Permeabilization of the mitochondrial outer membrane is the commitment step in intrinsic apoptosis. This process is tightly regulated by Bcl-2 family proteins that control formation of the megachannel mitochondrial apoptosis-induced channel (MAC) 2 in this membrane. MAC formation correlates with release of pro-apoptotic factors, including cytochrome c from the intermembrane space into the cytosol, and initiates apoptosis (1-7).MAC is absent from normal mitochondria but forms in the outer membrane early in apoptosis, reaching peak conductances of 1.5-5 nS. This channel is formed in the presence of the multidomain pro-apoptotic proteins Bax and/or Bak (8 -13), and may be composed of these proteins along with other components (14, 15). Unlike Bax, Bak is normally a resident of the mitochondrial outer membrane and is bound to VDAC2, another outer membrane protein (16). However, Bak is not available for oligomerization until another pro-apoptotic protein, like t-Bid, disrupts the interaction of Bak with VDAC2. In contrast, most Bax is located in the cytoplasm until an apoptotic signal induces the translocation of Bax to the outer membrane of mitochondria and eventual Bax oligomerization in this same membrane (14, 17).Bax and Bak have multiple putative transmembrane domains; the amphipathic helices 5 and 6 of Bax are predicted to form, at least in part, the pore of the cytochrome c release channel (18). Bax lacking helices 5 and 6 does not translocate to mitochondria nor cause cytochrome c release (19,20). Given the structural similarities between Bax and Bak, the same helices may be important in formation of the MAC pore by both proteins (21). Although Bax and Bak are certainly involved in MAC formation, the exact molecular composition of this channel remains unknown.In this study we report that Bax and Bak are functionally redundant with regard to MAC formation and cytochrome c release in mouse embryonic fibroblasts (MEF). This is true despite the fact that Bak normally resides in the outer membrane, whereas Bax is generally translocated to this membrane to induce MAC formation. Our experimental design bypasses Bax translocation and any underlying autocatalytic mechanism that might be involved (22). Instead, it focuses on formation of the MAC pore. Early MAC-associated conductance increments are relatively small, suggesting that Bax-dependent formation of the cytochrome c-permeable pore does not occur prior to membrane insertion of Bax. Mathematical modeling of the conductance changes indicates that, if MAC is a circular pore assembled by sequential addition of helices 5 and 6 from Bax and/or Bak monomers, the mature, cytochrome c transportcompetent pore is likely a 9 -10-mer of these proteins. ) cell lines were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 1% nonessential amino acids, and 1% L-glutamine (23,
EXPERIMENTAL PROCEDURES
