Spatiotemporal Regulation of Nascent Protein Targeting

Citation

Zhu, Zikun (2024) Spatiotemporal Regulation of Nascent Protein Targeting. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ybz3-4m48. https://resolver.caltech.edu/CaltechTHESIS:05302024-023258565

Abstract

Proper protein targeting to the correct cellular compartments is essential for maintaining the functionality and organization of all cells. However, the mechanisms that ensure newly synthesized proteins are accurately and efficiently directed to their specific cellular destinations remain unclear. Moreover, how protein targeting is coordinated with protein folding and other cellular processes, both spatially and temporally, is largely unknown.

In my thesis, I first demonstrated the mechanism of a nascent protein transport pathway in prokaryotes, mediated by a conserved ATPase SecA. Using a combination of ribosome profiling methods, I revealed the essential roles of SecA in recognizing and resolving the widespread accumulation of large periplasmic loops of inner membrane proteins in the cytoplasm during their cotranslational translocation, and in the cotranslational transport of secretory proteins with highly hydrophobic signal sequences. I also uncovered a function of the chaperone trigger factor (TF) in temporally regulating SecA engagement on secretory proteins. These findings elucidate the principles of SecA-driven cotranslational protein translocation and reveal a hierarchical network of protein export pathways in bacteria (Chapter 2).

The second part of research focused on the more complex protein sorting systems of eukaryotes, where I comprehensively investigated the mitochondrial protein delivery from the cytosol using selective ribosome profiling in human cells. I found that the cotranslational protein targeting to mitochondria is initiated late during translation, directed by an N-terminal presequence and the exposure of a complex globular fold in the nascent protein. This pathway does not favor membrane proteins, but is predominantly used by large, multidomain and topologically complex proteins, whose import efficiency is enhanced when targeted cotranslationally. My results indicate that the cotranslational targeting of mitochondrial proteins is fundamentally different from that of the endoplasmic reticulum (ER) proteins, highlighting the diversity and specificity of protein targeting mechanisms across cellular systems (Chapter 3).