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

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.

Figure 1

An efficient yeast internal-subunit eGFP labeling (YISEL) strategy. Taking CCT1 subunit as an example, in step 1, the PCR products of cct1 gene, HIS3 selection marker, and the sequence downstream the ORF of CCT1 subunit (~500 bp, now abbreviated as SDC1), with each fragment having a 20 bp overlapped sequence (blue square), were mixed with the linearized pUC19. The cloned DNA products were directly subjected to E. coli transformation for positive strain selection. In step 2, after obtaining the pUC19–cct1–HIS3–SDC1 plasmid (pUC19–C1HS), we amplified the cct1 gene upward the tag insertion site between K488 and P489 (referred as cct1-N), and the cct1 gene downward the tag insertion site together with the HIS3 selection marker and SDC1 from the pUC19–C1HS plasmid (referred as cct1-CD). In step 3, we mixed the amplified cct1-N, cct1-CD, eGFP, and the linearized pUC19, and subjected to E. coli transformation for positive strain selection. In step 4, we amplified the cct1-N–eGFP–cct1-CD sequence. Finally, in step 5, we transformed the purified PCR product of cct1-N–eGFP–cct1-CD to the haploid yeast, and select the positive strain.

Figure 2

Visualization of the inserted eGFP tag in reference-free 2D class averages. (A) For TRiC CCT1-eGFP in the both-ring closed conformation, we can visualize the exposed extra density corresponding to the inserted eGFP tag (indicated by yellow arrow) from the reference-free 2D class averages especially in the side views. (B,C) For TRiC CCT2-eGFP in the closed state and TRiC CCT5-eGFP in the open state, although shadow density exposed outside of TRiC can be observed in the reference-free 2D class averages, the feature is rather fuzzy and it is hard to identify which subunit the tag belongs to.

Figure 3

TRiC subunit identification through YISEL strategy combined with cryo-EM 3D reconstruction. (A) CCT2 subunit relative orientation determination in the closed state TRiC CCT2-eGFP map. Here eGFP was tagged to the A-domain of CCT2. The TRiC density is in grey, the extra density corresponding to eGFP tag in green, and the tag attached on-axis subunit in light blue. Since the tag density is usually weaker compared to that of TRiC, the map rendering threshold was lowered for better visualization of the tag density. (B,C) Direct subunit identification of CCT4 (B) and CCT5 (C) in the open NPP state TRiC CCT4-eGFP and TRiC CCT5-eGFP maps, respectively. Here eGFP was tagged to the I-domain of CCT4 or A-domain of CCT5. The adjacent Z-shaped on-axis subunit pair is in light blue. (D) CCT3 relative orientation determination in the closed state TRiC CCT3-CBP map. CBP was tagged to the A-I hinge region of CCT3. The on-axis subunit between the two tagged CCT3 subunits is in kaki and the extra density corresponding to CBP tag is in cyan.

Figure 4

TRiC subunit assignment in the open NPP state cryo-EM map. (A) In the open NPP state cryo-EM map of TRiC (EMD-9540), the subunits are numbered sequentially, starting from one of the on-axis subunits with the bent feature. Different subunits are in different colors. The previously determined subunits CCT1, CCT6, and CCT7 are also labeled in green. (B) Subunit assignment in the open NPP state TRiC map, determined by applying the YISEL strategy. Different views of the map were shown.