A switchable ceramide transfer protein for dissecting the mechanism of ceramide-induced mitochondrial apoptosis.

Mitochondrial translocation of ceramides triggers Bax-dependent apoptosis. To elucidate how ceramides activate Bax and commit cells to death, we developed a switchable version of the ceramide transfer protein CERT, sCERT. Upon its drug-induced recruitment to mitochondria, sCERT retains the ability to bind VAP proteins in the ER and catalyzes mitochondrial import of externally added fluorescent ceramides. Mitochondrial recruitment of sCERT also triggers mitochondrial translocation of Bax. The ability of mitochondria-bound sCERT to mediate ceramide import and Bax translocation requires both its START domain and ongoing ceramide biosynthesis. These data extend our previous finding that mistargeting of ER ceramides to mitochondria specifically activates Bax and establish sCERT as a novel tool to dissect the underlying mechanism in a time-resolved manner.