Abstract
Mitochondrial proteins such as cytochrome c, Smac/DIABLO and Omi/HtrA2 play important roles in the cell death pathways of mammalian cells. In Drosophila, the role of mitochondria in cell death is less clear. Here, we report the identification and characterization of the Drosophila ortholog of human Omi/HtrA2. We show that Drosophila Omi/HtrA2 is imported into the mitochondria where it undergoes proteolytic maturation to yield two isoforms, dOmi-L and dOmi-S. dOmi-L contains a canonical N-terminal IAP-binding motif (AVVS), whereas dOmi-S contains a distinct N-terminal motif (SKMT). DIAP1 was able to bind to both isoforms via its BIR1 and BIR2 domains. This resulted in cleavage of the linker region of DIAP1 between the BIR1 and BIR2 domains and further degradation of the BIR1 domain by the proteolytic activity of dOmi. The binding of DIAP1 to dOmi also resulted in DIAP1-mediated polyubiquitination of dOmi, suggesting that DIAP1 could target dOmi for proteasomal degradation. Consistent with this, expression of DIAP1 in Drosophila eye discs protected them from dOmi-induced eye ablation, indicating that DIAP1 plays an important role in protecting cells from the potentially lethal effects of dOmi. The ability of IAPs to bind to and ubiquitinate mitochondrial proteins such as dOmi may be a key conserved function to counterbalance the lethal effects of these proteins if accidentally released into the cytosol.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- BIR:
-
baculovirus inhibitor of apoptosis repeat
- DIABLO:
-
direct IAP-binding protein with low pI
- DIAP1:
-
Drosophila inhibitor of apoptosis protein 1
- DCP1:
-
Drosophila caspase 1
- dOmi:
-
Drosophila Omi
- DRICE:
-
Drosophila interleukin-1β-converting enzyme
- Dronc:
-
Drosophila Nedd2-like caspase
- GMR:
-
Glass multimer reporter
- GST:
-
glutathione-S-transferase
- IAP:
-
inhibitor of apoptosis protein
- IBM:
-
IAP-binding motif
- IMAGE:
-
Integrated Molecular Analysis of Genomes and their Expression
- MTS:
-
mitochondrial targeting sequence
- PAGE:
-
polyacrylamide gel electrophoresis
- PSORT:
-
protein sorting
- RING:
-
really interesting new gene
- Smac:
-
second mitochondrial-derived activator
- TM:
-
transmembrane
- WT:
-
wild type
References
Tsujimoto Y . Cell death regulation by the Bcl-2 protein family in the mitochondria. J Cell Physiol 2003; 195: 158–167.
Kornbluth S, White K . Apoptosis in Drosophila: neither fish nor fowl (nor man, nor worm). J Cell Sci 2005; 118: 1779–1787.
Varkey J, Chen P, Jemmerson R, Abrams JM . Altered cytochrome c display precedes apoptotic cell death in Drosophila. J Cell Biol 1999; 144: 701–710.
Igaki T, Miura M . Role of Bcl-2 family members in invertebrates. Biochim Biophys Acta 2004; 1644: 73–81.
Abdelwahid E, Yokokura T, Krieser RJ, Balasundaram S, Fowle WH, White K . Mitochondrial disruption in Drosophila apoptosis. Dev Cell 2007; 12: 793–806.
Chai J, Yan N, Huh JR, Wu JW, Li W, Hay BA et al. Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination. Nat Struct Biol 2003; 10: 892–898.
Christich A, Kauppila S, Chen P, Sogame N, Ho SI, Abrams JM . The damage-responsive Drosophila gene sickle encodes a novel IAP binding protein similar to but distinct from reaper, grim, and hid. Curr Biol 2002; 12: 137–140.
Srinivasula SM, Datta P, Kobayashi M, Wu JW, Fujioka M, Hegde R et al. Sickle, a novel Drosophila death gene in the reaper/hid/grim region, encodes an IAP-inhibitory protein. Curr Biol 2002; 12: 125–130.
Wing JP, Karres JS, Ogdahl JL, Zhou L, Schwartz LM, Nambu JR . Drosophila sickle is a novel grim-reaper cell death activator. Curr Biol 2002; 12: 131–135.
Yan N, Wu JW, Chai J, Li W, Shi Y . Molecular mechanisms of DrICE inhibition by DIAP1 and removal of inhibition by Reaper, Hid and Grim. Nat Struct Mol Biol 2004; 11: 420–428.
Vaux DL, Silke J . Mammalian mitochondrial IAP binding proteins. Biochem Biophys Res Commun 2003; 304: 499–504.
Shi Y . A conserved tetrapeptide motif: potentiating apoptosis through IAP-binding 1. Cell Death Differ 2002; 9: 93–95.
Tenev T, Ditzel M, Zachariou A, Meier P . The antiapoptotic activity of insect IAPs requires activation by an evolutionarily conserved mechanism. Cell Death Differ 2007; 14: 1191–1201.
Tenev T, Zachariou A, Wilson R, Ditzel M, Meier P . IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. Nat Cell Biol 2005; 7: 70–77.
Hays R, Wickline L, Cagan R . Morgue mediates apoptosis in the Drosophila melanogaster retina by promoting degradation of DIAP1. Nat Cell Biol 2002; 4: 425–431.
Wilson R, Goyal L, Ditzel M, Zachariou A, Baker DA, Agapite J et al. The DIAP1 RING finger mediates ubiquitination of Dronc and is indispensable for regulating apoptosis. Nat Cell Biol 2002; 4: 445–450.
Yoo SJ, Huh JR, Muro I, Yu H, Wang L, Wang SL et al. Hid, Rpr and Grim negatively regulate DIAP1 levels through distinct mechanisms. Nat Cell Biol 2002; 4: 416–424.
Ryoo HD, Bergmann A, Gonen H, Ciechanover A, Steller H . Regulation of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1. Nat Cell Biol 2002; 4: 432–438.
Holley CL, Olson MR, Colon-Ramos DA, Kornbluth S . Reaper eliminates IAP proteins through stimulated IAP degradation and generalized translational inhibition. Nat Cell Biol 2002; 4: 439–444.
Hegde R, Srinivasula SM, Zhang Z, Wassell R, Mukattash R, Cilenti L et al. Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts inhibitor of apoptosis protein–caspase interaction. J Biol Chem 2002; 277: 432–438.
Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K, Takahashi R . A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol Cell 2001; 8: 613–621.
Verhagen AM, Silke J, Ekert PG, Pakusch M, Kaufmann H, Connolly LM et al. HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins. J Biol Chem 2002; 277: 445–454.
Martins LM, Iaccarino I, Tenev T, Gschmeissner S, Totty NF, Lemoine NR et al. The serine protease Omi/HtrA2 regulates apoptosis by binding XIAP through a reaper-like motif. J Biol Chem 2002; 277: 439–444.
Chai J, Du C, Wu JW, Kyin S, Wang X, Shi Y . Structural and biochemical basis of apoptotic activation by Smac/DIABLO. Nature 2000; 406: 855–862.
Srinivasula SM, Datta P, Fan XJ, Fernandes-Alnemri T, Huang Z, Alnemri ES . Molecular determinants of the caspase-promoting activity of Smac/DIABLO and its role in the death receptor pathway. J Biol Chem 2000; 275: 36152–36157.
Srinivasula SM, Gupta S, Datta P, Zhang Z, Hegde R, Cheong N et al. Inhibitor of apoptosis proteins are substrates for the mitochondrial serine protease Omi/HtrA2. J Biol Chem 2003; 278: 31469–31472.
Yang QH, Church-Hajduk R, Ren J, Newton ML, Du C . Omi/HtrA2 catalytic cleavage of inhibitor of apoptosis (IAP) irreversibly inactivates IAPs and facilitates caspase activity in apoptosis 58. Genes Dev 2003; 17: 1487–1496.
Suzuki Y, Takahashi-Niki K, Akagi T, Hashikawa T, Takahashi R . Mitochondrial protease Omi/HtrA2 enhances caspase activation through multiple pathways. Cell Death Differ 2004; 11: 208–216.
Martins LM, Morrison A, Klupsch K, Fedele V, Moisoi N, Teismann P et al. Neuroprotective role of the Reaper-related serine protease HtrA2/Omi revealed by targeted deletion in mice. Mol Cell Biol 2004; 24: 9848–9862.
Jones JM, Datta P, Srinivasula SM, Ji W, Gupta S, Zhang Z et al. Loss of Omi mitochondrial protease activity causes the neuromuscular disorder of mnd2 mutant mice. Nature 2003; 425: 721–727.
Meier P, Finch A, Evan G . Apoptosis in development. Nature 2000; 407: 796–801.
Ditzel M, Meier P . Ubiquitylation in apoptosis: DIAP1's (N-)en(d)igma. Cell Death Differ 2005; 12: 1208–1212.
Vaux DL, Silke J . IAPs RINGs and ubiquitylation. Nat Rev Mol Cell Biol 2005; 6: 287–297.
Igaki T, Suzuki Y, Tokushige N, Aonuma H, Takahashi R, Miura M . Evolution of mitochondrial cell death pathway: proapoptotic role of HtrA2/Omi in Drosophila. Biochem Biophys Res Commun 2007; 356: 993–997.
Challa M, Malladi S, Pellock BJ, Dresnek D, Varadarajan S, Yin YW et al. Drosophila Omi, a mitochondrial-localized IAP antagonist and proapoptotic serine protease. EMBO J 2007; 26: 3144–3156.
Scott FL, Denault JB, Riedl SJ, Shin H, Renatus M, Salvesen GS . XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs. EMBO J 2005; 24: 645–655.
Verhagen AM, Kratina TK, Hawkins CJ, Silke J, Ekert PG, Vaux DL . Identification of mammalian mitochondrial proteins that interact with IAPs via N-terminal IAP binding motifs. Cell Death Differ 2007; 14: 348–357.
Ditzel M, Wilson R, Tenev T, Zachariou A, Paul A, Deas E et al. Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis. Nat Cell Biol 2003; 5: 467–473.
Martins LM, Turk BE, Cowling V, Borg A, Jarrell ET, Cantley LC et al. Binding specificity and regulation of the serine protease and PDZ domains of HtrA2/Omi. J Biol Chem 2003; 278: 49417–49427.
Li W, Srinivasula SM, Chai J, Li P, Wu JW, Zhang Z et al. Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi. Nat Struct Biol 2002; 22: 22.
Plun-Favreau H, Klupsch K, Moisoi N, Gandhi S, Kjaer S, Frith D et al. The mitochondrial protease HtrA2 is regulated by Parkinson's disease-associated kinase PINK1. Nat Cell Biol 2007; 9: 1243–1252.
Alnemri ES . HtrA2 and Parkinson's disease: think PINK? Nat Cell Biol 2007; 9: 1227–1229.
Hay BA, Maile R, Rubin GM . P element insertion-dependent gene activation in the Drosophila eye. Proc Natl Acad Sci USA 1997; 94: 5195–5200.
Acknowledgements
We thank Bruce Hay for the anti-DIAP1. We also thank the Bloomington Stock Center for Drosophila stocks. This study was supported by NIH grants GM076176 (ESA), CA78890 (ESA), and GM50231 (JBJ), and NSF grant IOB-0416760 (JBJ). FSK is supported by NRSA award T32-CA09678.
Author information
Authors and Affiliations
Corresponding author
Additional information
Edited by G Melino
Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd)
Supplementary information
Rights and permissions
About this article
Cite this article
Khan, F., Fujioka, M., Datta, P. et al. The interaction of DIAP1 with dOmi/HtrA2 regulates cell death in Drosophila. Cell Death Differ 15, 1073–1083 (2008). https://doi.org/10.1038/cdd.2008.19
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/cdd.2008.19
Keywords
This article is cited by
-
Wg/Wnt1 and Erasp link ER stress to proapoptotic signaling in an autosomal dominant retinitis pigmentosa model
Experimental & Molecular Medicine (2023)
-
Loss of Drosophila i-AAA protease, dYME1L, causes abnormal mitochondria and apoptotic degeneration
Cell Death & Differentiation (2016)
-
Apoptosis in Drosophila: which role for mitochondria?
Apoptosis (2016)
-
Mitochondrial fusion is regulated by Reaper to modulate Drosophila programmed cell death
Cell Death & Differentiation (2011)
-
Drosophila HtrA2 is dispensable for apoptosis but acts downstream of PINK1 independently from Parkin
Cell Death & Differentiation (2009)
