DIRECT ANALYSIS AND SIGNIFICANCE OF CARDIOLIPIN TRANSVERSE-DISTRIBUTION IN MITOCHONDRIAL INNER MEMBRANES

The distribution of cardiolipin across the inner mitochondrial membran e was directly determined by using the ability of the fluorescent dye 10-N-nonyl-3,6-bis(dimethylamino)acridine (10-N-nonyl acridine orange) to form dimers when it interacts with the diacidic phospholipid. Two independent methods were employed: (a) a spectrophotometric measuremen t of 10-N-nonyl acridine orange binding to isolated rat liver mitochon dria, mitoplasts and inside-out submitochondrial particles, and (b) a flow-cytometric analysis of specific red fluorescence, emitted when tw o dye molecules are bound to one membrane cardiolipin; the stoichiomet ry of 10-N-nonyl acridine orange binding to phosphatidylserine and pho sphatidylinositol, 1 mol dye/mol phospholipid, prevented dye dimerisat ion and subsequent red-fluorescence appearance. 57% total cardiolipin was present in the outer leaflets of inner membranes of isolated organ elles, a distribution confirmed by saturation measurements for mitopla sts and inside-out submitochondrial particles. The same asymmetry was directly observed in situ with mitochondrial membranes of quiescent L1 210 cells, and with mitochondrial membranes of respiring yeasts. Never theless, alterations in ATP synthesis and inhibition of mitochondrial protein synthesis revealed that cardiolipin distribution was apparentl y tightly correlated with mitochondrial membrane assembly and activity .