2026-09-24T03:13:38Zhttps://api.repository.cam.ac.uk/server/oai/request

oai:www.repository.cam.ac.uk:1810/3453002025-12-19T21:37:06Zcom_1810_263984com_1810_221767com_1810_256067col_1810_263986

Translational tuning pre-emptively modulates protein folding and secretory pathway defects 10.17863/CAM.92723 Binnian, Imogen Elizabeth, Miller Cells use multiple mechanisms to ensure the accurate synthesis of the proteome, including translational and post-translational regulation. My thesis investigates how translation can be modulated to pre-emptively protect against accumulation of aberrant membrane proteins caused by misfolding or secretory pathway disruption. Yor1, the yeast homolog of mammalian CFTR, encodes an ABC transporter that acts as a drug pump to extrude the mitochondrial toxin, oligomycin. A genome wide screen was previously performed in our lab to identify factors that specifically contribute to biogenesis of a misfolded version of this protein, Yor1-F. The main focus of my thesis work is to investigate translation regulators required for Yor1-F biogenesis, in particular the translation initiation repressor, Eap1. Loss of Eap1 significantly impairs synthesis of Yor1-F, whilst loss of the yeast translation initiation factor eIF4G is beneficial. Synthesis defects in eap1 cells can be rescued by reducing ribosome abundance, or by impairing the RQC pathway. This suggests ribosome collisions as a causative factor for reduced Yor1 biogenesis in the absence of Eap1. I further show that mRNAs encoding polytopic membrane proteins globally show low ribosome abundance, with Yor1 amongst the lowest. I propose that cells have evolved to modulate ribosome abundance on transcripts encoding proteins with challenging folding needs in order to reduce the risk of ribosome collisions. I also explored Eap1 function in the context of defects associated with mutations in Sec24, a COPII coat protein that generates ER-derived transport vesicles. Loss of Eap1 and other translation regulators exacerbates various Sec24 growth phenotypes. I further show that translation at the ER is repressed in cells where COPII vesicle formation is impaired by Sec24 mutation and propose that this response from the cell pre- emptively reduces the protein load in the ER to prevent cellular stress. Finally, I move my work into human cells and investigate the impact of knock down of an Eap1 ortholog, 4E-HP, on biogenesis of CFTR, using a flow cytometry assay. Knock down of 4E- HP does not have a detrimental impact on CFTR synthesis, suggesting that 4E-HP is not directly analogous to Eap1 and that mammalian cells likely have a more nuanced approach to regulating ribosome abundance and preventing collisions during translation of transmembrane proteins. Overall, I show that translation in yeast is modulated to manage protein folding and secretory pathway defects, in order to reduce the burden on the ER and enable cell recovery. University of Cambridge 2022-05-17 Thesis Doctoral Doctor of Philosophy (PhD) eng Medical Research Council https://www.repository.cam.ac.uk/handle/1810/345300 https://www.repository.cam.ac.uk/bitstreams/91404eda-fe34-467c-b9a4-6e00fc4b1290/download 2f51c4452b012075a4a77de3bf19d168 https://www.rioxx.net/licenses/all-rights-reserved/ Translation Protein quality control