In-depth and 3-Dimensional Exploration of the Budding Yeast Phosphoproteome

Abstract

Phosphorylation is one of the most dynamic and widespread post-translational modifications regulating virtually every aspect of eukaryotic cell biology. Here we present a comprehensive phosphoproteomic dataset for budding yeast, comprised of over 30,000 high confidence phosphorylation sites identified by mass spectrometry. This single dataset nearly doubles the size of the known phosphoproteome in budding yeast and defines a set of cell cycle-regulated phosphorylation events. With the goal of enhancing the identification of functional phosphorylation events, we performed computational positioning of phosphorylation sites on available 3D protein structures and systematically identified events predicted to regulate protein complex architecture. Results reveal a large number of phosphorylation sites mapping to or near protein interaction interfaces, many of which result in steric or electrostatic "clashes" predicted to disrupt the interaction. Phosphorylation site mutants experimentally validate our predictions and support a role for phosphorylation in negatively regulating protein-protein interactions. With the advancement of Cryo-EM and the increasing number of available structures, our approach should help drive the functional and spatial exploration of the phosphoproteome.

DNA damage kinase signaling: checkpoint and repair at 30 years

“…Looking ahead, it will be crucial to develop more efficient approaches for identifying functional phosphorylation events within the large phosphoproteomic datasets. One possibility is to use structural prediction analysis to identify phosphorylation events that are likely to impair protein function (such as disrupting protein-protein or protein-DNA interactions) (preprint: Lanz et al, 2019). In conclusion, a holistic understanding of the role and action of DNA damage signaling kinases will require powerful technologies capable of quantitatively monitoring the full extent of the phosphoproteome in combination with systematic approaches for functional and mechanistic analyses.…”

Yeast Rgd3 is a phospho-regulated F-BAR-containing RhoGAP involved in the regulation of Rho3 distribution and cell morphology

“…Earlier high-throughput work has shown that Rgd3 contains an SH3-domain binding motif at its C-terminus capable of being recognized by the SH3-domain containing factors Bbc1, Lsb1, Lsb3, and Ysc84, all of which participate in actin organization in endocytosis ( Figure 7D) (Tonikian et al, 2009). Moreover, the serine and threonines flanking the proline residues of this motif have been found to be phosphorylated in a recent yeast whole phospho-proteome analysis (Lanz et al, 2019). We therefore explored the phenotype of cells in which the serine and threonine residues in Rgd3 were mutated to either alanines (AA/AA mutant) to preclude phosphorylation or aspartic and glutamic acids (DE/EE mutant) to mimic phosphorylation.…”

Canonical and novel non-canonical activities of the Holliday junction resolvase Yen1

“…5B and C). In this sense, previous phosphomapping analyses of Yen1 revealed that there are at least 20 S/T residues at non-CDK consensus sites phosphorylated in vivo (67,68,93). Since the Yen1 ON preps we employed were not in vitrodephosphorylated, we wanted to assess if phosphorylation at those non-CDK consensus sites could also be contributing to the regulation of Yen1 activity.…”