1. Cytochrome c and ATP hydrolysis are required for Apaf‐1 to interact with procaspase‐9.
Cytochrome c and ATP hydrolysis are required for Apaf‐1 to interact with procaspase‐9. (A) Procaspase‐9 interacts with endogenous Apaf‐1 in a cytochrome c and dATP‐dependent manner. Equal amounts of 293T cytosolic extracts containing procaspase‐9 (C287S)‐HA were incubated with rabbit anti‐HA antibody and protein A/G–agarose beads (1:1) under the indicated conditions at 4°C for 2 h. Then the beads were pelleted and washed four times with 1 ml NP‐40 buffer. ‘++’ indicates that 5 mM ATP‐γS was included in the extract. (B) Procaspase‐9 interacts with Apaf‐1M368L in the absence of cytochrome c and dATP. The experiments were performed as in (A) except that procaspase‐9(C287S)‐HA‐containing extracts were mixed with lysates containing Apaf‐1, Apaf‐1M368L or Apaf‐1K160R. (C) A double‐point mutant, Apaf‐1M368L/K160R fails to interact with procaspase‐9. Expression of wt and mutant Apaf‐1 proteins in total lysates is shown in the lower panels in (B) and (C).
Yuanming Hu et al. EMBO J. 1999;18:
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