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在芽酵母中对BioID标记系统的表征和探索Ccr4-Not复合体的相互作用体

Jeffrey Pfannenstein¹, Misha Tyryshkin¹, Moira E Gulden¹

¹Center for Eukaryotic Gene Regulation and Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.

G3 (Bethesda, Md.)

|September 13, 2024

概括

新的TurboID酶可以改善酵母中的蛋白质标记,但需要仔细控制才能进行准确的近距离标记研究. 这项研究为在Saccharomyces cerevisiae中增强的BioID实验提供了工具和策略.

科学领域:

分子生物学分子生物学
生物化学生化学
酵母遗传学酵母遗传学

背景情况:

原始的BioID系统使用修饰的Escherichia coli生物酶 (BirA*),由于在温度低于37°C的低活性,在芽酵母等生物体中有效性有限.
虽然存在人工衍生品,但对于近距离标签实验,需要对酵母进行全面的比较和优化工具.

研究的目的:

为了比较Saccharomyces cerevisiae中各种生物酶酶的活性.
开发和验证用于酵母中的靠近依赖生物化 (BioID) 的多功能工具.
使用优化的BioID方法研究Ccr4-Not基因调控复合体的相互作用体.

主要方法:

开发一种载体套件,用于比较酵母中的生物素结合酶活性.
评估TurboID和其他生物素结合酶变体对蛋白质标记效率的评估.
化学诱导的BioID系统的实施,使用拉巴素诱导相互作用.
对BioID控制的描述,以减轻非特异性标签.

主要成果:

在酵母中,TurboID酶显示了蛋白质标记效率最高的酵母.
观察到TurboID的低构成标签,可能是由于培养基中的生物素.
为了解决非特异性标签问题,制定了一种表达自由BioID酶作为对照物的策略.
探索了Ccr4-Not复杂相互作用体,揭示了主要的细胞质mRNA调节器.

结论:

波ID变种对于酵母中的近距离标签是有效的,但需要仔细的实验设计来管理背景标签.
开发的工具和控制策略提高了在酵母中的BioID实验的可靠性.
这项研究提供了关于酵母细胞内Ccr4-Not复合物的局部化和功能的见解.

关键词:

生物ID BioID 是一个生物ID.Ccr4-不是没有在mRNA衰变过程中.接近性标签的标签.

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