The multidomain proapoptotic molecules Bax and Bak are directly activated by heat

During apoptosis, engagement of the mitochondrial pathway involves a decisive event characterized by the release of mitochondrial intermembrane space proteins, such as cytochrome c. This permeabilization of the mitochondrial outer membrane depends on activation and oligomerization of multidomain Bcl-2-family proteins Bax or Bak. Although specific members of the Bcl-2 family can activate these proapoptotic proteins, we found that heat directly activated Bax or Bak to induce cytochrome c release. A preparation of mitochondria heated at 43 degrees C released cytochrome c in association with Bak oligomerization, and Bcl-xL prevented these events. Similarly, heat induced the oligomerization of recombinant Bax, conferring an ability to permeabilize mitochondria. Compared with wild-type cells, bax(-/-)bak(-/-) mouse embryonic fibroblasts and mitochondria isolated from these cells were resistant to heat-induced cytochrome c release. Cytosol from untreated cells inhibited heat-activated Bax or Bak; however, depletion of cytosolic Bcl-xL ablated this protection. Although mitochondria heated in the presence of cytosol did not release cytochrome c, they displayed a dramatic increase in sensitivity to permeabilization by the BH3-only protein Bid. Additionally, a peptide corresponding to the BH3 domain of Puma counteracted the inhibitory effect of cytosol and permitted heat-activated Bak to permeabilize the mitochondria. Therefore, heat represents a condition under which multidomain proapoptotic proteins are activated, and this activation is regulated by both antiapoptotic and BH3-only members of the Bcl-2 family. Our results support an emerging paradigm, wherein the activation of Bax or Bak and the blockade of antiapoptotic Bcl-2 proteins are pivotal steps in the mitochondrial pathway of apoptosis.