Evaluating the raftophilicity of rhodopsin photoreceptor in a patterned model membrane
Evaluating the raftophilicity of rhodopsin photoreceptor in a patterned model membrane
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评估图案化模型膜中视紫红质感光体的亲筏性
DOI：
10.1016/j.bpj.2015.10.015
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发表时间：
2015
影响因子：
3.4
通讯作者：
K.
中科院分区：
文献类型：
--
作者：
Tanimoto;Y.;Okada;K.;*Hayashi;F.;Morigaki;K.
细胞膜中的脂筏被认为影响各种膜功能，包括通过G蛋白偶联受体（GPCR）的信号传导。然而，脂筏对GPCR功能的调节作用仍然知之甚少，部分原因是缺乏定量评估膜蛋白对脂筏亲和力（raftophilicity）的方法。在这里，我们描述了一种测量脊椎动物光感受器中代表性GPCR的亲脂性的方法，即，视紫红质（Rh）及其同源G蛋白转导素（Gt）。我们产生了基板支撑的平面脂质双层，具有图案化的区域的液体有序（Lo）和液体无序（Ld）的膜域。我们将Rh和Gt重组到图案化的膜中，观察它们的横向分布和扩散。移动的和功能性Rh分子可以通过快速稀释溶解的Rh来重构，通过优化重构条件，包括腔室设计、蛋白质/去污剂浓度和溶液混合。测定了Rh和G在富Lo区和富Li区的分配系数和扩散系数。Rh和Gt主要定位于Ld相，表明它们与脂筏的亲和力较低。图案化模型膜提供了一个强大的和可扩展的平台，系统和定量研究生物膜，包括视网膜盘膜的脂筏的功能作用。
Lipid 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 andGtinto 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 andGtin theLo-rich andLd-rich regions. Both Rh andGtwere predominantly localized in theLdphase, 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.