Evolutionary studies have suggested that between 35% to 65% of sites are constrained and therefore functional 4, 11. It has been speculated that a significant fraction of phosphosites may have no function 4, consistent with the fast rate of evolutionary change in phosphorylation detected across species 4, 7– 10. This vast extent of phosphorylation in eukaryotes has opened a debate on its physiological relevance 4– 7, fueled by the fact that even for yeast we currently know the functional roles for less than 5% of these sites 2.

Recent estimates suggest that up to 75% of the yeast and human proteomes are phosphorylated, with over 10,000 and 150,000 non-redundant phosphosites described to date in the two organisms, respectively 2, 3. In the past decade, mass spectrometry (MS)-based discovery of protein phosphorylation sites (phosphosites) has drastically changed our understanding of the extent of protein phosphorylation 1. Regulation by phosphorylation allows cells to modulate protein activity, interactions and localization. The relay of information from sensors to effector proteins often occurs via reversible protein post-translational modification (PTM), including protein phosphorylation. This high-throughput approach allows for functionally characterizing individual phosphosites at scale.Ĭells are constantly sensing and adapting to changes in environmental conditions. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. A high fraction exhibited phenotypes not seen in the corresponding gene deletion suggestive of a gain-of-function effect. We inferred their function based on the similarity of their growth profiles with that of gene deletions, and validated a subset by thermal proteome profiling and lipidomics. Of these phosphosites, 42% exhibited growth phenotypes, suggesting that these are more likely functional. We generated 474 yeast strains with mutations in specific phosphosites that were screened for fitness in 102 conditions, along with a gene deletion library.

Here, we devised a chemical genetic approach to study the functional relevance of phosphosites in S. However, less than 5% of thousands of recently discovered phosphosites have been functionally annotated. Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes.

Secondary structure of protein download