First published online August 7, 2003; 10.1104/pp.103.024620
Plant Physiology 133:274-286 (2003)
© 2003 American Society of Plant Biologists
BIOENERGETICS AND PHOTOSYNTHESIS
New Insights into the Respiratory Chain of Plant Mitochondria. Supercomplexes and a Unique Composition of Complex II1
Holger Eubel,
Lothar Jänsch and
Hans-Peter Braun*
Institut für Angewandte Genetik, Universität Hannover, Herrenhäuser Strasse 2, D-30419 Hannover, Germany (H.E., H.-P.B.); and Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, 38124 Braunschweig, Germany (L.J.)
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 III2. 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 III4I2 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.
1 This work was supported by the Fonds der Chemischen Industrie.
* Corresponding author; e-mail braun{at}genetik.uni-hannover.de; fax 49511-7623608.
Received April 1, 2003;
returned for revision April 22, 2003;
accepted May 27, 2003.
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