Coordination Between Mammalian Nascent Protein Targeting and Cotranslational Chaperones

Citation

Hsieh, Hao-Hsuan (2023) Coordination Between Mammalian Nascent Protein Targeting and Cotranslational Chaperones. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/x61p-vf31. https://resolver.caltech.edu/CaltechTHESIS:07252022-075028264

Abstract

Protein biogenesis starts with ribosome synthesizing nascent polypeptide chain. Ribosome is a major hub for multiple pathways including membrane targeting, chaperones, chemical modification, and quality control. All these pathways need to coordinate with each other spatially on the ribosomal surface and temporally within the translation elongation window. Accumulating data start to point to more intricate interaction and coordination between different pathways beyond the simple competition traditionally presumed.

In Chapter 1, I demonstrate the coordination between a cotranslational chaperone, NAC, and the ER targeting machinery, SRP. NAC and SRP can bind to the same ribosome simultaneously despite overlapping binding sites, allowing NAC to change conformation of SRP specifically to the NC sequences. This allostery enhances the specificity of SRP-SR association, explaining the long-observed effect of NAC modulating ER targeting specificity.

In Chapter 2, I dig deeper into the mechanism of NAC regulating SRP. Based on cryo-EM structures, NAC domain sits on top of the ribosomal tunnel exit, potentially sensing the identity of NC, and is anchored by positively charged NACβ N-terminal tail. NAC-UBA domain is the key to recruiting SRP and coordinating the substrate handover to SRP.

In Chapter 3, I focus on the cotranslational HSP40/HSP70 system of RAC. Ribosome binding of RAC stimulates its cochaperone activity to activate HSP70 ATP hydrolysis. Ribosome sensing by RAC is related to the NBD of HSPA14. RAC-stimulated HSP70 engagement to NC keeps it in a folding-competent unfolded state before HSP70 dissociation.

Taken together, this study advances the experimental and theoretical tools to studying cotranslational pathways associated with the mammalian ribosome and demonstrates the interesting question of coordination between cotranslational pathways.

Thesis Files