Cardiolipin is synthesized on the matrix side of the inner membrane in rat liver mitochondria.

In the mitochondrial inner membrane, cardiolipin is a specific lipid component associated with various protein complexes. The assembly of such complexes has been studied, and it seems that most protein subunits enter the inner membrane from the matrix side, but nothing is known about the path of cardiolipin. In this paper, the topography of cardiolipin biosynthesis is investigated. Cardiolipin synthase, a membrane-bound protein, could not be released by sonication or 1 M KCl. In sucrose density gradient subfractionation, cardiolipin synthase co-migrated with the inner membrane marker cytochrome oxidase. no indication was obtained for a preferential localization of this enzyme at contact sites between the outer and inner membranes. Protease digestion experiments showed that cardiolipin synthase exposed protease-susceptible domains mainly to the matrix side of the inner membrane. In intact mitochondria, the Mn(2+)-dependent stimulation of cardiolipin synthesis was abolished when the Mn2+ influx into the matrix was blocked by ruthenium red. 1-Decanoyl-sn-glycero-3-phosphorylcholine, a water-soluble inhibitor of cardiolipin synthase, was only effective after disintegration of mitochondria. The metabolic precursor of cardiolipin, CDP-diacylglycerol, was synthesized by an inner membrane enzyme whose protease-susceptible domains were mainly exposed to the matrix side. It is concluded that cardiolipin is synthesized in the inner leaflet of the mitochondrial inner membrane.