Two-dimensional native electrophoretic analysis of respiratory supercomplexes from Yarrowia lipolytica

Mitochondria of the strictly aerobic yeast Yarrowia lipolytica contain respiratory complex I with close functional and structural similarity to the mammalian enzyme. Unlike mammalian mitochondria, however, Yarrowia mitochondria have been thought not to contain supercomplexes. Here, we identify respiratory supercomplexes composed of complexes I, III and IV also in Y lipolytica. Evidence for dimeric complex I suggests further association of respiratory supercomplexes into respiratory strings or patches. Similar supercomplex organization in Yarrowia and mammalian mitochondria further makes this aerobic yeast a useful model for the human oxidative phosphorylation system. The analysis of supercomplexes and their constituent complexes was made possible by 2-D native electrophoresis, i.e. by using native electrophoresis for both dimensions. Digitonin and blue-native electrophoresis were generally applied for the initial separation of supercomplexes followed by less mild native electrophoresis variants in the second dimension to release the individual complexes from the supercomplexes. Such 2-D native systems are useful means to identify the constituent proteins and their copy numbers in detergent-labile physiological assemblies, since they can reduce the complexity of supramolecular systems to the level of individual complexes.