Evaluating the Raftophilicity of Rhodopsin Photoreceptor in a Patterned Model Membrane

AbstractLipid rafts in the cell membrane are believed to affect various membrane functions, including the signaling by G-protein coupled receptors (GPCRs). However, the regulatory roles of lipid rafts on GPCRs’ functions are still poorly understood, partially owing to the lack of the methods to quantitatively evaluate the affinity of membrane proteins to lipid raft (raftophilicity). Here, we describe a methodology to gauge the raftophilicity of a representative GPCR in vertebrate photoreceptor, i.e., rhodopsin (Rh), and its cognate G protein transducin (Gt) by using a patterned model membrane. We generated a substrate-supported planar lipid bilayer that has patterned regions of liquid-ordered (Lo) and liquid-disordered (Ld) membrane domains. We reconstituted Rh and Gt into the patterned membrane and observed their lateral distribution and diffusion. Mobile and functional Rh molecules could be reconstituted through the rapid dilution of solubilized Rh, by optimizing the reconstitution conditions including the chamber design, protein/detergent concentrations, and solution mixing. We determined the partition and diffusion coefficients of Rh and Gt in the Lo-rich and Ld-rich regions. Both Rh and Gt were predominantly localized in the Ld phase, suggesting their low affinity to lipid rafts. Patterned model membrane offers a robust and scalable platform for systematically and quantitatively studying the functional roles of lipid rafts in biological membranes including retinal disk membranes