| Summary: | The molecular mechanisms of apoptosis are evolutionarily-conserved with caspases being the chief executioners of this process. Though key regulators of apoptosis, including caspases, inhibitor of apoptosis (IAP) proteins, and IAP antagonists exist in both mammals and flies, there are reportedly mechanistic differences in the way the apoptotic process is executed. One of the differences pertains to the importance of mitochondrial permeabilization for caspase activation. Herein, we demonstrate that dOmi, a Drosophila homologue of the serine protease Omi/HtrA2, is a developmentallyregulated mitochondrial intermembrane space protein that undergoes processive cleavage in situ to generate two distinct inhibitor of apoptosis (IAP) binding motifs. Depending upon the pro-apoptotic stimulus, mature dOmi is then differentially released into the cytosol, where it binds selectively to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC. This interaction alone, however, is insufficient to promote apoptosis, as dOmi fails to displace the effector caspase DrICE from the BIR1 domain in DIAP1. Rather, dOmi alleviates DIAP1 inhibition of all caspases by proteolytically degrading DIAP1 and induces apoptosis both in cultured cells and in the developing fly eye. Thus, we demonstrate for the first time in flies that mitochondrial permeabilization not only occurs during apoptosis, but also results in the release of a bona fide pro-apoptotic protein. DIAP1, in addition to being regulated by dOmi, is also regulated by RINGdependent autoubiquitination and by the N-end rule degradation (NERD) pathway. Despite decreasing the cellular levels of DIAP1, the NERD pathway enhances its antiapoptotic function through an unknown mechanism(s). Herein, we show for the first time that the NERD pathway facilitates trans-ubiquitination and degradation of IAP antagonist bound to DIAP1. Indeed, Grim is trans-ubiquitinated in an Ubr1-dependent manner and requires its interaction specifically with the BIR1 domain of DIAP1. These results demonstrate that similar to RING domain-dependent ubiquitination, the NERD pathway regulates not only the levels of DIAP1, but also of the levels of IAP antagonists bound to it.
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