{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/345300"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/345300","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Translational tuning pre-emptively modulates protein folding and secretory pathway defects","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Binnian, Imogen"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Elizabeth, Miller"],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-05-17","date_published":"2022-05-17","updated_at":"2026-07-24T01:33:25Z","subjects":["Translation","Protein quality control"],"languages":["eng"],"rights":[],"rights_urls":["https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.92723","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Elizabeth, Miller"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Medical Research Council"]},{"key":"dc:creator","label":"Author","values":["Binnian, Imogen"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2022-05-17"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/345300"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Translation","Protein quality control"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.92723"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/91404eda-fe34-467c-b9a4-6e00fc4b1290/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["2f51c4452b012075a4a77de3bf19d168"]},{"key":"dc:title","label":"Title","values":["Translational tuning pre-emptively modulates protein folding and secretory pathway defects"]}]}],"canonical_facts":{"dc:contributor.advisor":["Elizabeth, Miller"],"dc:contributor.sponsor":["Medical Research Council"],"dc:creator":["Binnian, Imogen"],"dc:date.issued":["2022-05-17"],"dc:description.abstract":["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."],"dc:format.checksum.md5":["2f51c4452b012075a4a77de3bf19d168"],"dc:identifier.doi":["10.17863/CAM.92723"],"dc:identifier.uri":["https://www.repository.cam.ac.uk/bitstreams/91404eda-fe34-467c-b9a4-6e00fc4b1290/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/345300"],"dc:rights":["https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["Translation","Protein quality control"],"dc:title":["Translational tuning pre-emptively modulates protein folding and secretory pathway defects"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T01:33:25Z"}