Newly synthesized proteins are born as unfolded polypeptides emerging from the ribosomal exit tunnel. Folding these nascent chains into nati
Newly synthesized proteins are born as unfolded polypeptides emerging from the ribosomal exit tunnel. Folding these nascent chains into native conformations is crucial for protein functionality and preventing off-pathway interactions that trigger misfolding and jeopardize proteome stability. However, achieving the correct 3D structure is a major challenge for nascent chains exposed to high concentrations of molecules in the cytosol. General ribosome-associated chaperones assist co-translational folding of a wide variety of nascent peptides. It is unclear whether this “one-size-fits-all” system ensures the expression of proteins with challenging folding paths or if specialized ribosome-associated chaperones manage the folding of such demanding clients. In Study I, we investigated how the Hsp70 chaperone regulates Hsf1, a transcription factor that mediates the cellular response to proteotoxic stress. We demonstrated that Hsp70 directly binds to Hsf1, keeping it in a latent state under non-stress conditions. Protein misfolding, particularly of newly synthesized proteins, titrates Hsp70 away, activating Hsf1 and inducing the stress response. Thus, Hsp70 availability in response to misfolded proteins is a key regulatory mechanism of Hsf1 activity. In Study II, we identified a specialized ribosome-associated chaperone, Chp1, that assists in the co-translational folding of eEF1A, a highly abundant multidomain GTPase critical for mRNA translation into proteins. Deleting Chp1 leads to rapid proteolysis of eEF1A, widespread protein aggregation, and activation of the Hsf1-mediated stress response. Finally, in Study III, we elucidated how Chp1 assists in eEF1A folding and the ordered sequence of chaperone actions in the eEF1A folding pathway. We found that Chp1 binds to the α3 helix in the switch I region of the eEF1A G-domain, crucial for nucleotide binding, delaying the nucleotide-guided folding of the G-domain. As eEF1A domain II synthesis begins, the substrate is transferred to the downstream chaperone Zpr1 for final maturation. Our results provide insight into the molecular mechanisms of co-translational protein folding and its impact on proteome stability, as well as on the regulation of Hsf1, the central mediator of the response to proteotoxic stress in eukaryotic cells.
