FIGURE 6.
Cdc6 blocks apoptosome assembly by forming complexes with cytc-bound Apaf-1 in reconstituted system. A, cytc-triggered caspase-9 and caspase-3 activation assay was performed with a mitochondrion-free cytosol preparation in the presence or absence of Cdc6, Cdc6WB, or Cdc6Cy as described under “Experimental Procedures.” The products were directly analyzed for activated caspase-9 and caspase-3 or immunoprecipitated (IP) with the anti-caspase-9 or anti-Cdc6 antibody and analyzed for coprecipitated Apaf-1, Cdc6 or caspase-9, and cytc as in Fig. 5. B, in vitro cytc-triggered stable complex formation between recombinant Apaf-1 and Cdc6. The cytc-triggered complex formation assay was performed with E. coli-expressed affinity-purified Apaf-1, baculovirus-produced double affinity-purified Cdc6, and varying amounts of cytc (1, 2, and 4 μg). The products were analyzed as in A. C, Cdc6 binds to a monomer of cytc-activated Apaf-1. Upper panel, cytc-triggered caspase-9 activation and Cdc6 binding assay was carried out with 40 μg of free or Sepharose bead-conjugated cytc and a mitochondrion-free cytosol preparation in the presence or absence of Cdc6 as in A. After reaction, the production of activated caspase-9 (Asp315-cleaved) and the amounts of unbound and bead-bound Apaf-1 and Cdc6 or those in the reaction mixture were determined by immunoblotting. Lower panel, cytc-triggered Apaf-1 and Cdc6 binding assay was carried out with 40 μg of Sepharose bead-conjugated cytc in the presence or absence of E. coli-produced affinity-purified C-terminally histidine hexamer-tagged Apaf-1 and baculovirus-produced double affinity-purified C-terminally histidine hexamer-tagged Cdc6. The amounts of bead-bound and -unbound Apaf-1 and Cdc6 were determined by regular immunoblotting with specific antibodies. In parallel, the relative amounts of bead-bound Apaf-1 and Cdc6 were quantified by immunoblotting with an anti-histidine hexamer antibody (Ab). Two bands above the Cdc6 are N-terminally viral protein-fused C-terminally histidine hexamer-tagged Cdc6 proteins that were generated by transcription from an upstream promoter(s) in the baculovirus vector and affinity-purified together with Cdc6 protein. Casp, caspase.