Research Institute for Interdisciplinary Science (RIIS), Okayama University, Okayama, Japan.
Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Methods Mol Biol. 2021;2251:195-204. doi: 10.1007/978-1-0716-1142-5_14.
A large proportion of proteins are expected to interact with cellular membranes to carry out their physiological functions in processes such as membrane transport, morphogenesis, cytoskeletal organization, and signal transduction. The recruitment of proteins at the membrane-cytoplasm interface and their activities are precisely regulated by phosphoinositides, which are negatively charged phospholipids found on the cytoplasmic leaflet of cellular membranes and play critical roles in membrane homeostasis and cellular signaling. Thus, it is important to reveal which proteins interact with phosphoinositides and to elucidate the underlying mechanisms. Here, we present two standard in vitro methods, liposome co-sedimentation and co-flotation assays, to study lipid-protein interactions. Liposomes can mimic various biological membranes in these assays because their lipid compositions and concentrations can be varied. Thus, in addition to mechanisms of lipid-protein interactions, these methods provide information on the possible specificities of proteins toward certain lipids such as specific phosphoinositide species and can hence shed light on the roles of membrane interactions on the functions of membrane-associated proteins.
预计很大一部分蛋白质将与细胞膜相互作用，以在膜运输、形态发生、细胞骨架组织和信号转导等过程中发挥其生理功能。蛋白质在膜-细胞质界面的募集及其活性受到磷酸肌醇的精确调节，磷酸肌醇是存在于细胞膜细胞质小叶上的带负电荷的磷脂，在膜稳态和细胞信号转导中发挥关键作用。因此，揭示哪些蛋白质与磷酸肌醇相互作用并阐明其潜在机制非常重要。在这里，我们介绍了两种标准的体外方法，脂质体共沉淀和共浮选测定法，用于研究脂质-蛋白质相互作用。在这些测定中，脂质体可以模拟各种生物膜，因为它们的脂质组成和浓度可以改变。因此，除了脂质-蛋白质相互作用的机制外，这些方法还提供了有关蛋白质对某些脂质（如特定的磷酸肌醇种类）可能具有特异性的信息，从而揭示了膜相互作用对膜相关蛋白质功能的作用。
