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Convergence of ubiquitylation and phosphorylation signaling in rapamycin-treated yeast cells.

Iesmantavicius V, Weinert BT, Choudhary C - Mol. Cell Proteomics (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.

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Proteome, phosphoproteome, and ubiquitylome analysis of rapamycin-treated yeast.A, experimental outline. Exponentially growing yeast cells were metabolically labeled with lysine0 (light), lysine4 (medium), or lysine8 (heavy). Rapamycin was added to 0.2 mm, and cells were harvested at the indicated time points. Equal amounts of proteins were mixed and digested under denaturing conditions using endoproteinase Lys-C. Phosphorylated peptides were enriched using TiO2-based chromatography, and di-Gly-modified (ubiquitylated) peptides were enriched using anti-di-Gly monoclonal antibody. All peptides were fractionated with micro-SCX prior to analysis using reversed phase liquid chromatography–tandem mass spectrometry (LC-MS/MS). B, overlap between biological replicates for proteome, phosphoproteome, and ubiquitylome. The Venn diagrams indicate the number (n) of sites or proteins identified in each experiment and the overlap between biological replicates.
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Figure 1: Proteome, phosphoproteome, and ubiquitylome analysis of rapamycin-treated yeast.A, experimental outline. Exponentially growing yeast cells were metabolically labeled with lysine0 (light), lysine4 (medium), or lysine8 (heavy). Rapamycin was added to 0.2 mm, and cells were harvested at the indicated time points. Equal amounts of proteins were mixed and digested under denaturing conditions using endoproteinase Lys-C. Phosphorylated peptides were enriched using TiO2-based chromatography, and di-Gly-modified (ubiquitylated) peptides were enriched using anti-di-Gly monoclonal antibody. All peptides were fractionated with micro-SCX prior to analysis using reversed phase liquid chromatography–tandem mass spectrometry (LC-MS/MS). B, overlap between biological replicates for proteome, phosphoproteome, and ubiquitylome. The Venn diagrams indicate the number (n) of sites or proteins identified in each experiment and the overlap between biological replicates.

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).


Convergence of ubiquitylation and phosphorylation signaling in rapamycin-treated yeast cells.

Iesmantavicius V, Weinert BT, Choudhary C - Mol. Cell Proteomics (2014)

Proteome, phosphoproteome, and ubiquitylome analysis of rapamycin-treated yeast.A, experimental outline. Exponentially growing yeast cells were metabolically labeled with lysine0 (light), lysine4 (medium), or lysine8 (heavy). Rapamycin was added to 0.2 mm, and cells were harvested at the indicated time points. Equal amounts of proteins were mixed and digested under denaturing conditions using endoproteinase Lys-C. Phosphorylated peptides were enriched using TiO2-based chromatography, and di-Gly-modified (ubiquitylated) peptides were enriched using anti-di-Gly monoclonal antibody. All peptides were fractionated with micro-SCX prior to analysis using reversed phase liquid chromatography–tandem mass spectrometry (LC-MS/MS). B, overlap between biological replicates for proteome, phosphoproteome, and ubiquitylome. The Venn diagrams indicate the number (n) of sites or proteins identified in each experiment and the overlap between biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125731&req=5

Figure 1: Proteome, phosphoproteome, and ubiquitylome analysis of rapamycin-treated yeast.A, experimental outline. Exponentially growing yeast cells were metabolically labeled with lysine0 (light), lysine4 (medium), or lysine8 (heavy). Rapamycin was added to 0.2 mm, and cells were harvested at the indicated time points. Equal amounts of proteins were mixed and digested under denaturing conditions using endoproteinase Lys-C. Phosphorylated peptides were enriched using TiO2-based chromatography, and di-Gly-modified (ubiquitylated) peptides were enriched using anti-di-Gly monoclonal antibody. All peptides were fractionated with micro-SCX prior to analysis using reversed phase liquid chromatography–tandem mass spectrometry (LC-MS/MS). B, overlap between biological replicates for proteome, phosphoproteome, and ubiquitylome. The Venn diagrams indicate the number (n) of sites or proteins identified in each experiment and the overlap between biological replicates.
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).

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus