Development of a yeast internal-subunit eGFP labeling strategy and its application in subunit identification in eukaryotic group II chaperonin TRiC/CCT.
Development of a yeast internal-subunit eGFP labeling strategy and its application in subunit identification in eukaryotic group II chaperonin TRiC/CCT.
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酵母内部亚基 eGFP 标记策略的开发及其在真核 II 族伴侣蛋白 TRiC/CCT 亚基鉴定中的应用
DOI：
10.1038/s41598-017-18962-y
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发表时间：
2018-02-05
影响因子：
4.6
通讯作者：
Cong Y
中科院分区：
文献类型：
--
作者：
Zang Y;Wang H;Cui Z;Jin M;Liu C;Han W;Wang Y;Cong Y
在多组分复合物中的非耐药性亚基分配对于彻底理解机器及其功能至关重要。真核II组伴侣蛋白TRiC/CCT折叠约10%的胞质蛋白，对维持细胞内稳态很重要。TRiC由两个环组成，每个环具有八个同源但不同的亚基。通过中等或低分辨率的cryo-EM图谱鉴定大分子机器（如TRiC）的非耐药性亚基仍然具有挑战性。本文提出了一种酵母内亚基eGFP标记策略YISEL，该策略通过同源重组的方式将eGFP标签快速引入到靶亚基的内部位置，并使标签标记的蛋白质在内源水平上表达。通过这种方法，确保了标记效率和标签占有率，并且插入的标签与融合到末端的标签相比通常移动的较少。它还可用于在酵母蛋白质的内部位置进行其他标签的生物工程。通过应用我们的YISEL策略并结合cryo-EM 3D重建，我们明确识别了TRiC cryo-EM图中的所有亚基，证明了该策略在其他具有挑战性的复合物中准确有效的亚基识别中的广泛应用潜力。
Unambiguous subunit assignment in a multicomponent complex is critical for thorough understanding of the machinery and its functionality. The eukaryotic group II chaperonin TRiC/CCT folds approximately 10% of cytosolic proteins and is important for the maintenance of cellular homeostasis. TRiC consists of two rings and each ring has eight homologous but distinct subunits. Unambiguous subunit identification of a macromolecular machine such as TRiC through intermediate or low-resolution cryo-EM map remains challenging. Here we present a yeast internal-subunit eGFP labeling strategy termed YISEL, which can quickly introduce an eGFP tag in the internal position of a target subunit by homologous recombination, and the tag labeled protein can be expressed in endogenous level. Through this method, the labeling efficiency and tag-occupancy is ensured, and the inserted tag is usually less mobile compared to that fused to the terminus. It can also be used to bio-engineer other tag in the internal position of a protein in yeast. By applying our YISEL strategy and combined with cryo-EM 3D reconstruction, we unambiguously identified all the subunits in the cryo-EM map of TRiC, demonstrating the potential for broad application of this strategy in accurate and efficient subunit identification in other challenging complexes.
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