 nmol O2 min–1 mg–1 mitochondrial protein under state III conditions). In contrast, activity of potato stem mitochondria reproducibly was 40% to 50% lower under the same conditions. Mitochondria prepared from both organs had comparable state II respiration. Alternative respiration was low in mitochondrial isolations from potato stems and even lower in tuber mitochondria. We conclude that all mitochondrial fractions contained highly active organelles, but that mitochondria prepared from freshly harvested potato tubers exhibited highest state III respiration.
Optimization of Protein Solubilizations for Supercomplex Characterizations
To allow optimal visualization of the novel mitochondrial supercomplexes, isolated mitochondria from potato tubers and stems were solubilized by varying concentrations of digitonin (Fig. 3
). As previously reported (Eubel et al., 2003
Compositions of Newly Discovered Mitochondrial Supercomplexes
Two-dimensional BN/SDS-PAGE was carried out to characterize the subunit compositions of the novel mitochondrial protein supercomplexes (Fig. 4
). High protein amounts had to be loaded onto the gels to overcome their low abundance and to obtain information on subunits of these supercomplexes. The 850-kD complex of potato tuber mitochondria contains subunits of complexes III and IV and most likely has III2IV composition (Fig. 4A). The 1,200-kD complex could not be detected on our 2D gels. The 1,850-, 2,200-, and 3,000-kD complexes of potato tuber mitochondria all contain the subunits of the I1III2 supercomplex and additionally the Cox2 protein, which is the most dominant subunit of complex IV on BN gels (Fig. 4A). Further subunits of complex IV probably are present but could not be detected because they overlap with subunits of the complexes I and III on our gels. Due to low abundance, densitometric measurements of individual protein spots did not allow resolution of the stoichiometry of the protein complexes within these supercomplexes. However, based on the apparent molecular masses on the BN gels, the 1,850-, 2,200-, and 3,000-kD supercomplexes probably have I1III2IV1, I1III2IV2, and I1III2IV4 compositions, which would be in accordance with findings on respiratory supercomplexes in mammalian mitochondria (Schägger and Pfeiffer, 2000
Slightly different results were obtained upon resolution of mitochondrial protein complexes from potato stems by 2D BN/SDS-PAGE (Fig. 4B). First of all, about 50% of monomeric complex IV was not in the larger IVa (350 kD) but in the IVb form (270 kD), which could not be detected in the potato tuber mitochondrial fraction. Since the mitochondrial fractions from tubers and stems were treated equally, artificial generation of this smaller version of monomeric complex IV during mitochondrial isolations and/or BN-PAGE seems unlikely. As reported previously for Arabidopsis and bean, complex IVb lacks at least one 30-kD subunit, which was identified as being homologous to Cox6b proteins from yeast and mammals (Eubel et al., 2003 ). The 850- and 1,850-kD supercomplexes containing complex IV are also present in potato stem mitochondria and additionally two slightly smaller supercomplexes of 770 and 1,770 kD, which probably include complex IVb instead of complex IVa. The complex IV-containing 2,200- and 3,000-kD supercomplexes could not be found in mitochondrial isolations from potato stems. Instead, the 3,000-kD I2III4 supercomplex is present, which previously was described for Arabidopsis (Eubel et al., 2003).
To further investigate the structure of the newly discovered complex IV-containing supercomplexes from potato, 2D gel electrophoreses were repeated using 2D BN/BN-PAGE (Schägger and Pfeiffer, 2000 2D BN/BN-PAGE of mitochondrial fractions from potato tuber (Fig. 5A ) confirmed all results obtained by 2D BN/SDS-PAGE: the 850-kD supercomplex consists of complexes III and IV and the 1,850-, 2,200-, and 3,000-kD supercomplexes of complexes I, III, and IV. Separation of all these supercomplexes not only revealed occurrence of complex IVa but also presence of the smaller complex IVb. However, since monomeric complex IVb is absent in potato tuber mitochondrial fractions after digitonin solubilizations (Figs. 1A and 4A) but present after additional dodecylmaltoside treatment (Fig. 5A), this version of complex IV most likely is artificially generated during BN/BN-PAGE under the conditions applied. In contrast to 2D BN/SDS-PAGE, 2D BN/BN-PAGE allowed the analysis of the 1,200-kD supercomplex present in potato tuber mitochondria. Like the 850-kD complex, this supercomplex only contains the complexes III and IV and probably has a III2IV2 composition.
Analysis of mitochondrial fractions from potato stems by BN/BN-PAGE (Fig. 5B) also confirmed the findings obtained by 2D BN/SDS-PAGE: the 850- and 1,850-kD complexes include complexes III + IVa and I + III + IVa (complex IVa is partially converted into complex IVb as reported for mitochondria from potato tubers). The 770- and 1,770-kD complexes seem to have the same composition but most likely include complex IVb instead of complex IVa. The complex IV-containing 1,200-, 2,200-, and 3,000-kD supercomplexes of potato tuber mitochondria are absent, but a 3,000-kD I2III4 complex is present. Interestingly, destabilization of the large complex IV-containing supercomplexes partially results in generation of the I1III2 but not of the III2IV1-2 supercomplexes (Fig. 5, A and B). We conclude that interactions between the complexes I and III are stronger than interactions between complexes III and IV.
To increase sensitivity, a 2D BN-BN gel for potato stem mitochondria was repeated and stained by in-gel activity measurements for cytochrome c oxidase. This measurement was not possible after polymerization of the 1D BN gel stripe into the sample gel of a second gel dimension, most likely because N,N,N',N'-tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) diffused into the gel stripe and destroyed enzymatic activities. However, fixation of the first gel dimension with agarose onto the second gel dimension proved to be compatible with this experimental approach. As shown in Figure 6 , all previously made conclusions on complex IV-containing supercomplexes could be confirmed. Indeed, the 850- and 1,850-kD complexes include complex IVa, which partially dissociates into complex IVb in the presence of dodecylmaltoside. In contrast, the 770- and 1,770-kD complexes only contain the smaller IVb version of the cytochrome c oxidase complex (Fig. 6).
Structure of Respiratory Supercomplexes in Plants, Animals, and Fungi
Besides the previously described I1III2 and I2III4 supercomplexes and dimeric ATP synthase, potato tuber mitochondria contain five additional respiratory supercomplexes of about 850-, 1,200-, 1,850-, 2,200-, and 3,000-kD, which include complex IV. The 850- and 1,200-kD complexes only contain complexes III and IV and probably have III2IV1 and III2IV2 compositions; the other three complex IV-containing supercomplexes additionally include complex I and most likely have I1III2IV1, I1III2IV2, and I1III2IV4 structures (Table I; Fig. 7
). Similar supercomplexes were found in potato stem mitochondria. However, all newly described supercomplexes are of rather low abundance, because they only contain about 10% of total complex IV upon digitonin solubilizations and analysis on BN gels. Using comparable conditions, nearly 100% of yeast complex IV is associated with dimeric complex III (Cruciat et al., 2000
The I1III2IV4 supercomplex was suggested to represent a basic unit for respiration in mammalian mitochondria termed respirasome (Schägger and Pfeiffer, 2000 ). Respirasomes are also present in plant mitochondria (Fig. 8
). However, only very minor amounts of complex IV form part of respirasomes in mammals and plants (<5%). On the other hand, these structures might be much more abundant in vivo and only destabilized under the experimental conditions used for their characterization. Indeed, low digitonin to protein ratios seem to allow solubilization of higher quantities of respirasomes in potato (Fig. 3). Possibly in vivo even larger structures than respirasomes are formed by oligomerization of supercomplexes. In fact, some very weak protein bands can be seen above 3,000 kD on the gels shown in Figure 7. The I2III4 supercomplex of plant mitochondria could be a building block of these proposed oligomeric structures.
Are Some Supercomplexes Artificially Formed during Protein Solubilizations?
So far, formation of specific respiratory supercomplexes by artificial aggregation cannot be completely excluded but is highly unlikely for several reasons: (1) all complex IV-containing supercomplexes proved to be active by in-gel activity measurements for cytochrome c oxidase; (2) higher abundance of complex IV-containing supercomplexes in potato tuber mitochondria in comparison to potato stem mitochondria correlated with higher state III respiration; (3) the five OXPHOS complexes could theoretically form 10 different heterodimeric supercomplexes (composed of two different monomeric complexes); however, only heterodimeric I-III and III-IV complexes were observed, which represent the only meaningful associations with respect to the physiology of the mitochondrial respiratory chain (besides II-III associations, which were not observed); and (4) several physiological data reviewed in the introduction section support specific supercomplex formations, like reconstitution, inhibitor titration, and flux control experiments (Hatefi and Rieske, 1967
Currently the mechanisms for supercomplex formation in mitochondria are only poorly understood. In yeast cardiolipin proved to be essential for supercomplex stability. Based on studies with yeast mutants deficient in individual subunits of OXPHOS complexes, some proteins possibly forming part of supercomplex interphases could be defined (Pfeiffer et al., 2003
Digitonin solubilization and BN-PAGE proved to be a powerful tool for the investigation of mitochondrial supercomplexes from plants. However, visualization of individual supercomplexes in mitochondrial fractions of plants very much depends on various factors:
We speculate that complex IV-containing supercomplexes are present in other plants depending on the physiological state of the organs used for mitochondrial isolations but might be of low abundance. Indeed, mitochondria prepared from Arabidopsis leaves revealed some very small amounts of complex IV-containing supercomplexes (H. Eubel and H.-P. Braun, data not shown).
Monomeric complex IV is represented by two different forms in plants (Jänsch et al., 1996 An even larger probably monomeric form of complex IV can be seen by activity stainings of BN gels in the 400-kD range (Fig. 1). This version of complex IV is not visible on Coomassie-stained BN gels (Fig. 4), and its identity so far remains a mystery. Possibly this form of complex IV is a chaperone-bound assembly intermediate of cytochrome c oxidase. Similarly, a slightly larger form of complex III (550 instead of 500 kD) can be seen on the 2D BN/BN gel in Figure 5, which is invisible on the corresponding first gel dimension and might also represent a chaperone-bound form of this respiratory complex. Further experiments have to be carried out to explain these observations.
Plant mitochondria exhibit several special features in comparison to mitochondria from heterotrophic eukaryotes. Due to the presence of numerous alternative oxidoreductases, the respiratory chain of plant mitochondria is very much branched (Vanlerberghe and McIntosh, 1997
Isolation of Mitochondria from Potato Tubers and Stems
Freshly harvested potato (Solanum tuberosum var. cilena) tubers were purchased directly from a local farmer. Half of them were stored in the cold (4°C); the other half were planted into soil and grown in the dark at 20°C. Mitochondria were prepared from stored tubers and from etiolated potato stems after 20 d. Plant material (200 g) was homogenized at 4°C using a Waring blender for 3 x 5 s, filtrated through four layers of muslin, and subsequently organelles were purified by differential centrifugations and Percoll density gradient centrifugation as outlined previously (Braun et al., 1992a
Mitochondrial samples of 500 µg (50 µg mitochondrial protein) were sedimented by centrifugation for 10 min at 14,000g, resuspended in 50 µL of digitonin solution (1%–10% digitonin/30 mM HEPES/150 mM potassium acetate/10% glycerol), and incubated for 20 min at 0°C. Afterwards samples were centrifuged for 10 min at 18,000g. Finally supernatants were supplemented with 5 µL of a Coomassie Blue solution (5% Coomassie Blue/750 mM aminocaproic acid) and directly loaded onto BN gels.
BN-PAGE was carried out as described previously (Schägger, 2001b
In-gel activity of cytochrome c oxidase was measured according to Zerbetto et al. (1997)
Oxidative phosphorylation of all mitochondrial preparations was analyzed using a Clark-type oxygen electrode with a reaction chamber of 2 mL (Oxygraph, Hansatech, Norfolk, England). Oxygen consumption of 10 mg mitochondria (1 mg mitochondrial protein) in reaction buffer (0.3 M mannitol, 10 mM K2HPO4 (pH 7.2), 10 mM KCl, 5 mM MgCl2) was measured after supplementation of succinate (15 mM), ADP (5 mM), KCN (5 mM), and salicylhydroxamic acid (SHAM; 7.5 mM). Mitochondrial oxygen consumption was calculated in nmol
We thank Dagmar Lewejohann for expert technical assistance and Leila Matter, Dennis Kahlisch, and Prof. Dr. Udo Schmitz for critical reading of the manuscript. Received December 19, 2003; returned for revision January 13, 2004; accepted January 13, 2004.
1 This work was supported by the Fonds der Chemischen Industrie and the Deutsche Forschungsgemeinschaft (grant BR 1829–7/1.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.038018. * Corresponding author; e-mail braun{at}genetik.uni-hannover.de; fax 49511–7623608.
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O2 min–1 mg protein–1. 
