Abstract
The mitochondrial processing peptidase (MPP) is a heterodimeric enzyme that forms part of the cytochrome c reductase complex from higher plants. Mitochondria from mammals and yeast contain two homologous enzymes: (i) an active MPP within the mitochondrial matrix and (ii) an inactive MPP within the cytochrome c reductase complex. To elucidate the evolution of MPP, the cytochrome c reductase complexes from lower plants were isolated and tested for processing activity. Mitochondria were prepared from the staghorn fern Platycerium bifurcatum, from the horsetail Equisetum arvense, and from the colorless algae Polytomella, and cytochrome c reductase complexes were purified by a micro-isolation procedure based on Blue-native polyacrylamide gel electrophoresis and electroelution. This is the first report on the subunit composition of a respiratory enzyme complex from a fern or a horsetail. The cytochrome c reductase complexes from P. bifurcatum and E. arvense are shown to efficiently process mitochondrial precursor proteins, whereas the enzyme complex from Polytomella lacks proteolytic activity. An evolutionary model is suggested that assumes a correlation between the presence of an active MPP within the cytochrome c reductase complex and the occurrence of chloroplasts.
Highlights
The mitochondrial cytochrome c reductase is a multisubunit enzyme of the respiratory chain that catalyzes reduction of cytochrome c by oxidation of ubiquinol
These molecular data can be explained by an evolutionary model, which was suggested recently [3, 17]. (i) Early in evolution, mitochondrial processing peptidase (MPP) developed starting from a preexisting bacterial protease and became part of the cytochrome c reductase complex; (ii) later in evolution, the two subunits of MPP became detached from the enzyme complex to allow independent regulation of protein processing and respiration in some organisms; (iii) the detachment was realized by gene duplications, because due to the co-evolution of cytochrome c reductase and MPP, the two subunits of MPP became indispensable for assembly of this respiratory protein complex
Isolation of Cytochrome c Reductase Complexes from S. tuberosum, Polytomella spp., P. bifurcatum, and E. arvense—The isolation of highly pure mitochondria from plants devoid of chloroplast contaminations is facilitated by the use of etiolated starting material like tubers or dark grown seedlings
Summary
Isolation of Mitochondria from Solanum tuberosum, Polytomella spp., P. bifurcatum, and E. arvense. Pure mitochondrial fraction was washed twice; resuspended in 0.4 M mannitol, 0.1% BSA, 1 mM EGTA, 0.2 mM PMSF, 10 mM KH2PO4, pH 7.2, at a concentration of 2 mg of mitochondrial protein/ml; and divided into aliquots of 200 l. Mitochondria formed two light brown bands in the 26% phase They were washed twice in resuspension buffer, solved at a concentration of 1 mg of mitochondrial protein/ml, and divided into aliquots of 600 l. The mitochondria were removed, washed twice, and solved in 0.4 M mannitol, 0.1% BSA, 1 mM EGTA, 0.2 mM PMSF, 10 mM KH2PO4, pH 7.2, at a concentration of ϳ1.5 mg of mitochondrial protein/ml. Starting points for sample preparation were the obtained aliquots of mitochondria from S. tuberosum, Polytomella spp., P. bifurcatum, and E. arvense. The mitochondrial protein complexes from each organism were resolved on a separate gel consisting of a stacking gel (4% acrylamide) comprising 10 slots and a separating gel (4.95–12.6% acrylamide), respectively
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