HELA cells PARL Immunoprecipitation part II

Mitochondria drive apoptosis by releasing pro-apoptotic proteins that promote caspase activation in the cytosol. The rhomboid-like protease PARL, an intramembrane cleaving peptidase in the inner membrane, regulates mitophagy and cell death pathways, but its role in apoptosis remained enigmatic. Here, we employed PARL-based proteomics to define its substrate spectrum. Our data identified the mitochondrial pro-apoptotic protein Smac/DIABLO as a PARL substrate. In apoptotic cells, Smac/DIABLO is released into the cytosol and promotes caspase activity by inhibiting IAPs. Intramembrane cleavage of Smac/DIABLO by PARL generates an amino terminal IAP-binding motif, which is required for its apoptotic activity. Loss of PARL impairs proteolytic maturation of Smac/DIABLO, which fails to bind XIAP. Smac/DIABLO peptidomimetics, downregulation of XIAP or cytosolic expression of cleaved Smac/DIABLO restores apoptosis in PARL-deficient cells. Our results discover a pro-apoptotic function of PARL and identify PARL-mediated Smac/DIABLO processing and cytochrome c release facilitated by OPA1 dependent cristae remodeling as two independent pro-apoptotic pathways in mitochondria. Please note that these data are overlapping with data uploaded under: PXD004914. Exclusively in this upload, you will find the S277A mutant immunoprecipitations.