Abstract
Prohibitins (PHBs) are ubiquitously expressed proteins in the mitochondrial inner membrane (MIM) that provide membrane scaffolds for both mitochondrial proteins and phospholipids. Eukaryotic PHB complexes contain two highly homologous PHB subunits, PHB1 and PHB2, which are involved in various cellular processes, including metabolic control through the regulation of mitochondrial dynamics and integrity. Their mechanistic actions at the molecular level, however, particularly those of PHB1, remain poorly understood. To gain insight into the mechanistic actions of PHB1, we established an overexpression system for the full-length recombinant protein using silkworm larvae and characterized its biophysical properties in vitro. Using recombinant PHB1 proteoliposomes reconstituted into MIM-mimicking phospholipids, we found that PHB1 forms an oligomer via its carboxy-terminal coiled-coil region. A proline substitution into the PHB1 coiled-coil collapsed its well-ordered oligomeric state, and its destabilization correlated with mitochondrial morphologic defects. Negative-staining electron microscopy revealed that homotypic PHB1-PHB1 interactions via the coiled-coil also induced liposome tethering with remodeling of the lipid membrane structure. We clarified that PHB1 promotes membrane fusion mediated by optic atrophy 1 (OPA1), a key regulator of MIM fusion. Additionally, the presence of PHB1 reduces the dependency of lipids and OPA1 for completing the fusion process. Our in vitro study provides structural insight into how the mitochondrial scaffold plays a crucial role in regulating mitochondrial dynamics. Modulating the structure and/or function of PHB1 may offer new therapeutic potential, not only for mitochondrial dysfunction but also for other cell-related disorders.