Mitochondrial cytochrome c oxidase and succinate dehydrogenase complexes contain plant specific subunits.
Respiratory oxidative phosphorylation represents a central functionality in plant metabolism, but the subunit composition of the respiratory complexes in plants is still being defined. Most notably, complex II (succinate dehydrogenase) and complex IV (cytochrome c oxidase) are the least defined in plant mitochondria. Using Arabidopsis mitochondrial samples and 2D Blue-native/SDS-PAGE, we have separated complex II and IV from each other and displayed their individual subunits for analysis by tandem mass spectrometry and Edman sequencing. Complex II can be discretely separated from other complexes on Blue-native gels and consists of eight protein bands. It contains the four classical SDH subunits as well as four subunits unknown in mitochondria from other eukaryotes. Five of these proteins have previously been identified, while three are newly identified in this study. Complex IV consists of 9-10 protein bands, however, it is more diffuse in Blue-native gels and co-migrates in part with the translocase of the outer membrane (TOM) complex. Differential analysis of TOM and complex IV reveals that complex IV probably contains eight subunits with similarity to known complex IV subunits from other eukaryotes and a further six putative subunits which all represent proteins of unknown function in Arabidopsis . Comparison of the Arabidopsis data with Blue-native/SDS-PAGE separation of potato and bean mitochondria confirmed the protein band complexity of these two respiratory complexes in plants. Two-dimensional Blue-native/Blue-native PAGE, using digitonin followed by dodecylmaltoside in successive dimensions, separated a diffusely staining complex containing both TOM and complex IV. This suggests that the very similar mass of these complexes will likely prevent high purity separations based on size. The documented roles of several of the putative complex IV subunits in hypoxia response and ozone stress, and similarity between new complex II subunits and recently identified plant specific subunits of complex I, suggest novel biological insights can be gained from respiratory complex composition analysis.
Millar
A Harvey
AH
Plant Molecular Biology Group, School of Biomedical and Chemical Sciences, University of Western Australia, Crawley, Western Australia 6009, Australia.
Eubel
Holger
H
Jänsch
Lothar
L
Kruft
Volker
V
Heazlewood
Joshua L
JL
Braun
Hans-Peter
HP
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D042963:Electron Transport Complex II / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D003576:Electron Transport Complex IV / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D007887:Fabaceae / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000235:genetics / Q000378:metabolism
D010940:Plant Proteins; D042963:Electron Transport Complex II; D003576:Electron Transport Complex IV