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THE JOURNAL OF BIOLOGICAL CHEMISTRY 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 275, No. 35, Issue of September 1, pp. 2720527211, 2000 Printed in U.S.A.

Disruption of the CED-9 CED-4 Complex by EGL-1 Is a Critical Step for Programmed Cell Death in Caenorhabditis elegans*
Received for publication, February 3, 2000, and in revised form, June 2, 2000 Published, JBC Papers in Press, June 8, 2000, DOI 10.1074/jbc.M000858200

Luis del Peso, Vctor M. Gonzalez , Naohiro Inohara, Ronald E. Ellis**, and Gabriel Nunez
From the Department of Pathology and Comprehensive Cancer Center and the **Department of Biology, The University of Michigan, Ann Arbor, Michigan 48109

In the nematode Caenorhabditis elegans, the apoptotic machinery is composed of four basic elements: the caspase CED-3, the Apaf-1 homologue CED-4, and the Bcl-2 family members CED-9 and EGL-1. The ced9(n1950) gain-of-function mutation prevents most, if not all, somatic cell deaths in C. elegans. It encodes a CED-9 protein with a glycine-to-glutamate substitution at position 169, which is located within the highly conserved Bcl-2 homology 1 domain. We performed biochemical analyses with the CED-9G169E protein to gain insight into the mechanism of programmed cell death. We find that CED-9G169E retains the ability to bind both EGL-1 and CED-4, although its affinity for EGL-1 is reduced. In contrast to the behavior of wild-type CED-9, the interaction between CED-9G169E and CED-4 is not disrupted by expression of EGL-1. Furthermore, CED-4 and CED9G169E co-localizes with EGL-1 to the mitochondria in mammalian cells, and expression of EGL-1 does not induce translocation of CED-4 to the cytosol. Finally, the ability of EGL-1 to promote apoptosis is impaired by the replacement of wild-type CED-9 with CED-9G169E, and this effect is correlated with the inability of EGL-1 to induce the displacement of CED-4 from the CED-9 CED-4 complex. These studies suggest that the release of CED-4 from the CED-9 CED-4 complex is a necessary step for induction of programmed cell death in C. elegans.

Programmed cell death is a conserved mechanism of cellular demise developed by animals to delete cells during development and to maintain tissue homeostasis (1). Genetic analyses in the nematode Caenorhabditis elegans have elucidated a genetic pathway that is essential for the control of cell death (2). Two genes, ced-3 and ced-4, are required for the killing process (2). The product of ced-3 is a caspase, a member of a family of cysteine proteases (3). CED-3 and homologous caspases are synthesized as zymogens that require processing at specific

* This work was supported by Grant CA-64556 from the National Institutes of Health, a grant from The J. P. McCarthy Foundation (to G. N.), and by Grant RPG-97-172-01-DDC from the American Cancer Society (to R. E. E.). 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. Supported by a training grant from the University of Michigan/NCI Program. These authors contributed equally to this work. Supported by a fellowship from the Spanish Ministry of Education and Science. Recipient of the Research Career and Development Award CA64421 from the National Institutes of Health. To whom correspondence should be addressed. Tel.: 734-764-8514; Fax: 734-647-9654; E-mail: Gabriel.Nunez@umich.edu.
This paper is available on line at http://www.jbc.org

sites for activation (4). Once activated, CED-3 in the nematode and related caspases in other organisms kill by targeting protein substrates in the dying cell (4). The product of ced-4 is homologous to mammalian Apaf-1 and functions as a positive regulator of CED-3 (5, 6). By contrast, ced-9 protects cells that normally survive from undergoing programmed cell death (7). The ced-9 product is homologous to Bcl-2 family members that promote survival in mammals (8). Biochemical analyses of CED-3, CED-4, and CED-9 have provided critical insight into the mechanism by which these proteins interact to regulate cell death in the nematode. CED-4 binds to CED-3 and promotes its proteolytic and enzymatic activation (9 13). By contrast, CED-9 associates with CED-4 and inhibits its ability to activate CED-3 (9, 1315). The precise mechanism by which CED-4 promotes the activation of CED-3 remains unclear but might involve CED-4 oligomerization, which could promote CED-3 autoactivation by bringing several CED-3 molecules into close proximity (16). Recent analyses in C. elegans showed that EGL-1 is a nematode homologue of mammalian pro-apoptotic Bcl-2 family members (17). Gain-of-function mutations of egl-1 cause two hermaphrodite-specific neurons to undergo programmed cell death, whereas a loss-of-function mutation prevents most, if not all, somatic cell deaths in the worm (17). EGL-1 belongs to a subfamily of Bcl-2 proteins that contain only one of four Bcl-2 homology (BH)1 domains, the BH3 domain (18). EGL-1 associates with and inhibits CED-9 through this BH3 domain (17, 19). Furthermore, in mammalian cells, the binding of EGL-1 to CED-9 results in CED-4 release from CED-9 and the translocation of CED-4 from mitochondria to the cytosol (19). Thus, EGL-1 might promote cell death by binding to CED-9 and releasing the cell death activator CED-4 from the CED9 CED-4 protein complex. However, it is unclear whether the critical event for the initiation of programmed cell death is the binding of EGL-1 to CED-9 or the release of CED-4 from the CED-9 CED-4 complex. The ced-9(n1950 gain-of-function) mutation prevents most, if not all, somatic cell deaths during C. elegans development (7, 20). This mutation results in a glycine to glutamate substitution at position 169, which is located within the highly conserved BH1 domain of CED-9 (20). Mutational analysis of mammalian anti-apoptotic Bcl-2 family members (21, 22), as well as determination of the three-dimensional structure of Bcl-XL in complex with a BH3 peptide (23), showed that the BH1 and BH3 domains of Bcl-XL are part of a hydrophobic pocket that mediates binding to the BH3 domain of pro-apoptotic Bcl-2 family members. In the present studies, we char1 The abbreviations used are: BH, Bcl-2 homology domain; mAb, mouse monoclonal antibody; pAb, polyclonal antibody; HA, hemagglutinin; HEK, human embryo kidney; wt, wild-type.

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Molecular Characterization of the Nematode CED-9G169E Protein

acterized the CED-9G169E protein to learn about the mechanism by which it inhibits programmed cell death in the nematode.
EXPERIMENTAL PROCEDURES

ReagentsM2, a monoclonal antibody (mAb) that recognizes the FLAG epitope, poly-L-lysine, and fluorescein isothiocyanate-conjugated goat anti-mouse IgG were purchased from Sigma; anti-Flag D-8 polyclonal antibody (pAb), anti-hemagglutinin (HA) Y-11 pAb, and anti-cMyc A-14 pAb were from Santa Cruz Biotechnology; anti-HA 12CA5 mAb was from Roche Molecular Biochemicals; anti-AU1 mAb was from Babco. Protein A/G-Sepharose 4B was purchased from Zymed Laboratories Inc. Mitotracker Red CMXRos and rhodamine-conjugated goat anti-rabbit were from Molecular Probes. Plasmid ConstructionsThe expression plasmids producing epitopetagged CED-4, CED-3, CED-9, and EGL-1 have been described (13, 19). The CED-9G169E expression plasmids were generated by a polymerase chain reaction protocol, using the primers 5 -CCAATGTCTTATGAACGTTTGATAGGT and 5 -ACCTATCAAACGTTCATAAGACATTGG to amplify the ced-9(n1950) cDNA, which was then subcloned into the pcDNA3-HA or -FLAG plasmids, which are engineered to produce FLAG or HA epitope-tagged proteins (24). The authenticity of each construct was confirmed by dideoxy sequencing. Transfection, Immunoprecipitation, and ImmunoblottingHuman embryonic kidney cells (HEK293T) were cultured in Dulbeccos modified Eagles medium (Life Technologies, Inc.) suplemented with 10% fetal bovine serum (Hyclone). Culture dishes (3 106 cells/100-mm plate) were transfected by the calcium phosphate method with the indicated amounts of plasmids (see figure legends). Transfected cells were harvested by centrifugation at 4 C and lysed with 0.2% Nonidet P-40 lysis buffer (19). About 10% of each lysate was saved to assay for protein expression from transfected plasmids. The remaining lysate was immunoprecipitated with 5 g/ml mAb or 12 g/ml pAb against the epitope tags. Proteins were immunoprecipitated with protein A/GSepharose 4B, subjected to SDS-polyacrylamide gel electrophoresis, and immunoblotted with indicated antibodies. Proteins were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech). In Vitro Transcription and Translation of ProteinsCoupled in vitro transcription and translation was carried out with the TNT system from Promega according to the manufacturers instructions. Briefly, pcDNA3 plasmids producing FLAG-Egl-1 and HA-CED-9 (mutant and wt) were transcribed with T7 polymerase and translated in the presence of [35S]methionine using a rabbit reticulocyte lysate. 0.5 g of each of the plasmids or empty pcDNA3 was added so that the total amount of DNA was 2 g/reaction. Proteins were inmmunoprecipitated with the indicated mAb, and immunoprecipitated proteins were resolved in 12% SDS-polyacrylamide gel electrophoresis gels. Cell Immunostaining and Confocal MicroscopyHEK293T cells (0.5 106 cells/well on 6-well plates) were cultured on poly-L-lysinecoated cover slides and transfected with the indicated plasmids by the calcium phosphate method (19). After 24 h, cells were fixed with cold methanol for 20 min at 20 C. In experiments where mitochrondria were stained, 391 nM Mitotracker was added to the culture medium for 30 min prior to processing. To prevent nonspecific hybridization, fixed cells were blocked with 30% goat serum, 1% bovine serum albumin in phosphate-buffered saline. Cells were stained with 35 g/ml of the indicated mAb and 0.3 0.5 g/ml rabbit anti-HA pAb. Primary antibodies were labeled by incubation with fluorescein isothiocyanate-conjugated goat anti-mouse IgG and rhodamine-conjugated goat anti-rabbit IgG diluted 1:200. Subcellular localization of proteins was assessed by laser confocal microscopy (Bio-Rad) as described (15). Apoptosis AssaysHEK293T cells were cultured in 12-well plates (2.5 105 cells/well) and transfected with the indicated plasmids as described (19). Each transfection mixture contained 20 ng of pcDNA3- -galactosidase plasmid as a reporter. Apoptotic morphology of transfected cells was determined 24 h after transfection by analysis of at least 100 cells expressing -galactosidase, as described (19).
RESULTS

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FIG. 1. EGL-1 binds to wt CED-9 and mutant CED-9G169E. A, sequence alignment of different prosurvival Bcl-2 family members. The position of BH1 domain and boundaries of helices 4 and 5 in human Bcl-XL (29) are shown. Conserved glycine residue in the BH1 domain is boxed. CED9 CAEEL, CED-9 protein from C. elegans; CED9 CAEBR, CED-9 protein from C. briggsae; E1BS ADE02, E1B19K protein from human type II adenovirus. B, HEK293T cells were transiently transfected with plasmids producing FLAG-EGL-1 (1 g), Myc-CED-4 (1 g), and HA-CED-9 (0.5 g). pcDNA3 plasmid DNA was added so that the total amount of DNA was always 5 g. 24 h after transfection, cell lysates were immunoprecipitated (IP) with anti-FLAG pAb, and immunoprecipitates were immunoblotted with anti-c-Myc, anti-HA, or anti-FLAG mAb. Immunoprecipitated proteins are shown in the two top panels, and expression of proteins in total lysate is shown in the lower three panels. Molecular size markers (in kDa) are shown at the right of each panel. * indicates nonspecific band recognized by the antibody. IP, antibody used for immunoprecipitation. Blot, antibody used for immunoblotting.

EGL-1 Binding to CED-9 Is Not Prevented by the G169E MutationAnalysis of the ced-9(n1950) mutation revealed that this mutation affects the coding region of ced-9 and results in a single amino acid substitution at position 169 (Gly to Glu) in the CED-9 protein (20). The Gly169 residue lies in the conserved BH1 domain of CED-9, and this glycine is highly conserved among different prosurvival Bcl-2 family members (Fig. 1A). Because the BH3 region of pro-apoptotic family members is

thought to interact with a hydrophobic cleft that includes the conserved BH1 and BH3 regions of prosurvival family members (23), we reasoned that the CED-9G169E protein might prevent cell deaths because it lacks ability to bind EGL-1. To test this possibility, we investigated the association of wt CED-9 and mutant CED-9G169E with EGL-1, both in the presence and the absence of CED-4. In these experiments, we transiently co-transfected HEK293T cells with expression plasmids producing the relevant proteins and measured protein interactions by immunoprecipitation and immunoblotting. Significantly, immunoprecipitation of EGL-1 co-immunoprecipitated both wt CED-9 and mutant CED-9G169E (Fig. 1B). Moreover, the presence of CED-4 did not affect the binding of EGL-1 to either form of CED-9 (Fig. 1B). Hence, the ability of CED9G169E to prevent cell deaths cannot be explained by an inability to bind EGL-1. The Binding of EGL-1 to CED-9G169E Does Not Disrupt the CED-9 CED-4 Protein ComplexCED-9 appears to block cell deaths by binding to and inhibiting the activity of CED-4 (9, 1315). To measure the effect of EGL-1 on the ability of wt and mutant CED-9 to interact with CED-4, we expressed the relevant proteins in HEK293T cells and assessed the interactions by immunoprecipitation and immunoblotting. As shown in Fig. 2A, immunoprecipitation of wt CED-9 or mutant CED-9G169E co-immunoprecipitated CED-4. In addition, immunoprecipitation of CED-4 co-immunoprecipitated both wt CED-9 and CED9G169E (Fig. 2B). To learn whether EGL-1 could inhibit the association of CED-9G169E with CED-4, we assayed the interaction of wt or

Molecular Characterization of the Nematode CED-9G169E Protein

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FIG. 2. EGL-1 does not disrupt the interaction of CED-9G169E with CED-4. HEK293T cells were transiently transfected with plasmids producing FLAG-EGL-1 (1 g), Myc-CED-4 (1 g), and HA-CED-9 (0.5 g in A and B). pcDNA3 plasmid DNA was added so that the total amount of DNA was always 5 g. 24 h after transfection, cell lysates were immunoprecipitated (IP) with anti-HA mAb (A) or with anti-c-Myc pAb (B, right). Immunoprecipitated proteins and total lysates were resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-Myc, anti-HA, or antiFLAG mAb. Labels are as in Fig. 1B. The data shown are representative of at least three independent experiments. C, EGL-1 binds mutant CED-9G169E, but with reduced affinity, compared with its binding to wild-type CED-9. Plasmids producing wt HA-CED-9 (WT), mutant CED-9G169E (GE), and FLAG-EGL-1 in the indicated combinations were added to an in vitro transcription/translation reaction in the presence of [35S]methionine. One half of each reaction was immunoprecipitated with antiFLAG or anti-HA mAb. The wt and mutant CED-9 bands were quantitated by a phosphoimager (in cpm). Numbers below the last two lanes represent the ratio of values obtained with mutant CED-9G169E over wt CED-9 for each lane.

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mutant CED-9 with CED-4 in the presence of EGL-1. Both the wt and mutant CED-9 proteins associated with EGL-1 in cells co-expressing CED-4 (Fig. 2A). However, as reported earlier (19), expression of EGL-1 disrupted the association between wt CED-9 and CED-4, as assessed by immunoprecipitation of either CED-9 (Fig. 2A) or CED-4 (Fig. 2B). By contrast, expression of EGL-1 did not affect the interaction between CED9G169E and CED-4, as determined by immunoprecipitation of CED-9G169E (Fig. 2A) or CED-4 (Fig. 2B). Hence, EGL-1 can induce the release of the cell death activator CED-4 from the CED-9 CED-4 complex, but not from a complex formed by the mutant CED-9G169E and CED-4. The Affinity of EGL-1 to Bind CED-9G169E Is ReducedTo

compare directly the ability of wt CED-9 and CED-9G169E to associate with EGL-1, we synthesized EGL-1 and wt and mutant CED-9 in a cell-free system. EGL-1 was synthesized and translated in a reticulocyte lysate in the presence of similarly translated wt and/or mutant CED-9 and assessed the binding of both forms of CED-9 to EGL-1 in the same immunoprecipitate. The HA-tagged CED-9G169E protein shows a slower electrophoretic mobility than HA-tagged wt CED-9 (Figs. 1B and 2, A and B), allowing discrimination of both forms by SDS-polyacrylamide gel electrophoresis. Analysis of EGL-1 immunoprecipitates expressing wt CED-9 or mutant CED9G169E showed that EGL-1 binds to both forms of CED-9 (Fig. 2C). In the presence of both wt and mutant CED-9, EGL-1 preferentially

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FIG. 3. EGL-1 co-localizes with CED-9G169E at mitochondria in HEK293 cells but it does not induce translocation of CED-4 to the cytosol. AD, HEK293T cells were transiently transfected with plasmids producing AU1-EGL-1 (1 g), Flag-CED-4 (1 g), and HA-CED-9 (0.5 g). 24 h after transfection, cells were processed for immunostaining and analyzed by confocal microscopy as indicated under Experimental Procedures. Labels shown on top of the panels indicate transfected expression plasmids. For each sample, three images are shown. Labeled proteins are indicated at the bottom of the left and middle panels. The right panel (labeled as Merge) represents a computer-generated overlay of both images. For the analysis, green and red color were assigned arbitrarily to the left and right images, respectively. Yellow color indicates overlap. Mito. indicates cells stained with the mitochondrion selective dye Mitotracker. Cells labeled with Mitotracker were not stained for CED-9 with anti-HA pAb. The images shown are from representative fields of each sample. The experiment was performed at least three times, with similar results.

bound to wt CED-9 over mutant CED-9 (Fig. 2C), indicating that the affinity of CED-9G169E for EGL-1 is reduced when compared with that of wt CED-9. These results also demonstrate that both wt and mutant CED-9 compete for EGL-1 binding, indicating that both forms of CED-9 bind to a common site on EGL-1. EGL-1 Induces Translocation of CED-4 from Mitochondria to the Cytosol in the Presence of wt CED-9 but Not in the Presence of CED-9G169EIn HEK293T cells, CED-4 co-localizes with CED-9 at intracellular membranes but is translocated to the cytosol upon expression of EGL-1 (19). Based on the results shown in Fig. 2, we suspected that EGL-1 might not interfere with the ability of CED-9G169E to retain CED-4 at intracellular membrane sites. To test this hypothesis, cells were transfected with different combinations of plasmids producing epitope-tagged CED-4, CED-9 (wt or G169E), and EGL-1, and the subcellular localization of each protein was visualized by confocal microscopy. The labeling pattern of EGL-1 was cytoplasmic and granular and appeared identical to those of wt and mutant CED-9 (Fig. 3A). These results are consistent with our proposal that EGL-1 binds to both forms of CED-9. In the absence of EGL-1, the CED-4 protein also co-localized with wt and mutant CED-9 (Fig. 3B). However, the expression of EGL-1 in the presence of wt CED-9 changed the CED-4 label-

ing pattern from granular to diffuse (Fig. 3B). This result indicates that EGL-1 caused the translocation of CED-4 from intracellular membranes to the cytosol. By contrast, EGL-1 did not change the subcellular localization of CED-4 in cells that expressed the mutant CED-9G169E protein (Fig. 3B). Furthermore, simultaneous staining for EGL-1 and CED-4 revealed that these proteins co-localize in the presence of mutant CED9G169E but not in the presence of wt CED-9 (Fig. 3C). To show that these images are representative of our data, we quantified the localization of CED-4 in all the cells that we examined (Fig. 4). Our findings are consistent with the results presented in Fig. 2, which showed that EGL-1 disrupts the interaction between wt CED-9 and CED-4 but not that between CED9G169E and CED-4. In addition, these results indicate that CED-4 might form a ternary protein complex with EGL-1 and CED-9G169E, because all three proteins co-localize at intracellular membrane sites. Bcl-2 and Bcl-XL localize at several intracellular membranes, primarily to those of mitochondria (25, 26). To determine more precisely the subcellular localization of EGL-1, CED-4, and CED-9 in HEK293T cells, we performed co-labeling analyses for each nematode protein with Mitotracker, a mitochondrial dye. In the presence of wt or mutant CED-9 and the absence of EGL-1, CED-4 co-localized with Mitotracker, sug-

Molecular Characterization of the Nematode CED-9G169E Protein

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FIG. 3 continued

gesting that CED-4 resides primarily in the mitochondria in these cells. However, in the presence of EGL-1 and wt CED-9, CED-4 did not co-localize with Mitotracker (Fig. 3D), consistent with our proposal that EGL-1 induces transfer of CED-4 to the cytosol. By contrast, CED-4 did co-localize with Mitotracker when co-expressed with EGL-1 and mutant CED-9G169E (Fig. 3D). These observations agree with the results shown in Fig. 3B. Finally, we note that EGL-1 (in the presence of either wt or mutant CED-9), wt CED-9 itself, and mutant CED-9G169E always co-localized with Mitotracker.2 EGL-1 Expression Induces Apoptosis in the Presence of wt CED-9 but Not Mutant CED-9G169EWe have shown that EGL-1 is unable to disrupt the association of CED-9G169E with CED-4. We suspected that the inability of EGL-1 to displace CED-4 from the CED-9G169E CED-4 complex might prevent the activation of CED-3 by EGL-1. To study the regulation of CED-3 activity by wt and mutant CED-9, we tested the ability of EGL-1 to act through CED-4 and induce CED-3mediated apoptosis in the HEK293T cell system (13, 19). The HEK293T cells were co-transfected with a CED-3 expression plasmid and different combinations of plasmids producing CED-4, EGL-1, and CED-9 (either wt or mutant). A reporter plasmid producing -galactosidase was included in all transfection mixtures to quantitate the level of apoptosis (19). In Fig. 5, we show that enhancement of CED-3-induced apoptosis by CED-4 was prevented by either wt CED-9 or mutant CED9G169E. However, although EGL-1 blocked the protective effect of wt CED-9, it had no effect on the ability of CED2 L. del Peso, V. M. Gonzalez, and G. Nunez, unpublished observations.

9G169E to repress apoptosis (Fig. 5). These results are consistent with genetic studies in C. elegans, which showed that killing mediated by egl-1 requires ced-9 and is suppressed by the ced-9(n1950 gain-of-function) mutation (17). Thus, in HEK293T cells, the ability of EGL-1 to induce the release of CED-4 from CED-9 is correlated with its ability to induce CED-3-mediated apoptosis.
DISCUSSION

The ced-9(n1950) allele was identified in C. elegans as a gain-of-function mutation that causes most somatic cells that normally undergo programmed cell death to instead survive (7, 20). We have shown that this CED-9 mutant retains its ability to bind EGL-1 and CED-4, although its affinity for EGL-1 is reduced. Significantly, in contrast to wt CED-9, the protein complex formed by CED-4 and CED-9G169E is not disrupted by EGL-1. Furthermore, the ability of EGL-1 to induce apoptosis is impaired in cells expressing CED-9G169E when compared with cells expressing wt CED-9. These results suggest that the release of CED-4 from its inhibitor CED-9 is the critical step for activation of CED-3 and induction of programmed cell death in the nematode. Interestingly, the mutation equivalent to CED-9G169E in Bcl-2 or Bcl-XL prevents binding with the pro-apoptotic protein Bax (21, 23) but does not affect binding with the pro-apoptotic protein Bad (27). This result is significant because C. elegans EGL-1 and mammalian Bad belong to the BH3 only subgroup of pro-apoptotic Bcl-2 family members, but Bax does not fall into this group (28). Thus, the conserved glycine residue in the BH1 domain of anti-apoptotic Bcl-2 family members may be critical for binding with Bax, but not with BH3 only killers.

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complex with CED-4 and EGL-1, then the binding sites for these two proteins cannot overlap. Instead, EGL-1 might bind to CED-9 and induce a conformational change that disrupts its interaction with CED-4. In this model, the reduced affinity of EGL-1 for mutant CED-9 would impair the induction of the conformational change in CED-9. Another nonexclusive possibility is that the G169E mutation would alter CED-9 so that this conformational change could not occur, thereby preventing the release of CED-4 and the induction of cell death. How might this conformational change occur? The BH3 domain of proapoptotic proteins binds to a hydrophobic pocket in the Bcl-XL protein (23) that includes the BH1 and BH3 domains (29, 30). In addition to this hydrophobic pocket, two more hydrophobic surfaces have been identified in Bcl-XL (29, 30). One of them, the BH groove, is formed by the BH1 and BH2 domains of Bcl-XL (30). Mutation of residues located within the BH groove (Val135 3 Ala, Asn136 3 Ile, Trp137 3 Leu), but not mutation of nearby residues that lie outside of this hydrophobic pocket (Phe131 3 Val, Asp133 3 Ala), prevents the binding of CED-4 to Bcl-XL (9). Similarly, deletion of the BH1 and BH2 domains of CED-9 abolishes binding with CED-4 (9). Thus, CED-4 might bind to the BH groove of Bcl-XL and CED-9. The BH3 and BH hydrophobic pockets partially overlap in a small region close to residue Gly138 of Bcl-XL (30), which is equivalent to residue Gly169 in CED-9 (Fig. 1A). Hence, the binding of EGL-1 to the BH3 hydrophobic pocket of CED-9 might destabilize the neighboring CED-4 contact site on CED-9, thereby inducing the release of CED-4 from the CED-4 CED-9 complex. If so, the mutation G169E might slightly alter the EGL-1 binding site on CED-9, so that it does not interfere with the CED-4 binding region, thereby allowing simultaneous binding of both EGL-1 and CED-4 to CED-9. There is a high degree of functional and structural conservation in the apoptotic machinery between C. elegans and mammals. However, some differences are also apparent. Apaf-1, the mammalian homologue of CED-4, requires binding to cytochrome c to oligomerize and activate caspase-9 (31, 32). Interestingly, cytochrome c appears to bind to a region of Apaf-1 (WD repeats) that is not conserved in CED-4 (6). Thus, in mammalian cells, but perhaps not in the nematode, regulation of cytochrome c release from mitochondria plays a pivotal role in controlling caspase activity. There is convincing evidence that mammalian prosurvival Bcl-2 family members block cell death at least in part by preventing cytochrome c release from mitochondria (33, 34). However, it has also been shown that under certain conditions, prosurvival Bcl-2 family proteins are able to associate with Apaf-1 and CED-4 (9, 15, 35, 36) and that this interaction is prevented by BH3 only proteins (35). These results may explain the observation that mammalian anti-apoptotic Bcl-2 family members can partially substitute for CED-9 in C. elegans (37). Whether the interaction of Bcl-2 family members with Apaf-1 (or possibly with another mammalian CED-4 homologue) is relevant for the control of apoptosis in mammals or it is just a nonphysiological vestige of the ancient CED-9 CED-4 system remains to be determined. If the biochemical interaction between Bcl-2 family members and a mammalian CED-4 homologue parallels that between CED-9 and CED-4, then our studies would not only elucidate cell death in nematodes, but could indicate how the BH3 only proteins promote apoptosis in mammals. While this manuscript was in the reviewing process, it was shown that endogenous CED-4 and CED-9 co-localize to mitochondria and that CED-4 translocated to perinuclear regions during programmed cell death (38). The translocation of CED-4 was blocked by the ced-9(n1950) gain-of-function mutation (38). These results are consistent with the data presented in the current manuscript.

FIG. 4. Quantification of EGL-1 effect on CED-4 subcellular localization in the presence of wt CED-9 or mutant CED9G169E. HEK293T cells were transiently transfected with plasmids producing AU1-EGL-1 (0 2 g), Flag-CED-4 (1 g), and wt CED-9 (WT) (0.25 g) or mutant CED-9G169E (0.25 g). 24 h after transfection, cells were processed for immunostaining and analyzed by confocal microscopy as described under Experimental Procedures. The subcellular localization of CED-4 was scored blind. Results are based on at least 250 cells/sample, and represent the percentage of cells showing a diffuse labeling pattern (cytoplasmic), a granular pattern (mitochondrial), or cells showing both diffuse and granular labeling patterns. The results are representative of at least two independent experiments.

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FIG. 5. EGL-1 induction of apoptosis in the presence of wt CED-9 and mutant CED-9G169E. HEK293T cells (2.5 105 293T cells/well in 12-well plates) were transiently transfected with plasmids producing AU1-EGL-1 (50 150 ng/well), Flag-CED-4 (50 ng/well), HACED-3 (50 ng/well), and HA-CED-9 (100 ng/well). Each transfection mixture contained 25 ng of a reporter plasmid encoding -galactosidase. 24 h after transfection, cells were processed for -galactosidase staining and scored blind under microscope. To compare different experiments, we arbitrarily assigned the value of 1 to the percentage of apoptotic cells in samples transfected with CED-3 alone (dotted line). The percentage of apoptosis in the rest of the samples on each experiment was represented as a multiple of the amount induced by CED-3 alone. The basal percentage of apoptosis induced by CED-3 alone ranged from 8.4 to 23.7% for the different experiments. The results from three independent experiments are represented.

It was previously suggested that EGL-1 and CED-9 might compete for overlapping binding sites on CED-4 (17). However, our results suggest that the CED-9G169E protein may form a ternary protein complex with CED-4 and EGL-1. This possibility is supported by the finding that CED-9G169E immunoprecipitated with CED-4 in the presence of EGL-1 (Fig. 2B) and immunoprecipitation of mutant CED-9G169E co-immunoprecipitated both CED-4 and EGL-1 (Fig. 2A). Finally, when expressed together in HEK293T cells, CED-4, CED-9G169E, and EGL-1 all co-localize. If CED-9G169E does form a ternary

Molecular Characterization of the Nematode CED-9G169E Protein


AcknowledgmentsWe thank Y. Hu and P. Lucas for critical review of the manuscript and experimental suggestions.
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