A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast.

Autor: Fenech EJ; Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel., Cohen N; Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel., Kupervaser M; The de Botton Protein Profiling Institute of the Nancy and Stephen Grand Israel National Centre for Personalized Medicine, Weizmann Institute of Science, Rehovot, Israel., Gazi Z; Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel., Schuldiner M; Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Jazyk: angličtina
Zdroj: Molecular systems biology [Mol Syst Biol] 2023 Feb 10; Vol. 19 (2), pp. e11084. Date of Electronic Publication: 2023 Jan 18.
DOI: 10.15252/msb.202211084
Abstrakt: 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.
(© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)
Databáze: MEDLINE
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