Drp1-dependent mitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling

Cytochrome c release from the cristae into the cytoplasm constitutes the key step of intrinsic apoptosis (Frank et al., 2001; Detmer and Chan, 2007). A majority of total cytochrome c is encapsulated within the mitochondrial cristae folds that are connected to the intermembrane space (IMS) by relatively narrow structures named cristae junctions. At the early phase of intrinsic apoptosis, apoptotic signals induce cristae remodeling to redistribute cytochrome c into the IMS. Cytochrome c is then released into the cytoplasm through the mitochondrial outer membrane (MOM) pores generated by Bax and Bak, which are activated by BH3-only proteins such as proapoptotic truncated Bid (tBid), and initiates caspase cascade activation leading to cell death (Suen et al., 2008; Tait and Green, 2010). The mitochondrial inner membrane (MIM) profusion GTPase OPA1 plays a key role in maintaining healthy cristae junctions to protect cells from apoptosis; its oligomer stabilizes cristae morphology and prevents cristae remodeling and cytochrome c release (Olichon et al., 2003; Frezza et al., 2006; Varanita et al., 2015). Therefore, OPA1 down-regulation not only causes mitochondrial fragmentation but also alters cristae morphology, rendering cells susceptible to apoptosis. A current model indicates that the MIM-bound long forms of OPA1 (L-OPA1) and the processed soluble short forms (SOPA1) constitute high-molecular-weight OPA1 oligomers, and the Lto S-OPA1 balance is critical for maintaining cristae integrity; intrinsic apoptotic signals in vivo or incubation of isolated mitochondria with tBid induces the release of cytochrome c concomitant with stimulation of L-OPA1 processing to S-OPA1 and disassembly of OPA1 oligomers (Frezza et al., 2006; Jiang et al., 2014). So far, three MIM proteins are involved in cristae morphogenesis through the regulation of OPA1 function: prohibitin-1 and -2 (PHB1 and PHB2) and reactive oxygen species modulator protein 1 (ROMO1; Mgr2 in yeast). Prohibitins form large oligomeric structures with a membrane scaffold function and regulate cristae morphogenesis through OPA1 regulation (Merkwirth et al., 2008). Loss of PHB2 in PHB2−/− cells (which also induces PHB1 degradation) leads to selective loss of L-OPA1 isoforms, resulting in aberrant cristae morphogenesis and increased susceptibility to apoptosis. Re-expression of a noncleavable L-OPA1 mutant in PHB2−/− cells restores normal cristae structures and growth phenotypes, demonstrating that L-OPA1 is crucial for maintaining healthy cristae structures (Merkwirth et al., 2008). ROMO1, the MIM redox-regulated protein, is required for maintaining cristae junctions through the regulation of OPA1 oligomerization (Norton et al., 2014). The MOM proteins Fis1, Mff, MiD49/MIEF2, and MiD51/MIEF1 are reported to act as receptors of Drp1 in mammals, but recent studies revealed that Fis1 has little or no role in mitochondrial fission (Otera et al., 2010; Palmer et al., 2011; Zhao et al., 2011). During mitochondrial fission, ER Mitochondrial fission facilitates cytochrome c release from the intracristae space into the cytoplasm during intrinsic apoptosis, although how the mitochondrial fission factor Drp1 and its mitochondrial receptors Mff, MiD49, and MiD51 are involved in this reaction remains elusive. Here, we analyzed the functional division of these receptors with their knockout (KO) cell lines. In marked contrast to Mff-KO cells, MiD49/MiD51-KO and Drp1-KO cells completely resisted cristae remodeling and cytochrome c release during apoptosis. This phenotype in MiD49/51-KO cells, but not Drp1-KO cells, was completely abolished by treatments disrupting cristae structure such as OPA1 depletion. Unexpectedly, OPA1 oligomers generally thought to resist cytochrome c release by stabilizing the cristae structure were similarly disassembled in Drp1-KO and MiD49/51-KO cells, indicating that disassembly of OPA1 oligomers is not directly linked to cristae remodeling for cytochrome c release. Together, these results indicate that Drp1-dependent mitochondrial fission through MiD49/MiD51 regulates cristae remodeling during intrinsic apoptosis. Drp1-dependent mitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling