Stelter Philipp, Hurt Ed
Biochemie-Zentrum der Universität Heidelberg (BZH), Im Neuenheimer Feld, Heidelberg, Germany.
Methods Cell Biol. 2014;122:147-63. doi: 10.1016/B978-0-12-417160-2.00007-2.
The vast number of cellular proteins performs their roles within macromolecular assemblies and functional cell networks. Hence, an understanding of how multiprotein complexes are formed and modified during biogenesis is a key problem in cell biology. Here, we describe a detailed protocol for a nonradioactive pulse-chase in vivo-labeling approach. The method is based on the incorporation of an unnatural amino acid (O-methyl-tyrosine) by the nonsense suppression of an amber stop codon that quickly fuses an affinity tag of choice to a protein of interest. This affinity tag could be used to directly isolate the newly synthesized proteins and hence allows for the characterization of early complex biogenesis intermediates. Combined with a tetracycline controllable riboswitch in the 5'-UTR of the respective mRNA, this approach became a versatile tool to study dynamic protein assembly within cellular networks (Stelter et al., 2012). In the context of this volume, this method notably provides a suitable approach to study NPC, ribosome and mRNP biogenesis, or nuclear protein translocation. This chapter includes detailed protocols to track newly synthesized, epitope pulsed-chased proteins by western blot, their assembly within complexes using immunoprecipitation, and their subcellular localization using indirect immunofluorescence or subcellular fractionation. While these protocols use budding yeast as model system, this method can be adapted to other model systems.
大量的细胞蛋白质在大分子组装体和功能性细胞网络中发挥作用。因此，了解多蛋白复合物在生物发生过程中是如何形成和修饰的，是细胞生物学中的一个关键问题。在这里，我们描述了一种用于体内非放射性脉冲追踪标记方法的详细方案。该方法基于通过琥珀色终止密码子的无义抑制掺入非天然氨基酸（O-甲基酪氨酸），从而快速将选择的亲和标签与感兴趣的蛋白质融合。这种亲和标签可用于直接分离新合成的蛋白质，从而能够表征早期复合物生物发生中间体。结合各自mRNA 5'-UTR中的四环素可控核糖开关，该方法成为研究细胞网络内动态蛋白质组装的通用工具（Stelter等人，2012年）。在本卷的背景下，该方法特别提供了一种研究核孔复合体、核糖体和信使核糖核蛋白生物发生或核蛋白转运的合适方法。本章包括通过蛋白质印迹追踪新合成的、表位脉冲追踪的蛋白质、使用免疫沉淀法研究它们在复合物中的组装以及使用间接免疫荧光或亚细胞分级分离法研究它们的亚细胞定位的详细方案。虽然这些方案以芽殖酵母作为模型系统，但该方法可适用于其他模型系统。
