In vitro BioID: mapping the CENP-A microenvironment with high temporal and spatial resolution
In vitro BioID: mapping the CENP-A microenvironment with high temporal and spatial resolution
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DOI：
10.1091/mbc.e18-12-0799
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发表时间：
2019-05-15
影响因子：
3.3
通讯作者：
Earnshaw, William C.
中科院分区：
文献类型：
--
作者：
Remnant, Lucy;Booth, Daniel G.;Earnshaw, William C.
着丝粒位于浓缩染色体的初级缢缩处，充当调节染色体分离的平台。组蛋白 H3 变体 CENP-A 是着丝粒形成的基础。 CENP-A 指导高度动态的分子邻域的形成，由于现有技术的限制，其在有丝分裂过程中的时间表征仍然是一个挑战。 BioID 是一种利用“混杂”生物素连接酶（BirA118R 或 BirA*）来识别与目标融合蛋白非常接近的蛋白质的方法。如最初所述，表达 BirA* 融合体的细胞暴露于高生物素浓度 24 小时，在此期间连接酶将活化的生物素 (BioAmp) 转移到附近的其他蛋白质。然后可以通过基于链霉亲和素的纯化和质谱来表征蛋白质邻域。在这里，我们描述了该技术的进一步发展，允许在生理进程被“冻结”的裂解细胞的体外反应中仅几分钟内表征 CENP-A 相互作用子。这种方法被称为体外 BioID (ivBioID)，有可能研究任何结构蛋白的分子邻域，这些结构蛋白的相互作用在细胞周期中或响应细胞生理学的其他变化而变化。
The centromere is located at the primary constriction of condensed chromosomes where it acts as a platform regulating chromosome segregation. The histone H3 variant CENP-A is the foundation for kinetochore formation. CENP-A directs the formation of a highly dynamic molecular neighborhood whose temporal characterization during mitosis remains a challenge due to limitations in available techniques. BioID is a method that exploits a "promiscuous" biotin ligase (BirA118R or BirA*) to identify proteins within close proximity to a fusion protein of interest. As originally described, cells expressing BirA* fusions were exposed to high biotin concentrations for 24 h during which the ligase transferred activated biotin (BioAmp) to other proteins within the immediate vicinity. The protein neighborhood could then be characterized by streptavidin-based purification and mass spectrometry. Here we describe a further development to this technique, allowing CENP-A interactors to be characterized within only a few minutes, in an in vitro reaction in lysed cells whose physiological progression is " frozen." This approach, termed in vitro BioID (ivBioID), has the potential to study the molecular neighborhood of any structural protein whose interactions change either during the cell cycle or in response to other changes in cell physiology.