Activation of mitochondrial protease OMA1 by Bax and Bak promotes cytochrome c release during apoptosis

Intrinsic apoptotic stimuli initiate mammalian cells’ apoptotic program by first activating the proteins that have only Bcl-2 homology domain 3 (BH3), such as Bcl-2 interacting mediator of cell death (Bim) and truncated BH3 interacting death domain agonist (tBid), which in turn trigger conformational changes in BCL2-associated X (Bax) and BCL2-antagonist/killer (Bak) proteins that enable oligomer formation on the mitochondria, causing cytochrome c and other apoptogenic proteins in the intermembrane space to leak out. Leaked cytochrome c then initiates apoptotic caspase activation through a well-defined biochemical pathway. However, how oligomerized Bax and Bak cause cytochrome c release from mitochondria remains unknown. We report here the establishment of cell lines in which Bim or tBid can be inducibly expressed to initiate apoptosis in a controlled, quantitative manner. We used these cell lines to examine apoptotic events after Bax and Bak oligomerization but before cytochrome c release. The mitochondrial metalloprotease OMA1 was activated in this system in a Bax- and Bak-dependent fashion. Activated OMA1 cleaved the dynamin-like GTPase, optical nerve atrophy 1, an event that is critical for remodeling of mitochondrial cristae. Knockdown or knockout of OMA1 in these cells attenuated cytochrome c release. Thus it is clear that oligomerized Bax and Bak trigger apoptosis by causing both the permeabilization of the mitochondrial outer membrane and activation OMA1.Mitochondria in mammalian cells fulfill multiple functions. They are cells’ bio-energetic center, where reducing agents generated through the Krebs cycle transfer their electrons to oxygen in a manner mediated by the electron transfer chain, a process that builds a proton gradient across the inner membrane of mitochondria. The energy of this gradient is transferred into the high-energy bond of ATP by oxidative phosphorylation of ADP through the F1/F0 ATP synthase. During apoptosis, the sole water-soluble component of the electron transfer chain, cytochrome c, is released from the intermembrane space of mitochondria to the cytosol (1). Cytosolic cytochrome c binds to the Apaf-1 protein to promote the assembly of a heptamer complex named an “apoptosome”; this complex subsequently recruits procaspase-9, which autoactivates once on the apoptosome. The activated caspase-9 then cleaves and activates downstream caspase-3 and caspase-7, which subsequently cleave many intracellular substrates for apoptosis execution (2).In addition to cytochrome c, other proteins that normally are located in the mitochondrial intermembrane space also function in apoptosis. One such protein is second mitochondria-derived activator of caspase (Smac). When Smac is released, it binds to the BIR domain of inhibitors of apoptosis proteins to relieve their inhibition of the caspases directly or to cause their degradation (3, 4). Thus controlling the permeability of mitochondria for these apoptogenic proteins constitutes a key regulatory step for apoptosis.The B-cell leukemia/lymphoma 2 (Bcl-2) family of proteins constitutes a protein network that regulates the release of proteins such as cytochrome c and Smac (5, 6). BCL2-associated X (Bax) and BCL2-antagonist/killer (Bak), the proapoptotic members of the family with multiple Bcl-2 homology (BH) domains, form the core of the mitochondrial membrane permeability machinery that is activated by the proapoptotic proteins that have only the BH3 domain, a process that is inhibited by the proteins whose function is similar to that of Bcl-2 itself (7, 8). In response to apoptotic stimuli, BH3-only proteins, such as Bcl-2 interacting mediator of cell death (Bim), Puma, and truncated BH3 interacting death domain agonist (tBid) directly activate Bax/Bak and lift the inhibition of Bcl-2/Bcl-xl by forming stable heterodimers to sequester them from binding Bak/Bak (7, 9–13). Activated Bax and Bak initially form homodimers and then oligomers on the mitochondrial membrane (14–18). Bax/Bak oligomers are believed to form proteinaceous or lipidic pores on the mitochondrial outer membrane that allow the passage of proteins such as cytochrome c and Smac. Although the results of in vitro liposome leakage experiments support this model, there is no direct in vivo evidence to validate such a straightforward model (19–23).Moreover, increasing evidence indicates that the majority of cytochrome c in the mitochondrial intermembrane space is locked inside cristae by the protein complex containing optical nerve atrophy 1 (OPA1). The cristae must undergo reconfiguration to open up the neck of cristae for the bulk of cytochrome c to be released from the mitochondria after the outer membrane becomes permeable (24–26). The mitochondrial inner membrane fusion factor OPA1, a dynamin-like GTPase, plays a critical role in the remodeling of cristae. OPA1 presents in several spliced and proteolytic forms in mitochondria, and the maintenance of the relative amounts of each of these forms is known to be critical for stabilizing the cristae (27, 28). The longest form, L-OPA1, is cleaved in response to a variety of mitochondrial stresses, leading to the disassembly of OPA1-containing complexes and remodeling of the cristae (24, 26). It has been proposed that several different proteins cleave OPA1; these include the mitochondrial AAA proteases, presenilin-associated rhomboid-like protein (PARL), and the zinc metalloprotease OMA1 (overlapping activity with m-AAA protease) (26, 27, 29–31). However, the relationship between the pore formation on the mitochondrial outer membrane and OPA1 cleavage-mediated cristae remodeling during apoptosis, as well as the precise roles of those mitochondrial proteases in apoptosis, remain to be clarified.To dissect the molecular details of cytochrome c release induced by BH3-only proteins, we generated cell lines in which Bim or tBid can be inducibly expressed by adding doxycycline (Dox) into the culture medium. The expression of these proteins triggers apoptosis in a controlled and synchronized fashion. We used this cell-based system to characterize the mitochondrial response to the induction of Bim and tBid and found that OMA1 activation is an important step for apoptosis induction.