New insights into the respiratory chain of plant mitochondria. Supercomplexes and a unique composition of complex II.
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.
Eubel
Holger
H
Institut für Angewandte Genetik, Universität Hannover, Herrenhäuser Strasse 2, D-30419 Hannover, Germany.
Jänsch
Lothar
L
Braun
Hans-Peter
HP
D017360:Arabidopsis / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D004579:Electron Transport / Q000502:physiology; D042967:Electron Transport Complex I / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D042963:Electron Transport Complex II / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014450:Electron Transport Complex III / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D007887:Fabaceae / Q000378:metabolism; D005960:Glucosides / Q000494:pharmacology; D001467:Hordeum / Q000378:metabolism; D013058:Mass Spectrometry; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D017830:Octoxynol / Q000494:pharmacology; D010088:Oxidoreductases / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D010944:Plants / Q000378:metabolism*; D011487:Protein Conformation; D011198:Solanum tuberosum / Q000378:metabolism; D000099188:Alternative Oxidase
D004072:Digitonin; D042967:Electron Transport Complex I; D042963:Electron Transport Complex II; D014450:Electron Transport Complex III; D003576:Electron Transport Complex IV; D005960:Glucosides; D024101:Mitochondrial Proteins; D017830:Octoxynol; D010088:Oxidoreductases; D010940:Plant Proteins; D000099188:Alternative Oxidase; C040358:dodecyl maltoside