Cytochrome c and dATP-mediated Oligomerization of Apaf-1 Is a Prerequisite for Procaspase-9 Activation

To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we produced recombinant full-length Apaf-1 and purified it to complete homogeneity. Here we show using gel filtration that full-length Apaf-1 exists as a monomer that can be transformed to an oligomeric complex made of at least eight subunits after binding to cytochrome c and dATP. Apaf-1 binds to cytochromec in the absence of dATP but does not form the oligomeric complex. However, when dATP is added to the cytochromec-bound Apaf-1 complex, complete oligomerization occurs, suggesting that oligomerization is driven by hydrolysis of dATP. This was supported by the observation that ATP, but not the nonhydrolyzable adenosine 5′-O-(thiotriphosphate), can induce oligomerization of the Apaf-1-cytochrome c complex. Like the spontaneously oligomerizing Apaf-530, which lacks its WD-40 domain, the oligomeric full-length Apaf-1-cytochrome c complex can bind and process procaspase-9 in the absence of additional dATP or cytochrome c. However, unlike the truncated Apaf-530 complex, the full-length Apaf-1 complex can release the mature caspase-9 after processing. Once released, mature caspase-9 can process procaspase-3, setting into motion the caspase cascade. These observations indicate that cytochrome c and dATP are required for oligomerization of Apaf-1 and suggest that the WD-40 domain plays an important role in oligomerization of full-length Apaf-1 and the release of mature caspase-9 from the Apaf-1 oligomeric complex. To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we produced recombinant full-length Apaf-1 and purified it to complete homogeneity. Here we show using gel filtration that full-length Apaf-1 exists as a monomer that can be transformed to an oligomeric complex made of at least eight subunits after binding to cytochrome c and dATP. Apaf-1 binds to cytochromec in the absence of dATP but does not form the oligomeric complex. However, when dATP is added to the cytochromec-bound Apaf-1 complex, complete oligomerization occurs, suggesting that oligomerization is driven by hydrolysis of dATP. This was supported by the observation that ATP, but not the nonhydrolyzable adenosine 5′-O-(thiotriphosphate), can induce oligomerization of the Apaf-1-cytochrome c complex. Like the spontaneously oligomerizing Apaf-530, which lacks its WD-40 domain, the oligomeric full-length Apaf-1-cytochrome c complex can bind and process procaspase-9 in the absence of additional dATP or cytochrome c. However, unlike the truncated Apaf-530 complex, the full-length Apaf-1 complex can release the mature caspase-9 after processing. Once released, mature caspase-9 can process procaspase-3, setting into motion the caspase cascade. These observations indicate that cytochrome c and dATP are required for oligomerization of Apaf-1 and suggest that the WD-40 domain plays an important role in oligomerization of full-length Apaf-1 and the release of mature caspase-9 from the Apaf-1 oligomeric complex. Caspases, a highly conserved family of cysteine proteases that cleave their substrates after an aspartate residue, play fundamental roles in the initiation and execution of apoptosis (reviewed in Refs.1Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4145) Google Scholar, 2Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1941) Google Scholar, 3Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6178) Google Scholar, 4Cryns V. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1160) Google Scholar). Caspases are constitutively expressed in cells as single chain proenzymes that can be activated by proteolytic cleavage at specific internal aspartate residues within the procaspase polypeptide chain. Mature caspases can cleave their own proenzyme and other procaspases, suggesting that they operate in a protease cascade. Caspases have been divided into initiators and effectors, based on their place in the caspase cascade (1Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4145) Google Scholar, 2Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1941) Google Scholar, 3Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6178) Google Scholar, 4Cryns V. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1160) Google Scholar). The effectors (caspase-3, -6, and -7) are activated via the action of other caspases (i.e. initiators) and are responsible for the characteristic morphological changes of apoptosis. The initiators (caspase-8, -9, and -10) are activated by their own intrinsic autocatalytic activity with the help of other proteins with which they form complexes known as “apoptosomes” (5Green D.R. Cell. 1998; 94: 695-698Abstract Full Text Full Text PDF PubMed Scopus (1109) Google Scholar). Two apoptosomes that function to activate the initiator procaspases have been identified. The death receptor apoptosome is an oligomer that is formed upon ligation of death receptors such as Fas or tumor necrosis factor receptor 1 by their ligands (6Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5166) Google Scholar). This oligomer recruits procaspase-8 or -10 via the adaptor molecule FADD through homotypic protein-protein interactions, resulting in activation of these caspases by aggregation (7Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar, 8Yang X. Chang H.Y. Baltimore D. Mol. Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Another unrelated apoptosome is formed by Apaf-1 upon binding to cytochrome c, which is released from the mitochondria by various forms of apoptosis triggers (5Green D.R. Cell. 1998; 94: 695-698Abstract Full Text Full Text PDF PubMed Scopus (1109) Google Scholar, 9Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6255) Google Scholar). The Apaf-1-cytochrome c complex then recruits procaspase-9 in a dATP/ATP-dependent manner through a CARD-CARD 1The abbreviations used are: CARD, Caspase recruitment domain; DTT, dithiothreitol; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis; γ-S-ATP, adenosine 5′-O-(thiotriphosphate); AMC, 7-amino-4-methylcoumarin. interaction, resulting in its activation and presumably the release of mature caspase-9 from the apoptosome (5Green D.R. Cell. 1998; 94: 695-698Abstract Full Text Full Text PDF PubMed Scopus (1109) Google Scholar, 9Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6255) Google Scholar). A recent study from our laboratory demonstrated that a truncated Apaf-1 variant lacking the WD-40 repeat domain (Apaf-530) can activate procaspase-9 independent of cytochrome c and dATP through spontaneous oligomerization (10Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar). Interestingly, the truncated Apaf-1 was unable to release the mature caspase-9 from the complex, raising the possibility that the WD-40 repeats play a role in the release of mature caspase-9 from the Apaf-1 apoptosome (10Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar). To determine the role of cytochrome c and dATP and the function of the WD-40 repeats in the process of activation and release of caspase-9, we reconstituted an in vitro Apaf-1-caspase-9 activation system with purified recombinant full-length Apaf-1. We provide evidence that cytochrome c and dATP are required to promote oligomerization of Apaf-1 and that mature caspase-9 is released from the full-length Apaf-1 apoptosome but not from the truncated Apaf-530 complex. All the purification steps were carried out at 4 °C. Apaf-1L was expressed in Sf-9 cells by infecting the cells with recombinant Apaf-1 baculovirus. An S-100 extract was prepared from a 1-liter suspension culture of the infected cells in 25 mm HEPES buffer (pH 7.5) containing 300 mm NaCl, 10 mm KCl, 1.5 mm MgCl2, 10% glycerol, 0.1 mmDTT, 1 mmphenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, and 1 μg/ml leupeptin. 280 mg of total proteins were loaded onto a 2-ml bed volume column of Ni2+-nitriloacetic acid agarose (Novagen) at a flow rate of 0.05 ml/min in the presence of 10 mmimidazole. After washing the column with 2 × 20 ml of HEPES buffer containing 25 mm and 50 mm imidazole, respectively, bound proteins were eluted with a 30-ml gradient of 50–350 mm imidazole in HEPES buffer at a flow rate of 0.15 ml/min. The fractions containing Apaf-1L (∼90 mm-150 mm imidazole) were pooled and concentrated (Centricon-30; Amicon), and the final concentration of NaCl was adjusted to 20 mm in a final volume of 2.0 ml. Subsequently, the concentrated Apaf-1L sample (600 μg) was applied to an FPLC Mono Q column (1.0 ml; Amersham Pharmacia Biotech) at a flow rate of 0.05 ml/min. After washing the column with 10 ml of the HEPES buffer containing 20 mm NaCl, the protein was eluted with a 20-ml gradient of 20–300 mm NaCl at a flow rate of 0.2 ml/min. The peak fractions containing Apaf-1L were pooled and concentrated, and the concentration of NaCl was adjusted to 50 mm in a final volume of 0.5 ml (110 μg). Finally, 2 × 250 μl of the Mono Q purified Apaf-1L was loaded separately onto a Superose 12 FPLC column (Amersham Pharmacia Biotech) at a flow rate of 0.2 ml/min. 20-μl aliquots from each 250-μl fraction were separated by SDS-PAGE and analyzed by Western blotting with anti-Apaf-1 antibody. The peak fractions of Apaf-1L protein were pooled and concentrated (1 ml; 60 μg of protein), and the purity of the protein was verified by SDS gel electrophoresis and Coomassie staining. By comparison with gel filtration protein standards (Amersham Pharmacia Biotech), the peak fraction of Apaf-1L from the Superose 12 column corresponded to apparent molecular size of 125 kDa (this value was calculated by linear extrapolation from the calibration protein standards; data not shown). All oligomerization reactions of Apaf-1L were carried out by incubating Apaf-1 (3 μg) with or without cytochrome c (7 μg) or dATP (1 mm) or both at 4 °C for 70 min in a final volume of 100 μl of 25 mm HEPES buffer (pH 7.5) containing 50 mm NaCl, 10 mm KCl, 1.5 mmMgCl2, 10% glycerol, and 0.1 mm DTT (oligomerization buffer). In some experiments dATP was substituted with ATP (1 mm), or γ-S-ATP (1 mm). After oligomerization an additional 150 μl of the oligomerization buffer was added to each sample, and the reaction mixture was directly applied to a Superose 6 FPLC column at a flow rate of 0.2 ml/min. 45-μl aliquots of the 500-μl fractions were fractionated by SDS-PAGE and assayed for the presence of Apaf-1L protein by immunoblotting with anti-Apaf-1 antibody. Approximate molecular masses of the different forms of Apaf-1L protein were obtained by linear extrapolation from the calibration protein standards. Initially, 100 μl of in vitro translated35S-labeled pro-caspase-9 was fractionated on a 15-ml open column of Sephacryl S-400 HR (Amersham Pharmacia Biotech) at a flow rate of 0.05 ml/min. 15-μl aliquots of 200-μl fractions were separated on 10% polyacrylamide gels, and the elution peak of procaspase-9 was determined by autoradiography. 1 × 105 trichloroacetic acid counts of the partially purified procaspase-9 were incubated with 3.0 μg of pure Apaf-1L and 7.0 μg of cytochrome c in a final volume of 100 μl of the oligomerization buffer containing 1.0 mm dATP. The mixture was incubated at 30 °C for 45 min to allow processing of procaspase-9 followed by loading onto the Sephacryl S-400 column. The elution of caspase-9 forms and Apaf-1L protein were assessed by autoradiography and Western blotting with anti-Apaf-1 antibody, respectively. Similarly, 25 μg of affinity purified Apaf-530 protein (10Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar) were incubated with the partially purified procaspase-9 and fractionated on the same gel filtration column. These were performed as described previously (9Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6255) Google Scholar,11Srinivasula S.M. Ahmad M. Ottilie S. Bullrich F. Banks S. Wang Y. Fernandes-Alnemri T. Croce C.M. Litwack G. Tomaselli K.J. Armstrong R.C. Alnemri E.S. J. Biol. Chem. 1997; 272: 18542-18545Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). To obtain sufficient quantities of human Apaf-1 for functional and biochemical characterization, we engineered baculoviruses encoding two C-terminally His6-tagged Apaf-1 isoforms. One isoform (Apaf-1S) is identical in sequence to the published Apaf-1 (12Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2745) Google Scholar) and GenBankTM accession numberAF013263). The second isoform (Apaf-1L) has an additional WD-40 repeat at amino acid 812 relative to the initiator Met and was cloned from a Jurkat cDNA library (Fig.1 A). The human Apaf-1L is similar in structure to the recently cloned mouse Apaf-1, which also has an additional WD-40 repeat (13Cecconi F. Alvarez-Bolado G. Meyer B.I. Roth K.A. Gruss P. Cell. 1998; 94: 727-737Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar). The two Apaf-1 isoforms were expressed in Sf-9 cells by infecting the cells with their respective baculoviruses and then partially purified on Ni2+ affinity resin. Comparable amounts of the two proteins as determined by Western blotting (Fig. 1 B) were incubated with35S-labeled procaspase-9. As shown in Fig. 1 C, Apaf-1L, but not Apaf-1S, was capable of processing procaspase-9 in a cytochrome c and dATP-dependent fashion, suggesting that the additional WD-40 repeat might be critical for Apaf-1 stability and its overall tertiary structure. We observed that Apaf-1S is less soluble than Apaf-1L and that the majority of the expressed protein accumulates as insoluble occlusion bodies in Sf-9 cells. However, we did not see any precipitation of the soluble Apaf-1S during the incubation period with procaspase-9 not shown). on these data and on the published sequence of mouse Apaf-1, we that the human Apaf-1L isoform is the functional form of Apaf-1 in human cells. c and dATP are for activation of procaspase-9 (9Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6255) Google Scholar). However, their role in process to be recent demonstrated that of the WD-40 domain of Apaf-1 produced a constitutively Apaf-1 variant (Apaf-530) that can spontaneously and induce activation of procaspase-9 independent of cytochrome c and dATP (10Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar). the process of oligomerization to be critical for activation of we that cytochrome c and dATP oligomerization of full-length Apaf-1, by the of the WD-40 domain, it for To possibility we purified recombinant Apaf-1L to complete (Fig. 1 incubated it with or without cytochrome c or dATP or and then analyzed its elution by gel filtration on an FPLC column. We that cytochrome c or dATP or both induce oligomerization of Apaf-1, we be to the oligomeric form of Apaf-1 from its form on the of molecular size of Apaf-1L with dATP did not its elution from that of the buffer the buffer and eluted as single fraction (Fig. and C, The size of Apaf-1L in the peak fraction was suggesting that it is a monomer and that dATP is not sufficient to induce its of Apaf-1L with cytochrome c in a in the Apaf-1L elution (Fig. A The majority of Apaf-1L eluted in a peak fraction which to a size of In cytochrome c with Apaf-1L, that fraction a complex of cytochrome In to a peak of Apaf-1L eluted fraction cytochromec eluted fraction shown). These suggest that cytochrome c does not induce oligomerization of Apaf-1. Interestingly, of Apaf-1L with cytochrome dATP in a in the Apaf-1L elution (Fig. A and The majority of Apaf-1L eluted fraction The Apaf-1L eluted in two fractions 30 and which to cytochromec-bound Apaf-1L and Apaf-1L, respectively. The size of Apaf-1 in the peak fraction is This and the presence of cytochrome c in fraction suggest that the peak a oligomeric complex of Apaf-1L and cytochrome c. on the observed of oligomer and the cytochrome Apaf-1L monomer and that the oligomer is we calculated that oligomer at least eight of These data that Apaf-1 exists as a monomer and that binding of cytochrome c and dATP to Apaf-1 of an of cytochrome Apaf-1. To the gel filtration we performed experiments using and from cells with or Apaf-1L were and incubated with or without cytochrome c, or both and with a antibody. The were then fractionated by SDS gel electrophoresis and with a antibody. As in the presence of both cytochromec and dATP was a of the two Apaf-1L proteins with each other (Fig. 2 A of that was observed with cytochrome be of the presence of amounts of ATP or dATP in the was observed in the buffer or dATP To determine the activity of Apaf-1L in the peak fractions of the gel filtration experiments (Fig. we incubated of the peak fractions with procaspase-9 in the presence or absence of cytochrome c or dATP or As shown in the oligomeric Apaf-1L was capable of processing procaspase-9 without additional cytochrome c and dATP. The cytochrome complex on the other was capable of processing procaspase-9 when dATP was Apaf-1L from the buffer and and was capable of processing procaspase-9 when both dATP and cytochrome on these data and the gel filtration we suggest that cytochrome c can bind to Apaf-1 in the absence of dATP but induce its However, in the presence of the cytochrome complex form an oligomer that is capable of procaspase-9. This was by incubating the pooled peak (Fig. 2 with dATP and then it on Superose 6 column. As shown in dATP a complete in the elution of the cytochrome All Apaf-1L in peak eluted in a single peak fraction which to the oligomeric This observation that dATP is required for oligomerization of the cytochrome Apaf-1 This have cytochrome c release from the mitochondria of cells not be sufficient to induce oligomerization and activation of procaspase-9. sufficient dATP or ATP are can cytochrome c release from the mitochondria induce apoptosis. with purified Apaf-1 demonstrated that ATP in the presence of cytochrome c also induce activation of at a concentration than dATP (9Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6255) Google Scholar). However, of ATP or dATP by the nonhydrolyzable ATP γ-S-ATP suggesting that hydrolysis of the is for Apaf-1 function (9Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6255) Google Scholar). dATP is required to induce oligomerization of the cytochrome Apaf-1L (Fig. and we that ATP, but not γ-S-ATP, be to induce the same To we incubated purified Apaf-1L with cytochrome ATP, or γ-S-ATP and then analyzed its elution by gel filtration on an FPLC column. As dATP and ATP, but not γ-S-ATP, were to induce the of the Apaf-1L oligomeric complex, which eluted fraction 20 However, the of the oligomeric Apaf-1L by ATP was less than that by dATP. This is with observations that dATP is than ATP in procaspase-9 activation in and by purified Apaf-1 (9Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6255) Google Scholar, X. J. Wang X. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). This also that the of the dATP in activation of procaspase-9 by Apaf-1 on their to induce oligomerization of Apaf-1. that γ-S-ATP induce oligomerization of Apaf-1 its to induce activation of procaspase-9 by purified Apaf-1 (9Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6255) Google Scholar) and that hydrolysis of ATP or dATP might be critical in the oligomerization To determine purified Apaf-1L has an we incubated amounts of Apaf-1L with and the released was by liquid As shown in Fig. Apaf-1L was capable of ATP in a suggesting that Apaf-1 an To determine the of procaspase-9 after processing by the oligomeric Apaf-1-cytochrome c complex, we incubated procaspase-9 with Apaf-1L for 45 min at 30 °C in the presence of cytochrome c and dATP and then fractionated the complex by gel filtration on Sephacryl S-400 column. As shown in procaspase-9 with the Apaf-1 complex the mature caspase-9 eluted as a peak was Apaf-1 protein in the mature caspase-9 peak suggesting that the mature caspase-9 was released from the Apaf-1 complex after processing. The size of the oligomeric complex was the size of the released caspase-9 was The size of the released caspase-9 that it is a of two and two The size of a produced caspase-9 was also similar to the observed size of the released caspase-9 not shown). To the activity of caspase-9 in the two of the Sephacryl S-400 column (Fig. 4 we incubated peak fractions and or the pooled with fraction or the pooled peak was capable of processing to the and of (Fig. 4 C, This that the released caspase-9 is Interestingly, peak fraction or the pooled peak which the oligomeric complex, were also capable of processing after incubation with for 1 of peak fraction for 1 at 30 °C in processing of the procaspase-9 to the which its to process This that processing of procaspase-9 is important for its To the activity of the released caspase-9 the caspases that cleave the we incubated it with an extract from mouse in the presence of This extract was used to role out any of Apaf-1 to was used to the activity of the caspases in the extract that are activated by the released As shown in Fig. 4 the released caspase-9 at 1 concentration was capable of in activity in the extract with the buffer A of expressed caspase-9 was not to induce any activity in the However, the concentration of the caspase-9 to produced a similar activity to that observed with released These data are with a recent H.R. Dixit V.M. Salvesen G.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that produced and caspase-9 induce activity in without activation of the with cytochrome c and suggesting that Apaf-1 is required for activation of the our extract does not Apaf-1, does not to cytochrome c and and does not to of caspase-9, the activation of the activity by the released caspase-9 that the released caspase-9 is and we evidence suggesting that the truncated Apaf-530 processing of procaspase-9 but not release it from the oligomeric complex (10Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar). To directly we incubated procaspase-9 with Apaf-530 and then fractionated the complex by gel filtration on Sephacryl S-400 column (Fig. 4 Interestingly, both the mature caspase-9 and procaspase-9 in the same fractions that Apaf-530, suggesting that these proteins are with each other in the same complex. The size of oligomeric complex was which is than the mature These provide evidence that of the WD-40 domain the of Apaf-1 to release the mature caspase-9 from the oligomeric complex. the full-length Apaf-1 complex can release mature caspase-9, the Apaf-530 complex it is that can be when incubated with the but not with the To possibility we incubated with full-length Apaf-1L or Apaf-530 in the presence of procaspase-9 and cytochrome c and dATP. As both Apaf-1L and Apaf-530 were capable of processing procaspase-9 to the same was in the full-length Apaf-1L sample but not in the Apaf-530 sample (Fig. 4 and when purified Apaf-530 was added to it processing of procaspase-9 but processing of and This that Apaf-530 did bind mature caspase-9, it from processing These data provide evidence that mature caspase-9 is released after processing from the full-length Apaf-1 complex but not from the Apaf-530 complex. In we have demonstrated that Apaf-1 oligomerization upon binding to cytochrome c in a dATP-dependent This oligomeric complex can procaspase-9 directly and activate it and then release the mature caspase-9 from the complex to the caspase cascade. Apaf-1 as a death receptor that is activated upon binding to its cytochrome c, in the presence of dATP. We X. Wang for the anti-Apaf-1 and T. for mouse We also G. for