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J. Biol. Chem., Vol. 283, Issue 1, 367-379, January 4, 2008

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Mitochondrial Localization of Reaper to Promote Inhibitors of Apoptosis Protein Degradation Conferred by GH3 Domain-Lipid Interactions^*

From the Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

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 (GH3 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 GH3 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.

Received for publication, October 30, 2007

^* This work was supported in part by Grant R01GM061919 from the National Institutes of Health (to S. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Supported by Ruth L. Kirschstein National Research Service Award 5F32GM072165 for Individual Postdoctoral Fellows from the National Institutes of Health.

³ Supported by Ruth L. Kirschstein National Research Service Award 5F31GM073574 for Individual Predoctoral Fellows from the National Institutes of Health.

⁴ To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, Box 3813, Durham, NC 27713. Tel.: 919-613-8624; Fax: 919-681-1005; E-mail: kornb001{at}mc.duke.edu.

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