The Bcl-2 family of proteins controls the key decision point in the intrinsic mitochondrial pathway of apoptosis. The Bcl-2 family consists of the Bak/Bax effectors, the BH3-only proteins that can trigger Bak/Bax activation, and the prosurvival proteins, whose role is to oppose the other two factions. The lack of full-length recombinant BH3-only proteins has hampered the study of Bcl-2 protein function in vitro. To dissect the layers of regulation governing the Bcl-2 effector proteins Bak and Bax, we have used a panel of stable Bid chimeras that contain the BH3 domains of other BH3-only proteins. These Bid BH3 chimeras mimic the dynamic behaviours of their parent BH3-only proteins more closely than previously used synthetic peptides, as they are full-length membrane-targeted proteins.

We treated isolated mitochondria with a mixture of recombinant Bid BH3 chimeras and prosurvival Bcl-2 family proteins (Mcl-1 or Bcl-x_L), and found that certain chimeras promoted Bak:prosurvival protein complexes while others promoted cytochrome c release. The same preferences were seen with the parental BH3-only proteins on isolated mitochondria, as well as in cells. Thus, the promotion of Bak:Mcl-1 and Bak:Bcl-x_L complexes by some but not other BH3-only proteins may explain resistance to apoptosis in certain cancer cells.