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

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. Synopsis: This study presents a novel method for enhancing biotin‐specific signal relative to noise and a complete protein interaction toolbox, which harnesses the power of exogenous biotin ligases in discovering stable and transient protein interactors in yeast. TurboID is used in baker's yeast for the first time, uncovering transiently interacting protein substrates of the EMC insertase.ABOLISH is a novel method for reducing endogenous (background) biotinylation and enhancing signal detection from exogenous biotin ligases, such as TurboID.The BirA‐Avi system is implemented for pairwise protein interaction investigation.Five whole‐genome libraries are built, in which each yeast protein is N‐terminally tagged with different biotinylation machineries to enable full proteome interaction exploration. [ABSTRACT FROM AUTHOR]