Functional characterization of prohibitins by conditional inactivation in the mouse

Prohibitins comprise an evolutionary conserved and ubiquitously expressed family of membrane proteins implicated in a large variety of cellular processes. Large assemblies of PHB1 and PHB2 subunits are localized in the inner membrane of mitochondria, but various roles in other cellular compartments have also been proposed for both proteins. However, the function of prohibitins on the molecular level remains unclear. To investigate the physiological role of mammalian prohibitins, a mouse strain for the conditional, Cre/loxP-mediated inactivation of Phb2 was generated. Ubiquitous and brain-restricted deletion of Phb2 in vivo caused embryonic lethality indicating an essential role of Phb2 in mammalian development. To determine cellular functions of PHB2, a cell culture system was established allowing to define functional consequences of a Phb2 deletion. Mouse embryonic fibroblasts (MEFs) isolated from Phb2fl/fl embryos were transduced with cell-permeable Cre-recombinase to inactivate Phb2. Functional interdependence of prohibitin subunits was observed in MEFs after PHB2 depletion, illustrating the physiological relevance of the assembled prohibitin complex. The absence of prohibitins in MEFs leads to impaired cell proliferation and increased sensitivity towards apoptotic stimuli. This is accompanied by fragmentation of the mitochondrial network and defective morphogenesis of mitochondrial cristae. Complementation experiments attribute these defects to the loss of mitochondria-localized prohibitins indicating an essential requirement of the mitochondrial prohibitin complex in these processes. Loss of prohibitins affects the proteolytic cleavage of OPA1, a dynamin-like GTPase in the inner membrane essential for mitochondrial fusion, leading to the selective loss of long isoforms of OPA1. The specific expression of a long OPA1 isoform in prohibitin-deficient MEFs restores cristae morphogenesis, apoptotic resistance and partially cell proliferation, identifying impaired OPA1 processing as the primary cause for the cellular defects in the absence of prohibitins. These results identify a novel roles for mitochondrial prohibitins in cell proliferation and cristae formation by the proteolytic modulation of OPA1, suggesting a molecular interplay between cell growth and organelle morphogenesis