A GH3-like Domain in Reaper Is Required for Mitochondrial Localization and Induction of IAP Degradation

Abstract

Reaper is a potent pro-apoptotic protein originally identified in a screen for Drosophila mutants defective in apoptotic induction. Multiple functions have been ascribed to this protein, including inhibition of IAPs (inhibitors of apoptosis); induction of IAP degradation; inhibition of protein translation; and when expressed in vertebrate cells, induction of mitochondrial cytochrome c release. Structure/function analysis of Reaper has identified an extreme N-terminal motif that appears to be sufficient for inhibition of IAP function. We report here that this domain, although required for IAP destabilization, is not sufficient. Moreover, we have identified a small region of Reaper, similar to the GH3 domain of Grim, that is required for localization of Reaper to mitochondria, induction of IAP degradation, and potent cell killing. Although a mutant Reaper protein lacking the GH3 domain was deficient in these properties, these defects could be fully rectified by appending either the C-terminal mitochondrial targeting sequence from Bcl-x L or a homologous region from the pro-apoptotic protein HID. Together, these data strongly suggest that IAP destabilization by Reaper in intact cells requires Reaper localization to mitochondria and that induction of IAP instability by Reaper is important for the potent induction of apoptosis in Drosophila cells.

Mechanism of action of Drosophila Reaper in mammalian cells: Reaper globally inhibits protein synthesis and induces apoptosis independent of mitochondrial permeability

“…26 It has recently been reported that in insect cells the GH3 domain is required for localisation of Reaper to mitochondria. 41 However, in mammalian cells, we do not find constitutive co-localisation of Reaper with mitochondria. We did observe that full-length and DIBM Reaper redistributed from the cytosol to regions close to the nucleus throughout the apoptotic process, which resulted in a partial co-localisation with mitochondria.…”

Multifunctional reaper: sixty-five amino acids of fury

“…Reaper also has a similar hydrophobic sequence, critical for its ability to induce apoptosis and for its mitochondrial localization. Furthermore, Olson et al 57 showed that deletion of the GH3 domain disrupts Reaper-induced DIAP-1 degradation. This loss of function could be rescued by restoring mitochondrial localization by attaching the C-terminal mitochondrial-targeting sequence of Bcl-xL.…”

Regulation of apoptosis in Drosophila

“…18 Interestingly, Drosophila Reaper, Hid and Grim can effectively bind to vertebrate IAPs, promote XIAP self-conjugation both in vitro and in vivo and potently induce apoptosis in certain mammalian cell types. [49][50][51] Finally, small molecule IAPantagonists based on the IBM-IAP interaction are currently developed as anticancer therapeutics. 19,52,53 However, Reaper, Hid and Grim utilize additional domains besides the IBM for potent cell killing.…”