Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
The de Botton Protein Profiling Institute of the Nancy and Stephen Grand Israel National Centre for Personalized Medicine, Weizmann Institute of Science, Rehovot, Israel.
Mol Syst Biol. 2023 Feb 10;19(2):e11084. doi: 10.15252/msb.202211084. Epub 2023 Jan 18.
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.
鉴定稳定和瞬时相互作用对于理解蛋白质功能和调控至关重要。虽然评估稳定相互作用更为直接，但捕捉瞬时相互作用具有挑战性。近年来，涌现出许多复杂的工具来改进瞬时相互作用体的发现，其中许多利用进化的生物素连接酶进行邻近标记。然而，基于生物素化的方法在模式真核生物酿酒酵母中滞后，可能是因为存在几种丰富的、内源性生物素化的蛋白质。在这项研究中，我们优化了酵母中的稳健生物素连接方法，并通过创建一种专门用于下调内源性生物素化的技术来提高其灵敏度，我们将其称为 ABOLISH（Auxin-induced BiOtin LIgase diminiSHing）。我们使用内质网插入酶复合物（EMC）来证明我们的方法并揭示新的底物。为了使这些工具可用于系统探测稳定和瞬时相互作用，我们生成了五组全基因组菌株，其中每个酵母蛋白都被标记为每个经过测试的生物素化机制，其中一些是在 ABOLISH 系统的背景下。这个全面的工具包使整个酵母蛋白质组的功能相互作用组学成为可能。
