Center for Eukaryotic Gene Regulation and Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
Center for Proteome Analysis, Indiana University School of Medicine (IUSM), Indianapolis, IN 46202, USA.
G3 (Bethesda). 2024 Nov 6;14(11). doi: 10.1093/g3journal/jkae221.
The modified Escherichia coli biotin ligase BirA* was the first developed for proximity labeling of proteins (BioID). However, it has low activity at temperatures below 37°C, which reduces its effectiveness in organisms growing at lower temperatures, such as budding yeast. Multiple derivatives of the enzymes have been engineered, but a thorough comparison of these variations of biotin ligases and the development of versatile tools for conducting these experiments in Saccharomyces cerevisiae would benefit the community. Here, we designed a suite of vectors to compare the activities of biotin ligase enzymes in yeast. We found that the newer TurboID versions were the most effective at labeling proteins, but they displayed low constitutive labeling of proteins even in the absence of exogenous biotin, due to biotin contained in the culture medium. We describe a simple strategy to express free BioID enzymes in cells that can be used as an appropriate control in BioID studies to account for the promiscuous labeling of proteins caused by random interactions between bait-BioID enzymes in cells. We also describe chemically induced BioID systems exploiting the rapamycin-stabilized FRB-FKBP interaction. Finally, we used the TurboID version of the enzyme to explore the interactome of different subunits of the Ccr4-Not gene regulatory complex. We find that Ccr4-Not predominantly labeled cytoplasmic mRNA regulators, consistent with its function in mRNA decay and translation quality control in this cell compartment.
经改造的大肠杆菌生物素连接酶 BirA*是最初被开发用于蛋白质邻近标记（BioID）的酶。然而，它在 37°C 以下的温度下活性较低，这降低了它在生长温度较低的生物体中的有效性，例如 budding yeast。已经对该酶的多种衍生物进行了工程改造，但对这些生物素连接酶变体进行彻底比较，并开发用于在酿酒酵母中进行这些实验的通用工具将使该领域受益。在这里，我们设计了一套载体来比较酵母中生物素连接酶的活性。我们发现，较新的 TurboID 版本在标记蛋白质方面最有效，但即使在没有外源生物素的情况下，由于培养基中含有生物素，它们也会对蛋白质进行低组成型标记。我们描述了一种在细胞中表达游离 BioID 酶的简单策略，该策略可作为 BioID 研究中的适当对照，以解释由于细胞中诱饵-BioID 酶之间的随机相互作用而导致的蛋白质随机标记。我们还描述了利用雷帕霉素稳定的 FRB-FKBP 相互作用的化学诱导 BioID 系统。最后，我们使用该酶的 TurboID 版本来探索不同 Ccr4-Not 基因调控复合物亚基的互作组。我们发现 Ccr4-Not 主要标记细胞质 mRNA 调节剂，与它在该细胞区室中的 mRNA 降解和翻译质量控制功能一致。
