New insights into the composition, molecular mass and stoichiometry of the protein complexes of plant mitochondria

Abstract

Recently a powerful electrophoresis method for the native preparation and characterization of the respiratory protein complexes of mitochondria from fungi and mammals has been developed, which employs Coomassie dyes to introduce charge shifts on proteins (Schägger and von Jagow (1991) Anal. Biochem. 199, 223-231). The procedure, which is called 'blue native-polyacrylamide gel electrophoresis' (BN-PAGE), was modified and introduced for the analysis of mitochondria from higher plants. BN-PAGE of mitochondrial protein from potato allows the separation of nine distinct protein complexes between 100 and 1000 kDa and reveals novel results for their composition, molecular mass and stoichiometry. For the first time soluble mitochondrial protein complexes, like the HSP60 complex (750 kDa) and a complex of 200 kDa, which includes a formate dehydrogenase, are analysed by BN-PAGE. Complex I from potato (1000 kDa) is about 100 kDa larger than the corresponding enzyme from beef and can be resolved into more than 30 different subunits on a second gel dimension. The F1F0 ATP synthase (580 kDa) and the cytochrome c oxidase (160 kDa) from potato seem to contain more subunits than hitherto reported. Direct sequencing of subunits revealed that the F1 part of the F1F0 ATP synthase lacks the oligomycin sensitivity conferring protein (OSCP), which was reported to be present in F1 parts of dicotyledonous plants, but contains the ATPase inhibitory protein. N-terminal sequences of 16 mitochondrial proteins were obtained, several of which are presented for the first time from a plant source. BN-PAGE allows the preparation of mitochondrial protein complexes from gram amounts of plant tissue, as the procedure only requires milligram amounts of organelles. This potential of BN-PAGE is demonstrated by the separation and characterization of the mitochondrial enzyme complexes from Arabidopsis thaliana. Further analysis of organellar protein complexes by BN-PAGE will allow the generation of 'protein maps' from different tissues and developmental stages or from mutant plants.

Insights into the Composition and Assembly of the Membrane Arm of Plant Complex I through Analysis of Subcomplexes in Arabidopsis Mutant Lines

“…BN-PAGE in Gel Activity and Transfer-Blue-Native PAGE (BN-PAGE) was performed according to the method described by Jänsch et al (41). Mitochondrial proteins (100 g) were solubilized with dodecylmaltoside (1% (w/v) final) in 75 l of ACA buffer (750 mM aminocaproic acid, 0.5 mM EDTA, 50 mM TrisHCl, pH 7.0) and incubated 20 min at 4°C.…”

Changes in the Mitochondrial Proteome during the Anoxia to Air Transition in Rice Focus around Cytochrome-containing Respiratory Complexes

“…Blue-native PAGE analysis was carried out according to published methods (30), and second dimension SDS-PAGE was done by Tris-Tricine separations. Protein spots from polyacrylamide gels were excised and prepared for analysis by MS according to Sweetlove et al (31).…”

Remodeled Respiration in ndufs4 with Low Phosphorylation Efficiency Suppresses Arabidopsis Germination and Growth and Alters Control of Metabolism at Night

“…Blue Native-PAGE and Complex I Activity Assay Blue Native-PAGE was performed according to the method described by Jänsch et al (1996). Mitochondrial proteins (200 mg) were solubilized with dodecyl maltoside (1% [w/v] final) in ACA buffer (750 mM amino caproic acid, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.0) and incubated for 20 min at 4°C.…”