Mitochondrial protein FKBP8 captures PDZD8 to form mitochondria-ER contacts

SUMMARY Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in all cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types. PDZD8 tethering partner on the outer mitochondrial membrane (OMM) is unknown, limiting our understanding of MERCS formation and function. Here, by combining unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-electron microscopy (Cryo-EM) tomography, and correlative light-EM (CLEM), we identified the OMM protein FKBP8 as the tethering partner of PDZD8 at MERCS. Single molecule tracking of PDZD8 revealed highly dynamic diffusion properties along the ER contrasting with capture at contacts between ER and mitochondria. Overexpression of FKBP8 was sufficient to recruit PDZD8 along the OMM and narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrated their interdependence for MERCS formation. Our results identify a novel molecular complex tethering ER- mitochondria membranes in mammalian cells.