SUMMARY Mitochondria are functionally and physically associated with heterotypic membranes, yet little is known about how these interactions impact mitochondrial outer-membrane permeabilization (MOMP) and apoptosis. We observed that dissociation of heterotypic membranes from mitochondria inhibited BAK/BAX-dependent cytochrome c (cyto c) release. Biochemical purification of neutral sphingomyelinases that correlated with MOMP sensitization suggested that sphingolipid metabolism coordinates BAK/BAX activation. Using purified lipids and enzymes, sensitivity to MOMP was achieved by in vitro reconstitution of the sphingolipid metabolic pathway. Sphingolipid metabolism inhibitors blocked MOMP from heavy membrane preparations but failed to influence MOMP in the presence of sphingolipid-reconstituted, purified mitochondria. Furthermore, the sphingolipid products, sphingosine-1-PO4 and hexadecenal, cooperated specifically with BAK and BAX, respectively. Sphingolipid metabolism was also required for cellular responses to apoptosis. Our studies suggest that BAK/BAX activation and apoptosis are coordinated through BH3-only proteins and a specific lipid milieu that is maintained by heterotypic membrane-mitochondrial interactions

Background: BH3 mimetics are promising chemotherapeutics, but their full mechanisms of action are underexplored. Results: BAX, BID, and BIM directly influence BH3 mimetics to pharmacologically inhibit anti-apoptotic BCL-2 proteins. Conclusion: BH3 mimetics display differential activities that are dependent upon both anti-and proapoptotic BCL-2 members function. Significance: Understanding how BH3 mimetics function within the BCL-2 family repertoire will improve the utility of these drugs.

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