Development of a yeast internal-subunit eGFP labeling strategy and its application in subunit identification in eukaryotic group II chaperonin TRiC/CCT

• Published in: Scientific reports Vol. 8; no. 1; pp. 2374 - 9
• Main Authors: Zang, Yunxiang, Wang, Huping, Cui, Zhicheng, Jin, Mingliang, Liu, Caixuan, Han, Wenyu, Wang, Yanxing, Cong, Yao
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 05-02-2018; Nature Publishing Group
• Subjects: 101/28; 38/77; 631/337/470/1981; 631/535/1258/1259; 82/80; Cryoelectron Microscopy; Green Fluorescent Proteins - analysis; Green Fluorescent Proteins - genetics; Group II Chaperonins - analysis; Group II Chaperonins - genetics; Homeostasis; Homologous Recombination; Humanities and Social Sciences; Imaging, Three-Dimensional; Labeling; Macromolecules; Molecular Biology - methods; multidisciplinary; Protein Subunits - analysis; Protein Subunits - genetics; Saccharomyces cerevisiae - enzymology; Science; Science (multidisciplinary); Staining and Labeling - methods; Yeast; Yeasts
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-18962-y

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.