title: Mitochondrial localization of Reaper to promote inhibitors of apoptosis protein degradation conferred by GH3 domain-lipid interactions.
abstract: Morphological hallmarks of apoptosis result from activation of the caspase family of cysteine proteases, which are opposed by a pro-survival family of inhibitors of apoptosis proteins (IAPs). In Drosophila, disruption of IAP function by Reaper, HID, and Grim (RHG) proteins is sufficient to induce cell death. RHG proteins have been reported to localize to mitochondria, which, in the case of both Reaper and Grim proteins, is mediated by an amphipathic helical domain known as the GH3. Through direct binding, Reaper can bring the Drosophila IAP (DIAP1) to mitochondria, concomitantly promoting IAP auto-ubiquitination and destruction. Whether this localization is sufficient to induce DIAP1 auto-ubiquitination has not been reported. In this study we characterize the interaction between Reaper and the mitochondria using both Xenopus and Drosophila systems. We find that Reaper concentrates on the outer surface of mitochondria in a nonperipheral manner largely mediated by GH3-lipid interactions. Importantly, we show that mitochondrial targeting of DIAP1 alone is not sufficient for degradation and requires Reaper binding. Conversely, Reaper able to bind IAPs, but lacking a mitochondrial targeting GH3 domain (DeltaGH3 Reaper), can induce DIAP1 turnover only if DIAP1 is otherwise targeted to membranes. Surprisingly, targeting DIAP1 to the endoplasmic reticulum instead of mitochondria is partially effective in allowing DeltaGH3 Reaper to promote DIAP1 degradation, suggesting that co-localization of DIAP and Reaper at a membrane surface is critical for the induction of DIAP degradation. Collectively, these data provide a specific function for the GH3 domain in conferring protein-lipid interactions, demonstrate that both Reaper binding and mitochondrial localization are required for accelerated IAP degradation, and suggest that membrane localization per se contributes to DIAP1 auto-ubiquitination and degradation.
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Freel
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: D Ashley
authors.initials: DA
authors.lastname: Richardson
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Thomenius
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: Eugene C
authors.initials: EC
authors.lastname: Gan
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: Sarah R
authors.initials: SR
authors.lastname: Horn
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Olson
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710. Electronic address: kornb001@mc.duke.edu.
authors.forename: Sally
authors.initials: S
authors.lastname: Kornbluth
mesh_terms: D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D004330:Drosophila; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination; D014981:Xenopus
chemical_list: C111891:DIAP1 protein, Drosophila; D029721:Drosophila Proteins; D051034:Inhibitor of Apoptosis Proteins; D008081:Liposomes; D008563:Membrane Lipids; D011993:Recombinant Fusion Proteins; C086973:rpr protein, Drosophila; D049452:Green Fluorescent Proteins