New Insights into the Respiratory Chain of Plant Mitochondria. Supercomplexes and a Unique Composition of Complex II

Abstract

A project to systematically investigate respiratory supercomplexes in plant mitochondria was initiated. Mitochondrial fractions from Arabidopsis, potato (Solanum tuberosum), bean (Phaseolus vulgaris), and barley (Hordeum vulgare) were carefully treated with various concentrations of the nonionic detergents dodecylmaltoside, Triton X-100, or digitonin, and proteins were subsequently separated by (a) Blue-native polyacrylamide gel electrophoresis (PAGE), (b) two-dimensional Blue-native/sodium dodecyl sulfate-PAGE, and (c) two-dimensional Blue-native/Blue-native PAGE. Three high molecular mass complexes of 1,100, 1,500, and 3,000 kD are visible on one-dimensional Blue native gels, which were identified by separations on second gel dimensions and protein analyses by mass spectrometry. The 1,100-kD complex represents dimeric ATP synthase and is only stable under very low concentrations of detergents. In contrast, the 1,500-kD complex is stable at medium and even high concentrations of detergents and includes the complexes I and III 2 . Depending on the investigated organism, 50% to 90% of complex I forms part of this supercomplex if solubilized with digitonin. The 3,000-kD complex, which also includes the complexes I and III, is of low abundance and most likely has a III 4 I 2 structure. The complexes IV, II, and the alternative oxidase were not part of supercomplexes under all conditions applied. Digitonin proved to be the ideal detergent for supercomplex stabilization and also allows optimal visualization of the complexes II and IV on Blue-native gels. Complex II unexpectedly was found to be composed of seven subunits, and complex IV is present in two different forms on the Blue-native gels, the larger of which comprises additional subunits including a 32-kD protein resembling COX VIb from other organisms. We speculate that supercomplex formation between the complexes I and III limits access of alternative oxidase to its substrate ubiquinol and possibly regulates alternative respiration. The data of this investigation are available at http://www.gartenbau.uni-hannover.de/genetik/braun/AMPP.Structural basis for oxidative phosphorylation in mitochondria are five protein complexes termed NADH dehydrogenase (complex I), succinat dehydrogenase (complex II), cytochrome c reductase (complex III, which is a functional dimer), cytochrome c oxidase (complex IV), and ATP synthase (complex V). They were first characterized about 40 years ago by solubilizations of mitochondrial membrane proteins using detergents and differential precipitations or chromatographic separations. According to the popular "liquid state" model, the protein complexes of the respiratory chain are randomly arranged in the membrane and freely diffuse in lateral direction within the inner mitochondrial membrane (for review, see Rich, 1984). However, other results rather indicate an ordered association of these protein complexes forming larger structures. These socalled "supercomplexes" were first described for bacteria (Berry and Trumpower, 1985;Sone et al...

Interaction between Cytochrome caa3 and F1F0-ATP Synthase of Alkaliphilic Bacillus pseudofirmus OF4 Is Demonstrated by Saturation Transfer Electron Paramagnetic Resonance and Differential Scanning Calorimetry Assays

“…Although these interactions could be dynamic, this model differs significantly from the free diffusible model of electron transfer complexes derived from results of membrane fusing (33) and fluorescence recovery after photobleaching (FRAP) measurements (34). Rather, the current results are in line with a growing body of work that now suggests that some mitochondrial electron-transfer complexes specifically interact to form supermolecular structures called supercomplexes in organisms ranging from the yeast Saccharomyces cerevisiae (35,36), beef (19,29,35,37), and plants (38)(39)(40)(41)(42). Similar supermolecular structures have also been described for the respiratory chains of bacteria (43)(44)(45)(46)(47).…”

“…The roles that have been attributed to respiratory supercomplexes are substrate-channeling, catalytic enhancement, sequestration of reactive intermediates (35), stabilization of protein complexes (48), increasing the capacity of the inner mitochondrial membrane for protein insertion (36), and generating mitochondrial cristae morphology (49). Furthermore, the dynamic formation of such supercomplexes is speculated to serve some regulatory function in the energy generation in mitochondria from plants (39) and beef (19). It should be mentioned that supercomplex forms from two or more lipoprotein complexes isolated from detergent solublized membrane depends on the kind of detergent used and the method used in the detection.…”

Combined defects in oxidative phosphorylation and fatty acid β-oxidation in mitochondrial disease

“…Further research resulted in the isolation of a supercomplex containing CIII/CIV 2 in several strains of bacteria [10][11][12] and CI/CIII 2 , CIII 2 , CI 2 /CIV and CI/CIII 2 /CIV with varying stoichiometry in potato mitochondria [13,14]. In yeast, a CV dimer (CV 2 ) [15] and CIII 2 /CIV 1-2 supercomplex have also been detected [16].…”

Identification and Characterization of Respirasomes in Potato Mitochondria

“…Instead, two different forms of monomeric complex IV are visible on BN gels, which are termed complex IVa and IVb (about 300 and 220 kD in Arabidopsis). Complex IVa includes at least one additional subunit, which is homologous to the Cox6b protein from mammals and yeast (Eubel et al, 2003).…”

“…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). Three different supercomplexes were found in digitonin-solubilized mitochondrial fractions of Arabidopsis, potato (Solanum tuberosum), barley (Hordeum vulgare), and bean (Phaseolus vulgaris): (1) a 1,500-kD I 1 III 2 supercomplex; (2) a 3,000-kD I 2 III 4 supercomplex; and (3) a 1,100-kD dimeric ATP synthase complex.…”