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

Yunxiang Zang,Wenyu Han,Yao Cong,Zhicheng Cui,Caixuan Liu,Huping Wang,Yanxing Wang,Mingliang Jin

doi:10.1038/s41598-017-18962-y

Abstract

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.

Highlights

Many essential cellular processes, including transcription, translation, and protein folding and degradation, are carried out by macromolecular complexes
We have previously determined the locations of CCT1, CCT6, and CCT7 subunits in the open conformation cryo-electron microscopy (cryo-EM) map of TRiC in the nucleotide partially preloaded (NPP) state with the Z-shaped feature formed by an on-axis subunit pair[28], while the locations of the other five subunits remain to be pinpointed
To unambiguously locate all the subunits in the cryo-EM map of TRiC complex, we developed a yeast internal-subunit eGFP labeling (YISEL) strategy

Summary

Introduction

Many essential cellular processes, including transcription, translation, and protein folding and degradation, are carried out by macromolecular complexes. It is usually more difficult to introduce a tag in the internal position of a target protein than that in its terminus in yeast system Along this line, several methods have been developed, including bioengineering proteins by heterologous promoters with exogenous vectors[40], and modification of gene by homologous recombination. We present a yeast internal-subunit eGFP labeling (YISEL) strategy, which integrates a one-step cloning and homologous recombination, allowing us to efficiently introduce a tag in the internal position of one gene in the chromosome using a PCR-based strategy[43] Through this method, we can express the complex with internal eGFP labeled subunit in endogenous level, and the length of the linker bridging the tag and the protein is controllable. Combining our YISEL strategy with cryo-EM 3D reconstruction, we were allowed to unambiguously identify all the TRiC subunits in its open NPP state cryo-EM map, demonstrating the potential broad application of this method in precise subunit identification in other macromolecular complexes

Methods

Results

Conclusion