Asseck Lisa Yasmin, Grefen Christopher
Centre for Plant Molecular Biology, ZMBP-Developmental Genetics, University of Tübingen, Tübingen, Germany.
Methods Mol Biol. 2018;1794:49-60. doi: 10.1007/978-1-4939-7871-7_4.
The in vivo analysis of protein-protein interactions (PPIs) is a critical factor for gaining insights into cellular mechanisms and their biological functions. To that end, a constantly growing number of genetic tools has been established, some of which are using baker's yeast (Saccharomyces cerevisiae) as a model organism. Here, we provide a detailed protocol for the yeast mating-based split-ubiquitin system (mbSUS) to study binary interactions among or with full-length membrane proteins in their native subcellular environment. The system is based on the reassembly of two autonomously non-functional ubiquitin moieties attached to proteins of interest (POIs) into a native-like molecule followed by the release of a transcription factor. Upon its nuclear import, the activation of reporter gene expression gives a visual output via growth on interaction-selective media. Additionally, we apply a modification of the classical split-ubiquitin technique called CytoSUS that detects interactions of non-membrane/soluble proteins in their full-length form via translational fusion of an ER membrane anchor.
蛋白质-蛋白质相互作用（PPI）的体内分析是深入了解细胞机制及其生物学功能的关键因素。为此，已经建立了越来越多的遗传工具，其中一些工具使用面包酵母（酿酒酵母）作为模式生物。在这里，我们提供了基于酵母交配的分裂泛素系统（mbSUS）的详细方案，用于研究全长膜蛋白在其天然亚细胞环境中的二元相互作用或与其他蛋白的相互作用。该系统基于将附着在感兴趣蛋白质（POI）上的两个自主无功能的泛素部分重新组装成类似天然的分子，随后释放转录因子。转录因子核输入后，报告基因表达的激活通过在相互作用选择性培养基上生长给出视觉输出。此外，我们应用了一种对经典分裂泛素技术的改进，称为CytoSUS，它通过内质网膜锚定的翻译融合来检测全长形式的非膜/可溶性蛋白质的相互作用。
