Cardiolipin (CL) is an inner mitochondrial membrane phospholipid that contributes to optimal mitochondrial function and is gaining widespread attention in studies of mitochondria-mediated apoptosis. Divergent hypotheses describing the role of CL in cytochrome c release and apoptosis have evolved. We addressed this controversy directly by comparing the spontaneous- and Bax-mediated cytochrome c release from mitochondria isolated from two strains of Saccharomyces cerevisiae: one lacking CL-synthase and therefore CL (DeltaCRD1) and the other, its corresponding wild type (WT). We demonstrated by liquid chromatography-mass spectrometry that the main yeast CL species [(16:1)2(18:1)2] differs in fatty acid composition from mammalian CL [(18:2)4], and we verified the absence of the yeast CL species in the DeltaCRD1 strain. We also demonstrated that the mitochondrial association of Bax and the resulting cytochrome c release is not dependent on the CL content of the yeast mitochondrial membranes. Bax inserted equally into both WT and DeltaCRD1 mitochondrial membranes under conditions that lead to the release of cytochrome c from both strains of yeast mitochondria. Furthermore, using models of synthetic liposomes and isolated yeast mitochondria, we found that cytochrome c was bound more "loosely" to the CL-deficient systems compared with when CL is present. These data challenge recent studies implicating that CL is required for Bax-mediated pore formation leading to the release of proteins from the mitochondrial intermembrane space. In contrast, they support our recently proposed two-step mechanism of cytochrome c release, which suggests that CL is required for binding cytochrome c to the inner mitochondrial membrane.
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