A global map of protein–metabolite interactions in budding yeast reveals a novel role of a proteogenic dipeptide Ser-Leu in the regulation of a key glycolytic enzyme Pgk1.

Protein-metabolite interactions (PMIs) are of crucial importance for all cellular processes but remain understudied. Here, we applied a novel biochemical approach named PROMIS, to address the complexity of the protein-small molecule interactome in the model yeast Saccharomyces cerevisiae. By doing so, we provide a unique dataset, which can be queried for novel PMIs between 74 small molecules and 3982 proteins using a user-friendly interface. By interpolating PROMIS with the list of predicted PMIs, we provided experimental validation for 225 binding events. Remarkably, of the 74 small molecules co-eluting with proteins, 36 were proteogenic dipeptides. Targeted analysis of a representative dipeptide, Ser-Leu, revealed numerous protein interactors comprising chaperones, proteasomal subunits, and metabolic enzymes. We could further demonstrate that Ser-Leu binding increases activity of a glycolytic enzyme phosphoglycerate kinase (Pgk1). Consistent with the binding analysis, Ser-Leu supplementation leads to the acute metabolic changes and delays timing of a diauxic shift. Supported by the dipeptide accumulation analysis our work attests to the role of Ser-Leu as a metabolic switch at the interface of protein degradation and central metabolism.