Convergence of ubiquitylation and phosphorylation signaling in rapamycin-treated yeast cells.
Bottom Line: We found that proteome, phosphorylation, and ubiquitylation changes converged on the Rsp5-ubiquitin ligase, Rsp5 adaptor proteins, and Rsp5 targets.Furthermore, we found that permeases and transporters, which are often ubiquitylated by Rsp5, were biased for reduced ubiquitylation and reduced protein abundance.Collectively, these data reveal new insights into the global proteome dynamics in response to rapamycin treatment and provide a first detailed view of the co-regulation of phosphorylation- and ubiquitylation-dependent signaling networks by this compound.
Affiliation: From the ‡Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.Show MeSH
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Mentions: In this study we analyzed rapamycin-induced changes in protein, ubiquitylation, and phosphorylation abundance at two time points (1 h and 3 h) in the model organism S. cerevisiae (Fig. 1A). Proteome changes were quantified in an unbiased (non-hypothesis-driven) manner using a SILAC-based proteomic approach (44). Protein extracts from “light” (control, mock treated), “medium” (1 h, 200 nm rapamycin), and “heavy” (3 h, 200 nm rapamycin) SILAC-labeled yeast samples were combined in equal amounts and digested to peptides using Lys-C and trypsin. Di-Gly-modified peptides were enriched using a monoclonal antibody directed toward the di-Gly remnant (16, 17, 21). Phosphorylated peptides were enriched using TiO2-based metal affinity chromatography (32, 33). In order to reduce sample complexity, peptides were fractionated using microtip SCX columns (28, 45). Peptides were analyzed by means of high-pressure nano-flow reversed phase chromatography directly connected to a quadrupole-Orbitrap mass spectrometer (Q Exactive) (34, 35). Computational analysis of MS data was performed using MaxQuant (36, 37), allowing a maximum false discovery rate of 1%. We used stricter criteria for PTM analysis by requiring a minimum posterior error probability score of 0.01 and localization probability of 0.75 for phosphorylated peptides or 0.9 for di-Gly-modified peptides. From three biological replicates, we quantified 3590 proteins, 2299 di-Gly modification sites, and 8961 phosphorylation sites (supplemental Table S1).
Affiliation: From the ‡Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.