P01 - The role of prohibitin membrane scaffolds for the spatial organization of the mitochondrial inner membrane

The complex structure of the mitochondrial inner membrane (IM) is maintained by different multimeric protein assemblies, such as respiratory chain supercomplexes, ATP synthase oligomers or MICOS and OPA1 complexes. Moreover, membrane scaffolds of the prohibitin family are required to preserve the structural and functional integrity of mitochondria. Prohibitins form multimeric ring complexes in the IM, which are composed of two homologous subunits, PHB1 and PHB2. These PHB complexes are proposed to serve as lipid and membrane scaffolds defining functional IM domains. The tissue-specific ablation of prohibitins in mice revealed essential functions of prohibitins and identified disturbances in the cristae structure as an early pathogenic event in various tissues. However, how PHB membrane domains regulate cristae morphogenesis and the spatial organization of proteins and lipids in the IM has remained ill-defined and will be addressed in the present project.

We will use complementary cell biological, biochemical and structural approaches to unravel the role of prohibitins as membrane scaffolds on a molecular level. To visualize PHB membrane domains in intact cells under various conditions, we will establish CRISPR/Cas9 knock-in cell lines expressing tagged versions of PHB2 that allow multicolor super-resolution microscopy (gSTED), transmission electron microscopy (TEM) and correlative light- and electron microscopy (CLEM). Further experiments will examine the function and composition of PHB membrane domains. Complementing our previous interaction studies, we will use proximity proteomics and chemical cross-linking combined with mass spectrometry to identify proteins that constitute PHB membrane domains. The functional characterization of interacting proteins that were functionally linked to cristae morphogenesis, or to the assembly of the respiratory chain will be in the focus of these experiments. To further examine the role of prohibitins as putative lipid scaffolds, we will identify lipid species that are bound to PHB complexes by MS and initiate reconstitution experiments to analyze effects of prohibitins on the lateral distribution of lipids in membrane bilayers. Purified PHB complexes will be also characterized structurally by cryo-electron microscopy. Together, the proposed experiments will define the role of prohibitins as membrane scaffolds and provide new insight into the role of the spatial membrane organization for the structural and functional integrity of mitochondria.