Dynamics of chaperonin function in live human cells

The eukaryotic chaperonin TRiC/CCT plays an essential role in protein folding1-4. As shown in vitro, the cylindrical TRiC complex captures unfolded client proteins and facilitates their folding through ATP-regulated encapsulation5-8. However, the functional dynamics of the chaperonin system within the cellular environment remain largely unexplored. In this study, we developed single-particle tracking in live human cells to monitor the interactions of TRiC with newly synthesized proteins, as well as its interplay with the co-chaperone PFD. We found that PFD and TRiC engage nascent protein chains in repeated, brief probing events—typically lasting around one second—with PFD acting to recruit TRiC. Using actin as an obligate chaperonin client7, the co-translational interactions of PFD and TRiC increased in both frequency and lifetime during nascent chain elongation. Near the point of translation termination, PFD interactions lasted for ~6 seconds, enabling TRiC recruitment for post-translational completion of folding via multiple reaction cycles of ~2.5 seconds. Notably, the lifetime of TRiC cycles was 3-4 times longer when a folding-defective actin mutant was analyzed, indicating that the conformational state of the client protein modulates TRiC function. Mutant actin continued cycling on TRiC until being targeted for proteasomal degradation. Unexpectedly, TRiC remained confined near its client protein between successive binding cycles, suggesting that folding occurs within a localized 'protective zone.' Together, these findings offer the first detailed insights into the dynamic behavior and supramolecular organization of the chaperonin system in living cells. The methodology developed in this study offers a framework for systematically investigating protein folding within the intact cellular environment. Overall design: Selective Ribo-seq of TRiC and PFDN performed in U2OS cells