Article |
Address correspondence to Sharad Kumar, The Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, P.O. Box 14, Rundle Mall, Adelaide SA 5000, Australia. Tel.: 61-88-222-3738. Fax: 61-88-222-3139. E-mail: sharad.kumar{at}imvs.sa.gov.au
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Abstract |
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Key Words: DRONC; caspase activation; DRICE; apoptosis; apoptosome
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Introduction |
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DRONC is the only Drosophila caspase that contains a caspase recruitment domain, suggesting that it is the functional counterpart of CED-3 in Caenorhabditis elegans and caspase-9 in mammals (Dorstyn et al., 1999a). Genetic and gene ablation experiments have demonstrated that DRONC is essential for programmed cell death during development and is required for cell death in the fly eye, mediated by Reaper, Hid, and Grim (Hawkins et al., 2000; Meier et al., 2000; Quinn et al., 2000). DRONC interacts with and processes the effector caspase DRICE, suggesting that DRONC is an initiator caspase (Hawkins et al., 2000; Kumar and Doumanis, 2000; Meier et al., 2000). Furthermore, dronc transcript is massively up-regulated in larval salivary glands and midgut by the hormone ecdysone, which mediates programmed deletion of larval tissues during larval/pupal metamorphosis (Dorstyn et al., 1999a, b; Baehrecke, 2000). Although the biochemical mechanism of DRONC activation is not well understood, genetic studies demonstrate that DARK/Dapaf-1/HAC-1, the Drosophila CED-4/Apaf-1 homologue, is required for DRONC-mediated cell death (Quinn et al., 2000), suggesting that a DARKDRONC complex may be necessary for initial autocatalytic activation of DRONC. In support of this model, DRONC has been shown to interact with DARK in a caspase recruitment domaindependent manner (Quinn et al., 2000).
In mammals, cellular stress signals lead to the release of mitochondrial cytochrome c, which binds to and enables oligomerization of the adaptor protein Apaf-1 (Li et al., 1997; Zou et al., 1997, 1999). Apaf-1, in turn, recruits the precursor form of caspase-9, an apical caspase, and thereby promotes its proximity-induced autocatalytic activation (Li et al., 1997; Zou et al., 1997, 1999). The requirement of cytochrome c in caspase activation is well documented in mammals. In mice in which the cytochrome c gene has been deleted by homologous recombination, the caspase-9Apaf-1 pathway is severely impaired (Li et al., 2000). In contrast, in C. elegans, there is no published evidence that CED-4mediated CED-3 activation requires cytochrome c, suggesting that cytochrome c function in apoptosis may have evolved later in more complex organisms.
The role of cytochrome c in Drosophila caspase activation is not well established. Structurally, DARK is more similar to its mammalian counterpart Apaf-1 than to CED-4, in that it contains several WD40 repeats that are not found in CED-4, and it binds cytochrome c in vitro (Kanuka et al., 1999; Rodriguez et al., 1999; Zhou et al., 1999). However, it is unclear whether the Drosophila protein requires cytochrome c for its oligomerization. Kanuka et al. (1999) have shown that addition of cytochrome c and dATP to Drosophila embryo extracts results in a twofold enhancement of DEVDase activity, which was not seen in extracts prepared from dapaf-1 mutant embryos. The cytochrome c/dATPinduced caspase activity was also inhibited by an ATPase inhibitor, suggesting that the ATPase activity of Dapaf-1 may be necessary for its ability to mediate caspase activation (Kanuka et al., 1999).
There are conflicting reports of cytochrome c release from the mitochondria of Drosophila cells during apoptosis. In Drosophila SL2 cells, overexpression of rpr or treatment with staurosporine or cycloheximide causes an apparent release of cytochrome c into the cytosol (Kanuka et al., 1999). On the other hand, Varkey et al. (1999) demonstrated an alteration in cytochrome c conformation, as evidenced by display of an otherwise hidden epitope, in Drosophila tissues preceding apoptosis. This alteration occurs without release of the protein into the cytosol. In cell-free studies, caspase activation was triggered by mitochondria from apoptotic cells but not by those from healthy cells. These observations suggest that in the fly, the function of cytochrome c in caspase activation may be somewhat different from its role in mammalian cells. If cytochrome c is not released from mitochondria, it is unclear if it is required for caspase activation in vivo. The analysis of endogenous Drosophila caspases has been limited due to a lack of appropriate antibodies.
In this paper, we have investigated whether cytochrome c release from mitochondria is necessary for the activation of key Drosophila apoptotic caspases, i.e., DRONC and DRICE. Using specific antibodies, we demonstrate that during apoptosis, DRONC and DRICE are rapidly processed without any cytochrome c release from mitochondria. We also show that in cell-free extracts, both DRONC and DRICE are recruited to a >700-kD complex, which is presumably required for their activation.
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Results |
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Cytochrome c remains associated with mitochondria in dying BG2 cells
To confirm our cell fractionation experiments, we performed immunostaining of BG2 cells undergoing apoptosis. Staining with the mitochondrial marker MitoTracker showed punctate staining that was similar to that seen with cytochrome c staining (Fig. 4). Upon treatment with cycloheximide, there were clear morphological changes in treated cells that became evident at 4 h, however, the majority of the MitoTracker and cytochrome c staining was still punctate. These results suggest that after treatment with cycloheximide, mitochondria remain intact in dying cells and the majority of the cytochrome c remains associated with mitochondria (Fig. 4). Staining of cells with the cationic dye DiOC6 over 8 h indicated that mitochondrial membrane potential is not altered in BG2 cells after cycloheximide treatment (unpublished data). Staining of endogenous DRONC, using the affinity-purified DRONC antibody, showed that the majority of the protein is cytosolic with some punctate staining that appears to become more concentrated in the perinuclear regions of the cells treated with cycloheximide (Fig. 4). We also determined the localization of DRICE, a key downstream caspase activated by DRONC (Meier et al., 2000). We used an antibody for DRICE that detects only the processed form. As shown in Fig. 4, at 0 h, there was little or no DRICE staining in cells, but by 4 h, significant processed DRICE was detected in BG2 cells. At 8 h, considerably more active DRICE was evident, and in many cells it showed punctate staining or appeared to have accumulated in large aggregates (Fig. 4).
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To further explore the role of cytochrome c in the formation of the DRONC-containing complex, we immunodepleted cytochrome c from S100 fractions (Fig. 10 A). These fractions were then subjected to gel filtration experiments. When incubated at 27°C, a small fraction of DRONC was found in the high molecular mass complex (Fig. 10 B). Addition of cytochrome c and dATP caused a significant increase in the recruitment of DRONC to the >700-kD complex. Immunoblotting the fractions with the cytochrome c antibody showed that incubation of S100 at 27°C results in the recruitment of a significant proportion of cytochrome c to the >700-kD complex (Fig. 10 C). Interestingly, only dimeric (26 kD) cytochrome c was detected in the >700-kD complex. These results suggest that cytochrome c and dATP, at least in part, are responsible for the formation of the complex.
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Discussion |
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Treatment of cells with multiple death stimuli, including cycloheximide, factor withdrawal, ecdysone, or transfection of the Bax-like protein DEBCL, failed to induce the release of detectable cytochrome c. Our immunolocalization studies suggest that cytochrome c remained associated with mitochondria in cells undergoing apoptosis. Moreover, the mitochondria appeared remarkably intact even in late stage apoptotic cells. However, in some cells undergoing apoptosis there appeared to be an accumulation of mitochondria in the perinuclear region of the cells, which is similar to that reported in mammalian cells expressing truncated Bid (Li et al., 1998). Interestingly, a fraction of endogenous DRONC colocalized with mitochondria. Cell fractionation studies suggest that a significant proportion of the DRONC precursor and processed DRONC is associated with both the mitochondrial membrane fraction and the light membrane fraction. Therefore, it is possible that membrane recruitment of DRONC is important for its activation. Overexpression of DEBCL results in its localization to mitochondria (Igaki et al., 2000; Zhang et al., 2000). Again, in the DEBCL-overexpressing cells, a fraction of DRONC appeared to colocalize with mitochondria and DEBCL, suggesting that translocation of DRONC may be important for caspase activation.
If cytochrome c is not released from mitochondria during apoptosis, how may caspase activation occur in Drosophila cells? Previous work using cytochrome c monoclonal antibodies has shown that in Drosophila egg chambers and in SL2 cells, cytochrome c undergoes conformational changes within the mitochondria before the onset of apoptosis (Varkey et al., 1999). However, cytochrome c was not released from mitochondria during apoptosis, as we have found in our studies with BG2 and l(2)mbn cells. This is in contrast to apoptosis in mammalian cells where cytochrome c release from mitochondria is rapid and complete (Green and Reed, 1998; Goldstein et al., 2000). The work by Varkey et al. (1999) also demonstrates that mitochondria isolated from apoptotic SL2 cells can mediate caspase activation in vitro. These results suggest that although both mitochondria and cytochrome c play a role in apoptosis, in Drosophila, cytochrome c release from mitochondria does not occur. One possibility is that cytochrome c is released but remains tethered to the mitochondrial membrane (Varkey et al., 1999). In such a case, an apoptosome, albeit somewhat different from the mammalian apoptosome, may form in the vicinity of mitochondria. Our observations that in BG2 cells undergoing apoptosis, mitochondria become somewhat clustered and a significant proportion of DRONC colocalizes with mitochondria support this possibility. Because proapoptotic proteins such as DEBCL and Hid also localize to mitochondria, mitochondria clearly play a role in apoptosis in Drosophila.
We explored the possibility of apoptosome formation by gel filtration of BG2 cell extracts and testing for recruitment of DRONC to high molecular mass complexes. Incubation of BG2 cell extracts containing endogenous cytochrome c at 27°C37°C resulted in the recruitment of cytosolic DRONC to a complex of >700 kD. Furthermore, a large fraction of DRONC in the complex was in processed form, suggesting that recruitment to the putative apoptosome may be necessary for DRONC activation. Interestingly, a significant proportion of DRICE was also found in the >700-kD complex, most of which was fully processed when extracts were incubated at 37°C. As DRICE has been shown to directly interact with DRONC, the recruitment of DRICE may be required for the initial DRONC-mediated activation of DRICE. This prediction is consistent with the studies with caspase-9/Apaf-1 apoptosome, which also mediates the initial activation of caspase-3 (Bratton et al., 2001).
Given that cytochrome c is not released from mitochondria in Drosophila cells, yet cytochrome c addition to Drosophila cell extracts can enhance the formation of an apoptosome-like complex and caspase activation, it is reasonable to hypothesize that caspase activation in insects follows a more primitive mechanism, which may be the precursor to the caspase activation pathways in mammals. However, our data presented in this paper does not rule out the possibility that a factor(s) other than cytochrome c may be involved in caspase activation in Drosophila cells. In future studies, the elucidation of the exact nature of the Drosophila apoptosome may shed light on the mechanism of apoptosome formation and caspase activation.
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Materials and methods |
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Cell death and caspase processing assays
5 x 106 cells were washed in PBS and incubated with 20 µg/ml cycloheximide (Sigma-Aldrich) or were grown in medium depleted of FBS and insulin. Cells were harvested at various time points after apoptotic induction and the number of surviving cells was estimated by trypan blue exclusion. For immunoblotting with caspase antibodies, cells were centrifuged at 400 g for 5 min and pellets were resuspended in 50 µl PBS, mixed with an equal volume of 2x SDS protein loading buffer (100 mM Tris-HCl, pH 6.8, 200 mM DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol), and then boiled for 10 min before SDS-PAGE.
Caspase assays
Cells were harvested and resuspended in 100 µl caspase assay buffer (0.1 M Hepes, pH 7, 0.1% CHAPS, 10% PEG4000, 10 mM DTT, supplemented with 1x protease inhibitor cocktail; Roche Biochemicals). Cells were lysed by three cycles of freezethawing. Cell debris was centrifuged at 9,000 g for 10 min at 4°C. The cleared lysates (100 µg) were assessed for caspase activity by incubation with 100 µM of either DEVD-7-amino-4-methylcoumaride (DEVD-amc; Enzyme System Products) or VDVAD-amc (California Peptide Research) in a total volume of 40 µl in caspase assay buffer at 37°C for 30 min. We also tested caspase activity at 27°C because Drosophila cells grow at this temperature. However at 27°C, measured caspase activity was considerably lower. Reactions were transferred to acryl cuvettes (Sarstedt), and 3 ml water was added before analysis on a luminescence spectrometer (excitation 385 nm, emission 460 nm; PerkinElmer).
RNA interference
Regions of cDNA for dronc (nt 7811047), dark (nt 36033962), and the negative control mouse N4WBP5 (700-bp coding region; Harvey et al., 2002) were PCR amplified using appropriate primers, and cloned into pGEM-T Easy (Promega). Plasmids were linearized and RNA synthesized using T7 and SP6 Megascript kits (Ambion). Sense and antisense strands were annealed to generate dsRNA, and RNA quality was analyzed on agarose gel. dsRNA (37 nM) was added to cells in 1 ml serum-free media and mixed vigorously. Cells were incubated for 1 h followed by the addition of 2 ml of media supplemented with 10% FBS. Cells were incubated overnight and then treated with cycloheximide for 6 h before cell lysis and immunoblotting.
Cell fractionation
5 x 106 cells were harvested by centrifugation at 400 g for 5 min. Cell pellets were resuspended in 200 µl of lysis buffer A (20 mM Hepes-KOH, pH 7.5, 50 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT in 250 mM sucrose and supplemented with protease inhibitor cocktail) and incubated on ice for 5 min. Cells were homogenized by 10 strokes with a tight fitting pestle, and homogenates were centrifuged twice at 500 g for 10 min at 4°C. Pellets, consisting of unbroken cells and nuclei, were retained and resuspended in 100 µl PBS. The supernatants were centrifuged at 10,000 g for 15 min at 4°C, and the heavy membrane/mitochondrial pellets were washed in cold buffer, resuspended in 30 µl lysis buffer A, and stored at -70°C. Supernatants were further centrifuged at 100,000 g for 30 min at 4°C. The resulting pellets (P100) consisting of light membrane fractions were resuspended in 30 µl lysis buffer A and stored at -70°C. Resulting supernatants (S100) were also stored at -70°C. For protein gel analysis, 4x SDS protein loading buffer was added to each fraction and samples were resolved on 815% SDS-PAGE gels.
Affinity purification of DRONC antibodies
Anti-DRONC rabbit polyclonal sera was passed through a column of glutathione-S-transferase (GST) coupled to cyanogen bromideactivated Sepharose 4 (Amersham Pharmacia Biotech) to remove GST-specific antibodies. Flowthrough from this step was affinity purified against DRONC p14GST (Quinn et al., 2000) coupled to cyanogen bromideactivated Sepharose 4. Bound proteins were eluted into Tris-HCl (pH 8.6) with 100 mM glycine (pH 2.5) and dialyzed against PBS. Affinity-purified antibodies were diluted in 50% glycerol and stored at -20°C.
Generation and affinity purification of antiactive DRICE antibodies
Active DRICEspecific antibodies used in immunofluorescence studies (Figs. 48) were raised in rabbits using a synthetic octapeptide corresponding to the COOH terminus of the DRICE large subunit (QRSQTETD) conjugated with keyhole limpet hemocyanin as the immunogen (Covance). Active DRICEspecific antibodies were purified by sequential protein affinity purification. Antisera were first applied to an affinity column containing full-length DRICE with an inactivating active site mutation (C211A). The flowthrough from this column was applied to an affinity column containing the DRICE large subunit ending at the caspase cleavage site TETD (1230). Bound antibody was eluted using 100 mM glycine, pH 2.5. Immunoblotting of the eluted anti-DRICE antibodies showed that they were specific for the cleaved large subunit of active DRICE. They did not detect full-length DRICE or other caspases, including the closely related DCP-1 (unpublished data).
A second rabbit anti-DRICE polyclonal antibody, used for immunoblotting, was generated using a version of DRICE that extends from residue 80 to the COOH terminus, followed by a COOH-terminal 6x His tag as the immunogen. DRICE-reactive antibodies were purified by binding this serum with the antigen and washing extensively. DRICE-reactive antibodies were eluted as above for the DRICE cleavage-specific antibodies.
Immunoblotting
After SDS-PAGE, protein was transferred to a PVDF membrane (Dupont). The membranes were blocked in 5% skim milk (Diploma)PBS/0.05% Tween 20 solution overnight at 4°C. Blots were incubated with 0.5 µg/ml anticytochrome c mouse antibody 7H8.2C12 (BD PharMingen), 1.5 µg/ml affinity-purified anti-DRONC rabbit antibody, 0.1 µg/ml anti-HA mouse antibody (Roche Biochemicals), or 1.5 µg/ml anti-DRICE rabbit antibody, followed by incubation with the corresponding HRP-conjugated secondary antibody. Signals were visualized by ECL (Amersham Pharmacia Biotech).
Immunofluorescence
2 x 106 cells were grown on coverslips in 35-mm dishes. After apoptotic induction, cells were washed in PBS. For MitoTracker staining, cells were incubated with 300 nM MitoTracker red (Molecular Probes) in PBS with 1% FBS for 15 min at room temperature (RT). Coverslips were either mounted on slides in antifade solution (80% glycerol, 1% propylgalate) or were fixed with 3% paraformaldehyde for 10 min at RT, washed in PBS, and then permeabilized in 0.1% Triton X-100 for 10 min at RT before antibody staining. Cells were costained with anticytochrome c mouse antibody clone 6H2.B4 (BD PharMingen) at 2.5 µg/ml, affinity-purified anti-DRONC rabbit antibody at 4 µg/ml, and/or anti-HA high affinity rat antibody (Roche Biochemicals) at 1 µg/ml and antiactive DRICE antibody at 4 µg/ml. All antibodies were diluted in PBS (0.1% FBS) and incubated with cells overnight at 4°C. Cells were then washed three times in PBS and incubated with FITC-conjugated antimouse, rabbit, or rat secondary antibodies and rhodamine-conjugated antirabbit or mouse antibodies for 1 h at RT. Cells were again washed several times in PBS and coverslips were mounted onto slides in antifade solution. Cell staining was visualized under fluorescent microscopy.
Gel filtration chromatography
108 BG2 cells were resuspended in 200 µl buffer A (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT supplemented with protease inhibitor cocktail; Zou et al., 1999) and lysed by freezethaw. Cell debris was pelleted twice by centrifugation at 10,000 g for 20 min at 4°C. Supernatants were further centrifuged at 100,000 g for 30 min at 4°C. Resulting lysates (S100) were incubated at 4°C, 27°C, or 37°C alone or in the presence of 2 µg cytochrome c and 2 mM dATP. Proteins were then fractionated through a Superdex 200 column (Amersham Pharmacia Biotech). Fractions were collected 18 min after sample injection and a total of 42 fractions were collected at a rate of 0.4 ml/fraction/min. Aliquots from each fraction were resolved by electrophoresis through 815% polyacrylamide gels. For immunodepletion of cytochrome c, S100 lysates were incubated overnight at 4°C with an anticytochrome c antibody 6H2 (BD PharMingen). Antigenantibody complexes were removed by protein GSepharose, and cytochrome cdepleted extracts were fractionated through a Superdex 200 column after incubation at 27°C alone or in the presence of 2 µg cytochrome c and 2 mM dATP.
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Footnotes |
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Acknowledgments |
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This work was supported by a grant and a fellowship to S. Kumar from the National Health and Medical Research Council of Australia.
Submitted: 29 November 2001
Revised: 1 February 2002
Accepted: 4 February 2002
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References |
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---|
Brachmann, C.B., O.W. Jassim, B.D. Wachsmuth, and R.L. Cagan. 2000. The Drosophila bcl-2 family member dBorg-1 functions in the apoptotic response to UV-irradiation. Curr. Biol. 10:547550.[CrossRef][Medline]
Bratton, S.B., G. Walker, S.M. Srinivasula, X.M. Sun, M. Butterworth, E.S. Alnemri, and G.M. Cohen. 2001. Recruitment, activation and retention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP complexes. EMBO J. 20:9981009.
Cain, K., D.G. Brown, C. Langlais, and G.M. Cohen. 1999. Caspase activation involves the formation of the aposome, a large (700kDa) caspase-activating complex. J. Biol. Chem. 274:2268622692.
Cain, K., S.B. Bratton, C. Langlais, G. Walker, D.G. Brown, X.-M. Sun, and G.M. Cohen. 2000. Apaf-1 oligomerises into biologically active 700-kDa and inactive
1.4MDa apoptosome complexes. J. Biol. Chem. 275:60676070.
Colussi, P.A., L.M. Quinn, D.C.S. Huang, M. Coombe, S.H. Read, H. Richardson, and S. Kumar. 2000. DEBCL, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery. J. Cell Biol. 148:703714.
Dorstyn, L., P.A. Colussi, L.M. Quinn, H. Richardson, and S. Kumar. 1999a. DRONC, an ecdysone-inducible Drosophila caspase. Proc. Natl. Acad. Sci. USA. 96:43074312.
Dorstyn, L., S.H. Read, L.M. Quinn, H. Richardson, and S. Kumar. 1999b. DECAY, a novel Drosophila caspase related to mammalian caspase-3 and caspase-7. J. Biol. Chem. 274:3077830783.
Fraser, A.G., and G.I. Evan. 1997. Identification of a Drosophila melanogaster ICE/CED-3-related protease, drICE. EMBO J. 16:28052813.
Fraser, A.G., N.J. McCarthy, and G.I. Evan. 1997. DrICE is an essential caspase required for apoptotic activity in Drosophila cells. EMBO J. 16:61926199.
Green, D.R., and J.C. Reed. 1998. Mitochondria and apoptosis. Science. 281:13091312.
Gross, A., X.M. Yin, K. Wang, M.C. Wei, J. Jockel, C. Milliman, H. Erdjument-Bromage, P. Tempst, and S.J. Korsmeyer. 1999. Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death. J. Biol. Chem. 274: 11561163.
Harvey, K.F., L.M. Shearwin-Whyatt, A. Fotia, R.G. Parton, and S. Kumar. 2002. N4WBP5, a potential target for ubiquitination by the Nedd4 family of proteins, is a novel Golgi-associated protein. J. Biol. Chem. In press.
Harvey, N.L., T. Daish, K. Mills, L. Dorstyn, L.M. Quinn, S.H. Read, H. Richardson, and S. Kumar. 2001. Characterisation of the Drosophila caspase, DAMM. J. Biol. Chem. 276:2534225350.
Hawkins, C.J., S.J. Yoo, E.P. Peterson, S.L. Wang, S.Y. Vernooy, and B.A. Hay. 2000. The Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by Diap1, Hid and Grim. J. Biol. Chem. 275:2708427093.
Igaki, T., H. Kanuka, N. Inohara, K. Sawaamoto, G. Nunez, H. Okano, and M. Miura. 2000. Drob-1, a Drosophila member of the Bcl-2/CED-9 family that promotes cell death. Proc. Natl. Acad. Sci. USA. 97:662667.
Kluck, R.M., M.D. Esposito, G. Perkins, C. Renken, T. Kuwana, E. Bossy-Wetzel, M. Goldberg, T. Allen, M.J. Barber, D.R. Green, and D.D. Newmeyer. 1999. The pro-apoptotic proteins Bid and Bax cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol. J. Cell Biol. 147:809822.
Kumar, S., and P.A. Colussi. 1999. Prodomains-adaptors-oligomerization: the pursuit of caspase activation in apoptosis. Trends Biochem. Sci. 24:14.[CrossRef][Medline]
Li, H., H. Zhu, C.J. Xu, and J. Yuan. 1998. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell. 94:491501.[Medline]
Li, P., D. Nijhawan, I. Budihardjo, S.M. Srinivasula, M. Ahmad, E.S. Alnemri, and X. Wang. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 91:479489.[Medline]
McCall, K., and H. Stellar. 1998. Requirement for DCP-1 caspase during Drosophila oogenesis. Science. 279:230234.
Meier, P., J. Silke, S.J. Leevers, and G.I. Evan. 2000. The Drosophila caspase DRONC is regulated by Diap1. EMBO J. 19:598611.
Nechushtan, A., C.L. Smith, I. Lamensdorf, S.-H. Yoon, and R.J. Youle. 2001. Bax and Bac coalesce into novel mitochondria-associated clusters during apoptosis. J. Cell Biol. 153:12651276.
Quinn, L.M., L. Dorstyn, K. Mills, P.A. Colussi, P. Chen, M. Coombe, J.M. Abrams, S. Kumar, and H. Richardson. 2000. An essential role for the caspase DRONC in developmentally programmed cell death in Drosophila. J. Biol. Chem. 275:4041640424.
Rodriguez, A., H. Oliver, H. Zou, P. Chen, X. Wang, and J.M. Abrams. 1999. DARK is a Drosophila homolog of Apaf-1/CED-4 and functions in an evolutionarily conserved death pathway. Nat. Cell Biol. 1:272279.[CrossRef][Medline]
Rodriguez, J., and Y. Lazebnik. 1999. Caspase-9 and Apaf-1 form an active holoenzyme. Genes Dev. 13:31793184.
Song, Z., K. McCall, and H. Steller. 1997. DCP-1, a Drosophila cell death protease essential for development. Science. 275:536540.
Ui-Tei, K., M. Nagano, S. Sato, and Y. Miyata. 2000. Calmodulin-dependent and independent apoptosis in cells of a Drosophila neuronal cell line. Apoptosis. 5:133140.[CrossRef][Medline]
Varkey, J., P. Chen, R. Jemmerson, and J.M. Abrams. 1999. Altered cytochrome c display precedes apoptotic cell death in Drosophila. J. Cell Biol. 144:701710.
Wang, X. 2001. The expanding role of mitochondria in apoptosis. Genes Dev. 15:29222933.
Wei, M.C., T. Lindsten, V.K. Mootha, S. Weiler, A. Gross, M. Ashiya, C.B. Thompson, and S.J. Korsmeyer. 2000. tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev. 14:20602071.
Zhang, H., Q. Huang, N. Ke, S. Matsuyama, B. Hammock, A. Godzik, and J.C. Reed. 2000. Drosophila pro-apoptotic Bcl-2/Bax homologue reveals evolutionary conservation of cell death mechanisms. J. Biol. Chem. 275:2730327306.
Zou, H., W.J. Henzel, X. Liu, A. Lutschg, and X. Wang. 1997. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell. 90:405413.[Medline]
Zou, H., Y. Li, X. Liu, and X. Wang. 1999. An Apaf-1 cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J. Biol. Chem. 274:1154911556.