NEW INSIGHTS INTO THE COMPOSITION, MOLECULAR-MASS AND STOICHIOMETRY OF THE PROTEIN COMPLEXES OF PLANT-MITOCHONDRIA

Recently a powerful electrophoresis method for the native preparation and characterization of the respiratory protein complexes of mitochond ria from fungi and mammals has been developed, which employs Coomassie dyes to introduce charge shifts on proteins (Schagger and von Jagow ( 1991) Anal. Biochem. 199, 223-231). The procedure, which is called 'bl ue 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 ni ne distinct protein complexes between 100 and 1000 kDa and reveals nov el results for their composition, molecular mass and stoichiometry. Fo r the first time soluble mitochondrial protein complexes, like the HSP 60 complex (750 kDa) and a complex of 200 kDa, which includes a format e dehydrogenase, are analysed by BN-PAGE. Complex from potato (1000 kD a) 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 oxidas e (160 kDa) from potato seem to contain more subunits than hitherto re ported. Direct sequencing of subunits revealed that the F-1 part of th e F1F0 ATP synthase lacks the oligomycin sensitivity conferring protei n (OSCP), which was reported to be present in F-1 parts of dicotyledon ous plants, but contains the ATPase inhibitory protein. N-terminal seq uences of 16 mitochondrial proteins were obtained, several of which ar e presented for the first time from a plant source. BN-PAGE allows the preparation of mitochondrial protein complexes from gram amounts of p lant tissue, as the procedure only requires milligram amounts of organ elles. This potential of BN-PAGE is demonstrated by the separation and characterization of the mitochondrial enzyme complexes from Arabidops is thaliana. Further analysis of organellar protein complexes by BN-PA GE will allow the generation of 'protein maps' from different tissues and developmental stages or from mutant plants.