Raguz Nakin, ZrinkaSeisenbacher, GerhardPosas Garriga, FrancescSauer, Uwe2017-03-092017-03-092016Raguz Nakin Z, Seisenbacher G, Posas Garriga F, Sauer U. Untargeted metabolomics unravels functionalities of phosphorylation sites in Saccharomyces cerevisiae. BMC Systems Biology. 2016; 10:104. DOI: 10.1186/s12918-016-0350-81752-0509http://hdl.handle.net/10230/28193Background: Coordinated through a complex network of kinases and phosphatases, protein phosphorylation regulates essentially all cellular processes in eukaryotes. Recent advances in proteomics enable detection of thousands of phosphorylation sites (phosphosites) in single experiments. However, functionality of the vast majority of these sites remains unclear and we lack suitable approaches to evaluate functional relevance at a pace that matches their detection. Results: Here, we assess functionality of 26 phosphosites by introducing phosphodeletion and phosphomimic mutations in 25 metabolic enzymes and regulators from the TOR and HOG signaling pathway in Saccharomyces cerevisiae by phenotypic analysis and untargeted metabolomics. We show that metabolomics largely outperforms growth analysis and recovers 10 out of the 13 previously characterized phosphosites and suggests functionality for several novel sites, including S79 on the TOR regulatory protein Tip41. We analyze metabolic profiles to identify consequences underlying regulatory phosphorylation events and detecting glycerol metabolism to have a so far unknown influence on arginine metabolism via phosphoregulation of the glycerol dehydrogenases. Further, we also find S508 in the MAPKK Pbs2 as a potential link for cross-talking between HOG signaling and the cell wall integrity pathway. Conclusions: We demonstrate that metabolic profiles can be exploited for gaining insight into regulatory consequences and biological roles of phosphosites. Altogether, untargeted metabolomics is a fast, sensitive and informative approach appropriate for future large-scale functional analyses of phosphosites.application/pdfeng© The Author(s). 2016. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1186/s12918-016-0350-8PhosphorylationMetabolismPost-translational modificationTOR signalingOsmotic stressinfo:eu-repo/semantics/openAccess