A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast
Abstract Identification of both stable and transient interactions is essential for understanding protein function and regulation. While assessing stable interactions is more straightforward, capturing transient ones is challenging. In recent years, sophisticated tools have emerged to improve transie...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Springer Nature
2023-02-01
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| Series: | Molecular Systems Biology |
| Subjects: | |
| Online Access: | https://doi.org/10.15252/msb.202211084 |
| _version_ | 1797283678191091712 |
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| author | Emma J Fenech Nir Cohen Meital Kupervaser Zohar Gazi Maya Schuldiner |
| author_facet | Emma J Fenech Nir Cohen Meital Kupervaser Zohar Gazi Maya Schuldiner |
| author_sort | Emma J Fenech |
| collection | DOAJ |
| description | Abstract Identification of both stable and transient interactions is essential for understanding protein function and regulation. While assessing stable interactions is more straightforward, capturing transient ones is challenging. In recent years, sophisticated tools have emerged to improve transient interactor discovery, with many harnessing the power of evolved biotin ligases for proximity labelling. However, biotinylation‐based methods have lagged behind in the model eukaryote, Saccharomyces cerevisiae, possibly due to the presence of several abundant, endogenously biotinylated proteins. In this study, we optimised robust biotin‐ligation methodologies in yeast and increased their sensitivity by creating a bespoke technique for downregulating endogenous biotinylation, which we term ABOLISH (Auxin‐induced BiOtin LIgase diminiSHing). We used the endoplasmic reticulum insertase complex (EMC) to demonstrate our approaches and uncover new substrates. To make these tools available for systematic probing of both stable and transient interactions, we generated five full‐genome collections of strains in which every yeast protein is tagged with each of the tested biotinylation machineries, some on the background of the ABOLISH system. This comprehensive toolkit enables functional interactomics of the entire yeast proteome. |
| first_indexed | 2024-03-07T17:35:18Z |
| format | Article |
| id | doaj.art-a79bc5831a384b309229ad027be6364b |
| institution | Directory Open Access Journal |
| issn | 1744-4292 |
| language | English |
| last_indexed | 2024-03-07T17:35:18Z |
| publishDate | 2023-02-01 |
| publisher | Springer Nature |
| record_format | Article |
| series | Molecular Systems Biology |
| spelling | doaj.art-a79bc5831a384b309229ad027be6364b2024-03-02T17:05:00ZengSpringer NatureMolecular Systems Biology1744-42922023-02-01192n/an/a10.15252/msb.202211084A toolbox for systematic discovery of stable and transient protein interactors in baker's yeastEmma J Fenech0Nir Cohen1Meital Kupervaser2Zohar Gazi3Maya Schuldiner4Department of Molecular Genetics Weizmann Institute of Science Rehovot IsraelDepartment of Molecular Genetics Weizmann Institute of Science Rehovot IsraelThe de Botton Protein Profiling Institute of the Nancy and Stephen Grand Israel National Centre for Personalized Medicine Weizmann Institute of Science Rehovot IsraelDepartment of Molecular Genetics Weizmann Institute of Science Rehovot IsraelDepartment of Molecular Genetics Weizmann Institute of Science Rehovot IsraelAbstract Identification of both stable and transient interactions is essential for understanding protein function and regulation. While assessing stable interactions is more straightforward, capturing transient ones is challenging. In recent years, sophisticated tools have emerged to improve transient interactor discovery, with many harnessing the power of evolved biotin ligases for proximity labelling. However, biotinylation‐based methods have lagged behind in the model eukaryote, Saccharomyces cerevisiae, possibly due to the presence of several abundant, endogenously biotinylated proteins. In this study, we optimised robust biotin‐ligation methodologies in yeast and increased their sensitivity by creating a bespoke technique for downregulating endogenous biotinylation, which we term ABOLISH (Auxin‐induced BiOtin LIgase diminiSHing). We used the endoplasmic reticulum insertase complex (EMC) to demonstrate our approaches and uncover new substrates. To make these tools available for systematic probing of both stable and transient interactions, we generated five full‐genome collections of strains in which every yeast protein is tagged with each of the tested biotinylation machineries, some on the background of the ABOLISH system. This comprehensive toolkit enables functional interactomics of the entire yeast proteome.https://doi.org/10.15252/msb.202211084interaction profilingsubstrate discoveryTurboIDyeast libraries |
| spellingShingle | Emma J Fenech Nir Cohen Meital Kupervaser Zohar Gazi Maya Schuldiner A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast Molecular Systems Biology interaction profiling substrate discovery TurboID yeast libraries |
| title | A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast |
| title_full | A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast |
| title_fullStr | A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast |
| title_full_unstemmed | A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast |
| title_short | A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast |
| title_sort | toolbox for systematic discovery of stable and transient protein interactors in baker s yeast |
| topic | interaction profiling substrate discovery TurboID yeast libraries |
| url | https://doi.org/10.15252/msb.202211084 |
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