Schematic representation of events that take place distal to the entry of cytochrome c to the cytoplasm. The mitochondrial apoptosome is formed upon exit of cytochrome c from the mitochondrial intermembrane space, possibly triggered by either Bax or Bid. Upon activation, caspase-9 may disengage from the cytochrome c/Apaf-1 complex in order to activate downstream caspases, alternatively, downstream caspases may also become recruited to this apoptosome via suitable adaptor proteins.

Selective binding of caspase-9 to Apaf-1. Glutathione Sepharose–immobilized GST-Apaf-1^1-97 fusion protein (CED-3 homology domain) was assessed for its ability to precipitate the indicated [³⁵S]methionine-labeled caspases, as described in Materials and Methods. (A) The signal generated by 10% of the total input amount of each caspase is shown. (B) The total amount of each caspase precipitated by 6 μg of GST or GST-Apaf-1^1-97 fusion protein. Identical exposures of each gel are shown to enable direct comparison.

Cytochrome c–initiated activation of multiple caspases. [³⁵S]Methionine-labeled caspases, prepared by coupled in vitro transcription/translation (∼0.25–0.5 μl of translation reaction in a total reaction volume of 10 μl), were incubated with cytochrome c (50 μg/ml) in the presence or absence of Jurkat postnuclear extract, as indicated. Where no cell extract was added to the reaction, an appropriate volume of extract dilution buffer was substituted. Cell-free reactions were incubated for 3 h at 37°C and were then analyzed by SDS-PAGE followed by fluorography.

Depletion of caspase-9 from Jurkat extracts abolishes cytochrome c–initiated processing of all caspases. (A) Jurkat extracts were depleted of caspase-9 using glutathione Sepharose–immobilized GST-Apaf-1^1-97 fusion protein, or mock-depleted with GST, and were then assessed for their ability to support caspase processing, as indicated. Caspase processing was assessed in the presence of 50 μg/ml cytochrome c and 1 mM dATP. (B) Extracts were depleted of caspase-9 using protein A/G agarose– immobilized anti–caspase-9 antibody, or mock- depleted using a control antibody (anti–Rel A), as described in Materials and Methods. The same concentrations of cytochrome c/dATP were used as in A. Results are representative of three independent experiments.

Depletion of caspase-6 from Jurkat extracts abolishes cytochrome c/dATP-initiated processing of caspases-8 and -10, whereas depletion of caspase-7 has no effect. Jurkat extracts were depleted of caspase-6 (A) or caspase-7 (B) as described in Materials and Methods and were then assessed for their ability to support processing of the indicated ³⁵S-labeled caspases. Caspase processing was assessed after incubation of extracts for 2 h at 37°C in the presence of 50 μg/ml cytochrome c and 1 mM dATP. (C) To confirm that caspases were depleted, equal amounts of either mock- depleted, caspase-6–depleted, or caspase-7–depleted Jurkat extracts were loaded, followed by probing for caspase-6 or caspase-7 by Western blot, as indicated.

Differential inhibition of cytochrome c–initiated processing of caspases-2, -3, -6, -7, -8, and -10 by DEVD-CHO. The indicated [³⁵S]methionine-labeled caspases were incubated in Jurkat cell extracts in the presence or absence of 10 μg/ml bovine heart cytochrome c and the indicated concentrations of peptides or recombinant proteins. Peptides and recombinant proteins were added to the extracts 30 min before the addition of cytochrome c. After incubation at 37°C for 3 h, reactions were stopped and caspase processing was subsequently assessed by SDS-PAGE/fluorography.

Kinetics of activation of caspases-2, -3, -6, -7, -8, -9, and -10 in response to cytochrome c. Cell-free reactions were assembled containing the indicated [³⁵S]methionine-labeled caspases and were incubated with or without 50 μg/ml cytochrome c and 1 mM dATP, as indicated. Reactions were incubated at 37°C and samples were removed at the indicated times, followed by analysis of caspase processing by SDS-PAGE/fluorography. Cleavage products are denoted by arrows. To allow direct comparison, all reactions were set up in an identical manner. Results are representative of a minimum of three independent experiments.

Cytochrome c initiates apoptotic changes in Jurkat cell–free extracts. Rat liver nuclei were incubated at 37°C in postnuclear extracts of Jurkat cells, prepared as described in Materials and Methods, in the presence or absence of 50 μg/ml of bovine heart cytochrome c. (A) At the indicated times, 2-μl aliquots of extract were removed for analysis of nuclear morphology by Hoescht 33342 staining. Nuclei were scored as apoptotic if they exhibited chromatin condensation and nuclear fragmentation characteristic of apoptosis. Each data point represents counts on 300 nuclei. Triplicate determinations (± SEM) are shown from a representative experiment. (B) At the indicated times, samples of each cell-free reaction were taken for SDS-PAGE, followed by Western blotting and probing for the indicated proteins.

Titration of cytochrome c–initiated apoptosis in Jurkat cell extracts. (A) Rat liver nuclei were incubated in postnuclear extracts from Jurkat cells (75 μg protein in 20 μl) for 2 h at 37°C in the presence or absence of the indicated amounts of bovine heart cytochrome c. As a control, nuclei were also incubated in the highest concentration of cytochrome c in the absence of cell extract (CEB was substituted). Apoptosis was assessed by scoring nuclei with features of apoptosis, as described in Fig. 1. Results shown are from a representative experiment and were derived from counts performed on 300 nuclei from several fields. (B) Cell-free reactions, set up identically to those in A, were incubated for 2 h at 37°C followed by analysis by SDS-PAGE and Western blotting. Blots were then probed with antibodies directed against the indicated caspases and caspase substrates.

Cytochrome c initiates processing and activation of caspase-3 but not caspase-1 in Jurkat cell extracts. (A) Processing of caspase-3 (top) and caspase-1 (bottom) in Jurkat cell–free extracts incubated for the indicated times with or without cytochrome c (50 μg/ml). Caspase processing was assessed by Western blot. (B) Cell-free reactions were set up as in A except that [³⁵S]methionine-labeled caspase-3, prepared by coupled in vitro transcription/translation, was added to the extracts. At the indicated times, portions of the extract were removed and were separated by SDS-PAGE followed by direct autoradiography. (C) Schematic representation of cytochrome c–initiated caspase-3 processing. The pattern of processing was deduced from the data shown in A and B. (D) At the indicated time points, extracts were assessed for DEVD-pNA or YVAD-pNA hydrolyzing activity, as described in Materials and Methods. Cell-free reactions were set up as described above and were incubated in the presence (closed circles) or absence (open circles) of cytochrome c (50 μg/ml). At the indicated times, samples of extract were removed and were assessed for their ability to hydrolyze the peptides DEVD-pNA and YVAD-pNA. Results shown are representative of three separate experiments.

Depletion of caspase-3 from Jurkat extracts abolishes cytochrome c–initiated processing of caspases-2, -6, -8, and -10 and reveals a feedback amplification loop involving caspase-9. (A) Jurkat extracts were depleted of caspase-3 as described in Materials and Methods and were then assessed for their ability to support processing of the indicated ³⁵S-labeled caspases. Caspase processing was assessed after incubation of extracts for 2 h at 37°C in the presence of 50 μg/ml cytochrome c and 1 mM dATP. (B) Caspase-9 processing is impaired in the absence of caspase-3. (Top) Jurkat extracts were depleted of caspase-3, or were mock depleted using a control antibody, and incubated under similar conditions to A. (Bottom) Cell extracts were prepared from Jurkat or MCF-7 cells and were compared for their ability to process caspase-9 to its p37 and p35 forms in the presence of cytochrome c/dATP. Caspase-9 was detected by Western blot. (C) Schematic representation of the sites at which caspase-9 and -3 are likely to cleave the caspase-9 pro-form to produce the p35 and p37 processed forms of caspase-9. (D) Verification of caspase-3 depletion from Jurkat extracts. Equal amounts of mock-depleted or caspase-3–depleted extract (top) or antibody-coupled protein A/G agarose beads used in the immunodepletions (bottom) were loaded, followed by probing for caspase-3.