Mapping the proteomes of endoplasmic reticulum domains by proximity biotinylation
The endoplasmic reticulum (ER) is the largest organelle in the cell and is involved in a wide range of functions such as biogenesis of membrane and secretory proteins, calcium homeostasis and lipid synthesis. To be able to carry out these functions, the ER depends on an elaborate architecture consis...
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| Format: | Thesis |
| Language: | English |
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2022
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| _version_ | 1797110547991232512 |
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| author | Hahn, L |
| author2 | Carvalho, P |
| author_facet | Carvalho, P Hahn, L |
| author_sort | Hahn, L |
| collection | OXFORD |
| description | The endoplasmic reticulum (ER) is the largest organelle in the cell and is involved in a wide range of functions such as biogenesis of membrane and secretory proteins, calcium homeostasis and lipid synthesis. To be able to carry out these functions, the ER depends on an elaborate architecture consisting of a network of flat cisternae-like sheets and narrow tubules. The ER also assembles the nuclear envelope (NE) which is formed by two membranes, the inner and outer nuclear membrane (INM and ONM), that separate the cytoplasm from the nucleoplasm. Despite continuous with each other, these ER domains serve different functions. Consistent with their diverse roles, ER domains also appear to harbour distinct proteins. However, the complete proteomes of the various ER domains have not been systematically analysed and annotated. The primary objective of this project was to map the protein compositions of the distinct ER domains. To this aim, an APEX2-based proximity biotinylation assay coupled to subcellular fractionation was established and a data analysis pipeline developed. The assay was capable to identify many of the well-characterised INM, ER tubule and ER sheet proteins in their anticipated domain. Additionally, a range of bona fide proteins, that have not previously been shown to enrich at any of these domains, were identified. Therefore, these data provide a valuable resource to enhance our understanding of ER organisation. Additionally, the developed assay and data analysis pipeline can serve as a discovery platform to probe how the protein compositions of the ER domains change in response to disease-associated mutations or upon external perturbances. As an example, the effect of LMNA knockout on the protein composition of the inner nuclear membrane was investigated, whereby a small number of proteins were identified to be affected by depletion of A-type lamins. |
| first_indexed | 2024-03-07T07:56:19Z |
| format | Thesis |
| id | oxford-uuid:9afd1a8c-306f-44c2-a6f5-3371faa9d204 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:56:19Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | oxford-uuid:9afd1a8c-306f-44c2-a6f5-3371faa9d2042023-08-21T08:40:41ZMapping the proteomes of endoplasmic reticulum domains by proximity biotinylationThesishttp://purl.org/coar/resource_type/c_db06uuid:9afd1a8c-306f-44c2-a6f5-3371faa9d204ProteomicsNuclear membranesEndoplasmic reticulumEnglishHyrax Deposit2022Hahn, LCarvalho, PThe endoplasmic reticulum (ER) is the largest organelle in the cell and is involved in a wide range of functions such as biogenesis of membrane and secretory proteins, calcium homeostasis and lipid synthesis. To be able to carry out these functions, the ER depends on an elaborate architecture consisting of a network of flat cisternae-like sheets and narrow tubules. The ER also assembles the nuclear envelope (NE) which is formed by two membranes, the inner and outer nuclear membrane (INM and ONM), that separate the cytoplasm from the nucleoplasm. Despite continuous with each other, these ER domains serve different functions. Consistent with their diverse roles, ER domains also appear to harbour distinct proteins. However, the complete proteomes of the various ER domains have not been systematically analysed and annotated. The primary objective of this project was to map the protein compositions of the distinct ER domains. To this aim, an APEX2-based proximity biotinylation assay coupled to subcellular fractionation was established and a data analysis pipeline developed. The assay was capable to identify many of the well-characterised INM, ER tubule and ER sheet proteins in their anticipated domain. Additionally, a range of bona fide proteins, that have not previously been shown to enrich at any of these domains, were identified. Therefore, these data provide a valuable resource to enhance our understanding of ER organisation. Additionally, the developed assay and data analysis pipeline can serve as a discovery platform to probe how the protein compositions of the ER domains change in response to disease-associated mutations or upon external perturbances. As an example, the effect of LMNA knockout on the protein composition of the inner nuclear membrane was investigated, whereby a small number of proteins were identified to be affected by depletion of A-type lamins. |
| spellingShingle | Proteomics Nuclear membranes Endoplasmic reticulum Hahn, L Mapping the proteomes of endoplasmic reticulum domains by proximity biotinylation |
| title | Mapping the proteomes of endoplasmic reticulum domains by proximity biotinylation |
| title_full | Mapping the proteomes of endoplasmic reticulum domains by proximity biotinylation |
| title_fullStr | Mapping the proteomes of endoplasmic reticulum domains by proximity biotinylation |
| title_full_unstemmed | Mapping the proteomes of endoplasmic reticulum domains by proximity biotinylation |
| title_short | Mapping the proteomes of endoplasmic reticulum domains by proximity biotinylation |
| title_sort | mapping the proteomes of endoplasmic reticulum domains by proximity biotinylation |
| topic | Proteomics Nuclear membranes Endoplasmic reticulum |
| work_keys_str_mv | AT hahnl mappingtheproteomesofendoplasmicreticulumdomainsbyproximitybiotinylation |