The chaperonin barrel of mammalian cells is called TRiC. Previous findings suggested that TRiC begins to function early during the life of a new protein chain, as it is made on the ribosome, the machinery of protein synthesis (Figure 1). To visualize this process, we used human cells in culture and modified them in such a way that TRiC and the ribosome carry different fluorescent tags. We then used a microscopy method called single particle tracking to observe these interactions, determine their frequency and duration. We were also able to put a fluorescent tag on a specific client protein of TRiC, called actin.
We observed that TRiC engages the protein chains on ribosomes in short probing interactions of about 1 second. Another chaperone, called prefoldin (PFD), proved to be necessary to recruit TRiC to the growing protein chains. The folding process is completed when the protein is fully synthesized. We found that the interactions of TRiC with completed actin chains last for several seconds, consistent with the protein requiring encapsulation by chaperonin for folding.
In a related study we used cryo-electron tomography (cryo-ET) to analyze the bacterial chaperonin, called GroEL, and its lid-shaped cofactor, GroES. The cells are in a life-like, shock-frozen state in these experiments. We localized the positions of all GroEL/GroES complexes and ribosomes in three-dimensional reconstructions of the cell (Figure 2). Most importantly, we succeeded in visualising client protein inside the chaperonin barrel (Figure 3), demonstrating that the folding reaction indeed occurs in the enclosed chaperonin chamber.
Finally, we addressed the question how cells deal with certain mistakes in protein synthesis, whereby the ribosome erroneously extends the protein chain with additional amino acids a problem that occurs at a low but significant frequency. This generates protein chains that cannot fold properly and may be toxic to cells. We found that these aberrant proteins resemble a particular class of regular proteins that are embedded in a lipid membrane. Interestingly, the same chaperones that mediate the physiological membrane insertion process also recognize the faulty proteins, but then transfer these to a molecular machine called proteasome that shreds the protein chain into its amino acids. Thus, the machinery used for the production of certain membrane proteins is co-opted in a quality control process that removes faulty and potentially dangerous proteins.
