Institut Curie, PSL Research University, Cellular and Chemical Biology unit, Endocytic Trafficking and Intracellular Delivery team, U1143 INSERM, UMR3666 CNRS, 26 rue d'Ulm, Paris Cedex 05, France.
Institut Curie, PSL Research University, UMR 168, Centre de Recherche, Paris, France.
Methods Mol Biol. 2022;2507:1-18. doi: 10.1007/978-1-0716-2368-8_1.
Transmembrane proteins (or integral membrane proteins) are synthesized in the endoplasmic reticulum where most of them are core glycosylated prior to folding and in some cases assembly into quaternary structures. Correctly glycosylated, folded, and assembled transmembrane proteins are then shuttled to the Golgi apparatus for additional posttranslational modifications such as complex-type glycosylations, sulfation or proteolytic clipping. At the plasma membrane, the glycosylated extracellular domains are key to communicate with the cellular environment for a variety of functions, such as binding to the extracellular matrix for cell adhesion and migration, to neighboring cells for cell-cell interaction, or to extracellular components for nutrient uptake and cell signaling. Intracellular domains are essential to mediate signaling cascades, or to connect to cytosolic adaptors for internalization and intracellular compartmentalization. Despite its importance for the understanding of molecular mechanisms of transmembrane protein function, the determination of their structures has remained a challenging task. In recent years, their reconstitution in lipid nanodiscs in combination with high resolution cryo-electron microscopy has provided novel avenues to render this process more accessible. Here, we describe detailed protocols for the solubilization of heavily glycosylated αβ integrin from rat livers, its purification and reconstitution into nanodiscs. At the plasma membrane of many cells, including tumor metastases, this essential heterodimeric transmembrane protein mediates the communication between extracellular matrix and cytosolic cytoskeleton in processes of cell adhesion and migration. We expect that the protocols that are described here will provide new opportunities for the determination of the full structure of αβ integrin, as well as for the understanding of how interacting partners can regulate function and activity of this transmembrane protein.
跨膜蛋白（或整合膜蛋白）在粗面内质网中合成，其中大多数在折叠之前进行核心糖基化，在某些情况下还会组装成四级结构。正确糖基化、折叠和组装的跨膜蛋白随后被运送到高尔基体进行额外的翻译后修饰，如复杂型糖基化、硫酸化或蛋白水解剪接。在质膜上，糖基化的细胞外结构域是与细胞环境进行各种功能交流的关键，例如与细胞外基质结合以实现细胞黏附和迁移、与相邻细胞结合以实现细胞间相互作用，或与细胞外成分结合以实现营养摄取和细胞信号转导。细胞内结构域对于介导信号级联反应或连接细胞质衔接子以进行内化和细胞内区室化至关重要。尽管了解跨膜蛋白功能的分子机制非常重要，但确定其结构一直是一项具有挑战性的任务。近年来，将其在脂质纳米盘中的重建与高分辨率冷冻电子显微镜相结合，为实现这一过程提供了新的途径。在这里，我们描述了从大鼠肝脏中溶解高度糖基化的 αβ 整合素、纯化和重建到纳米盘中的详细方案。在许多细胞的质膜上，包括肿瘤转移，这种必需的异二聚体跨膜蛋白介导细胞外基质与细胞质骨架之间的通讯，在细胞黏附和迁移过程中发挥作用。我们预计，这里描述的方案将为确定 αβ 整合素的完整结构以及理解相互作用的伙伴如何调节这种跨膜蛋白的功能和活性提供新的机会。
