DIRECT VISUALIZATION OF A CARDIOLIPIN-DEPENDENT CYTOCHROME P450SCC-INDUCED VESICLE AGGREGATION

Cytochrome P450scc can be reconstituted successfully into large unilam ellar phospholipid vesicles by a combined octylglucoside dialysis/adso rption method. Freeze-fracture electron microscopy was used to analyze the morphology, distribution, and protein topology of the cytochrome P450scc vesicles in dependence on lipid composition. Particles were ob served only in close contact to the vesicle surface, probably represen ting tightly associated cytochrome P450scc at the outer vesicle surfac e. In cytochrome P450scc vesicles similar in lipid composition to the inner membrane of bovine mitochondria direct evidence by freeze-fractu ring was found for a specific cytochrome P450scc-induced aggregation o f the vesicles. The vesicle aggregation critically depends on the cont ent of the specific mitochondrial membrane constituent cardiolipin. Th e aggregation and thus the intervesicular contacts were observed to be inhibited by both addition of anti-cytochrome P450scc IgG and adrenod oxin. Enzymatic reduction of cytochrome P450scc in the liposomal membr ane by its electron transfer partners completely indicates an asymmetr ical localization in/at the outer side of the bilayer membrane. It is suggested that vesiculation of the inner mitochondrial membrane may be a consequence of the characteristic cardiolipin dependent cytochrome P450scc membrane topology: the cardiolipin binding, peripheral, non-bi layer-spanning integration as an oligomer in the outer leaflet of the membrane may play a role in the dynamics of formation and dissociation of intramitochondrial vesicles with a functional importance for stero idogenesis. (C) 1994 Academic Press, Inc.