Biology and mechanism of an ancestral opsin function

• Applicant: Professor Dr. Martin Göpfert
• Subject Area: Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 539452282

The visual opsin protein NINAE occurs in Drosophila photo- and mechanoreceptor cells. In photoreceptors it binds a light-sensitive chromophore, enabling visual transduction, whereas in mechanoreceptors it is chromophore-free. In Drosophila mutants lacking NINAE, both of these cell types degenerate. Photoreceptor degeneration seems caused by the toxicity of the free 3-hydroxy retinal chromophore, but the degeneration of mechanoreceptors is unexpected, implying that the opsin has a critical chromophore-independent function in these receptors. Precedent for such a function comes from bovine rod opsin that, when reconstituted into vesicles, scrambles phospholipids, facilitating their bi-directional translocation between inner and outer membrane layers, independently of retinal. The mechanism by which opsin scrambles lipids has been deduced from molecular dynamics (MD) simulations but remains to be tested experimentally. Also, the biological importance of opsin-mediated scrambling is unclear because elucidating it would necessitate genetic manipulations in the animals. In pilot studies, we discovered that NINAE shares the phospholipid scramblase activity of bovine rod opsin, and we now propose to use this fly protein to dissect how opsins scramble lipids and how this scrambling impacts cells. Firstly, wild-type NINAE protein will be extracted from the fly’s compound eye and reconstituted into liposomes to (i) systematically characterize its scramblase activity using fluorescence- and radioactivity-based assays. Secondly, protein residues suggested by MD simulations to be important for scrambling, will be mutagenized in the fly to test for effects on (ii) scrambling of the mutant protein after reconstitution in liposomes, and (iii) the structure and function of NINAE-expressing photo- and mechanoreceptors, using electrophysiology, immunohistology, and electron microscopy. Residues of bovine opsin that are predicted to be important for scrambling are largely conserved in NINAE, pointing to a common scrambling mechanism, and enabling a targeted mutagenesis approach. In addition to establishing an opsin function that, for Drosophila, is novel, the project promises a molecular understanding of how unconjugated opsins scramble phospholipids, how this scrambling impacts opsin-expressing sensory receptors, and why these cells degenerate without opsin. Addressing these questions is important for a general understanding of opsins as phospholipid scrambling might be the ancestral primary function of these light sensor proteins.

• DFG Programme: Research Grants
• International Connection: USA
• Partner Organisation: National Science Foundation (NSF)
• Cooperation Partner: Professor Dr. Anant Menon, Ph.D.