Rhodopsin-mediated phospholipid flipping

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DESCRIPTION (provided by applicant): Lipid trafficking in the retina is crucial for vision. Retinoids must move rapidly between photoreceptor cells and retinal pigment epithelial cells to regenerate the visual pigment rhodopsin after light impinges on the retina. This trafficking-dependent regeneration process is termed the visual cycle and is essential for continuous vision. Defects in lipid trafficking result in retinopathies; for example, Stargardt's macular dystrophy is caused by the inability to translocate a retinoid-phospholipid adduct across photoreceptor discs. We recently discovered a surprising new player in lipid transport within the retina: our biochemical reconstitution studies revealed that rhodopsin is an ATP-independent phospholipid translocator (flippase) capable of moving phospholipids rapidly across a membrane bilayer. This discovery provides the molecular basis for previous enigmatic observations of phospholipid flip-flop in disc membranes, and assigns a novel activity to rhodopsin in addition to its well-known function in phototransduction. Our goal in this application is to decipher the molecular mechanism by which rhodopsin flips lipids across a membrane bilayer. We propose to identify structural and dynamic features of rhodopsin's transmembrane helical bundle that are necessary for its flippase activity and also determine whether it is regulated by its membrane environment, specifically cholesterol and phospholipids with polyunsaturated acyl chains. We believe that rhodopsin's lipid flippase activity is critical for dic membrane homeostasis as it corrects the phospholipid imbalance caused by ATP-driven lipid transporters, including the Stargardt's disease transporter' ABCA4, that pump phospholipids from the lumen to the cytoplasmic face of discs. Our proposal to elucidate rhodopsin's flipping mechanism is highly significant because it will not only establish a new mechanistic paradigm in membrane transport but is also key to understanding lipid homeostasis in the retina, with implications for retinal degeneration. Mutations in rhodopsin are linked to retinitis pigmentosa, but the underlying disease-causing mechanism for many of the rhodopsin mutations is not known. Our proposed studies have the potential to reveal that some of the unexplained mutations affect flippase activity, thus clarifying aspects of this retinal disease that have remained unresolved for decades. Finally, rhodopsin is a prototypical G protein-coupled receptor (GPCR). As other GPCRs have been shown to have phospholipid flippase activity, our discoveries here will have implications beyond the visual system.