Rhodopsin is the visual pigment in photoreceptor cells of the retina and is responsible for the first events in the perception of light. It consists of the apo-protein opsin, a multi-spanning membrane protein, and retinal, a reversibly bound cofactor. Very recently, rhodopsin was demonstrated to be an ATP-independent phospholipid flippase capable of moving phospholipids rapidly across a membrane bilayer. This discovery provided the molecular basis for observations of bidirectional, ATP-independent phospholipid flip-flop in photoreceptor disc membranes and assigned a novel activity to rhodopsin in addition to its well-known function in phototransduction. Lipid trafficking in the retina is crucial for vision. Retinoids must move rapidly between photoreceptor cells and retinal epithelial cells to regenerate rhodopsin after photoactivation. Defects in lipid trafficking result in retinopathies; for example, Stargardts macular dystrophy is caused by the inability to translocate a retinoid-phospholipid adduct across photoreceptor discs. Bidirectional flip-flop, now known to be catalyzed by rhodopsin, resolves the problem of expansion of one phospholipid monolayer at the expense of the other caused by the unidirectional movement of lipids by the two ATP-dependent flippases ABCA4 (Quazi et al., 2012; Weng et al., 1999) and Atp8a2 (Coleman et al., 2009). Although rhodopsin is an extensively-studied protein, hardly anything is known about its structural or dynamic features how it might flip lipids. The proposal presented here is focused on the identification of structural and dynamic features of rhodopsins transmembrane domains that are necessary for its flippase activity. We hypothesize that lipid flipping is made possible by structural elements within rhodopsins transmembrane helical bundle and by dynamic movements of the transmembrane helices. To test this hypothesis we will investigate the effect of replacing specific residues within rhodopsins transmembrane helical bundle, particularly those that face membrane lipids to identify key residues needed for flipping. The mutagenized rhodopsins will be expressed in cultured cells and characterized after affinity purification. Flippase activity will be tested by a well-established fluorescent approach using proteoliposomes with 7-nitrobenz-2-oxa-1,3-diazol (NBD)-labeled lipids whose transbilayer orientation will be probed with the membrane impermeant reductant dithionite. In addition, the fluorescent-based data will be complemented by the establishment of a new flippase assay using natural phospholipids.
|Effective start/end date||24/11/14 → 23/05/17|