A search for ceramide binding proteins using bifunctional lipid analogs yields CERT-related protein StarD7

Ceramides are central intermediates of sphingolipid metabolism with dual roles as mediators of cellular stress signalling and mitochondrial apoptosis. How ceramides exert their cytotoxic effects is unclear and their poor solubility in water hampers a search for specific protein interaction partners. In here, we report the application of a photoactivatable and clickable ceramide analogue, pacCer, to identify ceramide binding proteins and unravel the structural basis by which these proteins recognize ceramide. Besides capturing ceramide transfer protein CERT from a complex proteome, our approach yielded CERT-related protein StarD7 as novel ceramide binding protein. Previous work revealed that StarD7 is required for efficient mitochondrial import of phosphatidylcholine (PC) and serves a critical role in mitochondria function and morphology. Combining site-directed mutagenesis and photoaffinity labelling experiments, we demonstrate that the START domain of StarD7 harbours a common binding site for PC and ceramide. While StarD7 lacks robust ceramide transfer activity in vitro, we find that its ability to shuttle PC between model membranes is specifically affected by ceramides. Besides demonstrating the suitability of pacCer as a tool to hunt for ceramide binding proteins, our data point at StarD7 as a candidate effector protein by which ceramides may exert part of their mitochondria-mediated cytotoxic effects.