OPA1-dependent cristae remodeling disassembles respiratory chain supercomplexes, triggering apoptotic mitochondrial dysfunction

Mitochondria are key organelles in intermediatig cellular metabolism, energy production and calcium homeostasis (Rizzuto et al., 2000; Danial and Korsmeyer, 2004). They also integrate and amplify apoptosis induced by several intrinsic stimuli (Green and Reed, 1998). Their structure is extremely complex, being bound by two membranes. The inner membrane (IMM) can be further divided in two distinct compartments, the so called “boundary membrane” and the cristae, separated from the former by narrow tubular junctions. Such a functional versatility and complexity is controlled by a growing family of “mitochondria-shaping” proteins that regulate fusion and fission events of mitochondrial membranes, ultimately affecting the morphology and ultrastructure of the organelle. Optic Atrophy 1 (OPA1), the homologue of S.cerevisiae Mgm1p, is the only dynamin-related protein identified in the inner membrane (IM) so far (Olichon et al., 2002). Our laboratory discovered that OPA1 promotes mitochondrial fusion by cooperating with MFN1 (Cipolat et al., 2004), and demonstrated that OPA1 has antiapoptotic activity, genetically distinguishable from its function in mitochondrial fusion (Frezza et al., 2006). In particular we proved that OPA1 keeps the cristae junctions tight by forming oligomers that contain two forms of OPA1, one soluble in the IMS, generated by intramembrane proteolytic cleavage by the rhomboid protease PARL, and the second inserted in the IMM. In the early steps of apoptosis, the pro-apoptotic BH3-only BCL-2 family member BID disrupts these OPA1-containing oligomers and causes a remodeling of cristae structure. Conversely, high levels of OPA1 stabilize them and prevent mobilization of cytochrome c from mitochondria (Frezza et al., 2006). Notably, EM analysis and EM tomography showed that OPA1-depleted cells harbour disorganized cristae whose shape is irregular (Frezza et al., 2006), an observation supported by other experimental evidence in different cellular models that point out the importance of OPA1 in cristae morphology (Olichon et al., 2003; Griparic et al., 2004). The cristae are key mitochondrial structures: they are the site of oxidative phosphorylation where the complexes of respiratory chain are localized. Recent structural and functional evidence demonstrated that in order to improve the efficiency of electron channelling, the individual respiratory chain complexes are organized into functional and dynamic supramolecular structures referred as supercomplexes (RCS). (Acin-Perez et al., 2008) These evidence gave rise to a novel model called “plasticity model” that integrates the old “fluid” and “solid” models that have been put forward to explain the organization of the electron transport chain . While the role of cristae remodelling in the amplification of the apoptotic cascade has been established (Scorrano et al., 2002; Frezza et al., 2006; Germain et al., 2005) its consequences on mitochondrial function are unknown. The aim of this PhD thesis was to investigate if apoptotic cristae remodelling had any effect on the activity and the structure of the mitochondrial respiratory chain, and on supercomplexes (RCS),that are preferentially located in the cristae. Since the multiple changes affect mitochondria during apoptosis, including the release of cytochrome c and the activation of feedback circuits that lead to the inactivation of mitochondrial respiration, we needed to identify an inducer of apoptosis that causes all the apoptotic changes except for cristae remodeling. Analyzing the aminoacidic structure of BID, we identify a stretch of aminoacid, corresponding to the α6-helix, that displays high homology with mastoparan, a wasp venom peptide that severely perturbs mitochondrial membranes (Pfeiffer et al., 1995) and that seemed a natural candidate to exert the membrane perturbation of the cristae remodeling. A BID mutant in two conserved Lysines (K157,158) of this domain (BIDKKAA) was able to target mitochondria as well as its wt counterpart but was less efficient in inducing cytochrome c release. Crosslinking experiments revealed that the mutation in the α6-helix does not impair the oligomerization of BAK, but reduces the ability of BID to distrupt the high molecular weight complexes of OPA1, resulting in a reduction of the apoptotic rate. Thus, we capitalized on this mutant to study the effects of cristae remodeling on mitochondria physiology. Interestingly, wt BID selectively impairs complex I dependent mitochondrial respiration which is higly influenced by RCS assembly. BlueNative page (BN PAGE) confirmed that BID impacts on RCS by preventing the individual complexes from being assembled into supercomplexes, without affecting complexes. These changes were abrogated by the BID mutant that is unable to induce cristae remodeling, but not by other mutants of BID that do not trigger activation of BAX, BAK. Since the molecular basis of the assembly and stabilization of RCS are unknown, the selective impairment of RCS by cristae remodeling pointed our attention to the role of OPA1, whose oligomers are targets of BID and control cristae bioegnesis. We turned to a genetic approach and analyzed the structure and the assembly of RCS in Opa1-/- cells. First dimension BN PAGE and SDS-second dimension page revealed that the structure of RCS in Opa1 deficient cells is dramatically affected. In particular a RCS assembly assay (Acin-Perez et al., 2008) confirmed that the ordered pattern of assembly is perturbed in Opa1-/- cells, resulting in anomalous composition of RCS. BN PAGE performed with radiolabeled mt-DNA proteins revealed that the overall amount of mitochondrial protein is less in Opa1-/- than in wt cells and it correlates with the fact that Opa1-/- cells have less mt-DNA copy number a feature shared also by Mfn1-/-,2-/- cells that on the contrary display organized cristae. Of note, BN PAGE and assembly assay demonstrated that the structure of RCS is not affected in Mfn1-/-,2-/- cells. The physiological importance of RCS disassembly is substantiated by an impairment of the growth rate of Opa1-/- cells or of cells lacking Bak and Bax expressing wt, but not KKAA BID when energy production cannot bypass mitochondrial respiration. In conclusion, our data indicate that the shape of the cristae is essential for the assembly of the RCS and underline OPA1 as molecular regulator of the RCS structure. Moreover our results deepen the role of cristae remodelling during apoptosis, demonstrating that cristae remodelling affects mitochondrial respiratory efficiency and hence the program of mitochondrial dysfunction during apoptosis.