A GH3-like domain in reaper is required for mitochondrial localization and induction of IAP degradation.
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-xL 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.
Olson
Michael R
MR
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Holley
Christopher L
CL
Gan
Eugene C
EC
Colón-Ramos
Daniel A
DA
Kaplan
Bruce
B
Kornbluth
Sally
S
D000818:Animals; D017209:Apoptosis; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D004331:Drosophila melanogaster / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D051034:Inhibitor of Apoptosis Proteins; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000737:chemistry*; D016296:Mutagenesis; D009479:Neuropeptides / Q000737:chemistry*; D010446:Peptide Fragments / Q000235:genetics; D017433:Protein Structure, Secondary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection; D051020:bcl-X Protein; D000097573:Red Fluorescent Protein
D061945:Caspase Inhibitors; D020169:Caspases; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D051034:Inhibitor of Apoptosis Proteins; D008164:Luminescent Proteins; D009479:Neuropeptides; D010446:Peptide Fragments; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051020:bcl-X Protein; D000097573:Red Fluorescent Protein; C111891:DIAP1 protein, Drosophila; C108881:grim protein, Drosophila; C086973:rpr protein, Drosophila