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

Abstract

The target of rapamycin (TOR) kinase senses the availability of nutrients and coordinates cellular growth and proliferation with nutrient abundance. Inhibition of TOR mimics nutrient starvation and leads to the reorganization of many cellular processes, including autophagy, protein translation, and vesicle trafficking. TOR regulates cellular physiology by modulating phosphorylation and ubiquitylation signaling networks; however, the global scope of such regulation is not fully known. Here, we used a mass-spectrometry-based proteomics approach for the parallel quantification of ubiquitylation, phosphorylation, and proteome changes in rapamycin-treated yeast cells. Our data constitute a detailed proteomic analysis of rapamycin-treated yeast with 3590 proteins, 8961 phosphorylation sites, and 2299 di-Gly modified lysines (putative ubiquitylation sites) quantified. The phosphoproteome was extensively modulated by rapamycin treatment, with more than 900 up-regulated sites one hour after rapamycin treatment. Dynamically regulated phosphoproteins were involved in diverse cellular processes, prominently including transcription, membrane organization, vesicle-mediated transport, and autophagy. Several hundred ubiquitylation sites were increased after rapamycin treatment, and about half as many decreased in abundance. We found that proteome, phosphorylation, and ubiquitylation changes converged on the Rsp5-ubiquitin ligase, Rsp5 adaptor proteins, and Rsp5 targets. Putative Rsp5 targets were biased for increased ubiquitylation, suggesting activation of Rsp5 by rapamycin. Rsp5 adaptor proteins, which recruit target proteins for Rsp5-dependent ubiquitylation, were biased for increased phosphorylation. Furthermore, we found that permeases and transporters, which are often ubiquitylated by Rsp5, were biased for reduced ubiquitylation and reduced protein abundance. The convergence of multiple proteome-level changes on the Rsp5 system indicates a key role of this pathway in the response to rapamycin treatment. 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.

Fig. 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.

Fig. 2.

The rapamycin-regulated proteome. A, identification of significantly regulated proteins. The column chart shows the distribution of SILAC ratios comparing rapamycin-treated cells (1 h) to control cells. A cutoff for significantly up- or down-regulated proteins was determined using two standard deviations from the median of the distribution. Proteins that were significantly up- or down-regulated are marked in red and blue, respectively. B, functional annotation of the rapamycin-regulated proteome. The bar chart shows the fraction of regulated proteins that were associated with GO terms that were significantly overrepresented among the down-regulated (blue) or up-regulated (red) proteins. Significance (p) was calculated with hypergeometric test.

Fig. 3.

Dynamics of the rapamycin-regulated phosphoproteome. A, identification of significantly regulated phosphorylation sites. The histogram shows the distribution of phosphorylation site SILAC ratios for 1h rapamycin/control (1h/ctrl) and the distribution of unmodified peptide SILAC ratios (red). The cutoff for regulated phosphorylation sites was determined based on two standard deviations from the median for unmodified peptides. Unregulated sites are shown in black, and regulated sites are shown in blue. The numbers of down-regulated and up-regulated phosphorylation sites is indicated. B, the bar chart shows the distribution of phosphorylation sites into seven clusters, where cluster zero represents unregulated sites. The clusters were generated through unsupervised clustering of SILAC ratios with the fuzzy c-means algorithm. C, six distinct temporal patterns were generated, and the match between the profile of the cluster and phosphorylation change is described by the membership value. D, the heatmap shows the clustering of GO terms associated with the temporal clusters from C. A more detailed description of the enriched GO terms is provided in supplemental Figs. S2H–S2M. E, sequence motifs for distinct clusters were generated using IceLogo and show the percent difference in amino acid frequency relative to unregulated sites at a p value cutoff of 0.05.

Fig. 4.

The rapamycin-regulated ubiquitylome. A, identification of significantly regulated ubiquitylation sites. The histogram shows the distribution of ubiquitylation site SILAC ratios for 1h rapamycin/control (1h/ctrl) and the distribution of unmodified peptide SILAC ratios (red). The cutoff for regulated ubiquitylation sites was determined based on two standard deviations from the median for unmodified peptides. Unregulated sites are shown in black, and regulated sites are shown in blue. The numbers of down-regulated and up-regulated ubiquitylation sites is indicated. B, the bar chart shows the distribution of ubiquitylation sites into five clusters, where cluster zero represents unregulated sites. The clusters were generated through unsupervised clustering of SILAC ratios with the fuzzy c-means algorithm. C, four distinct temporal patterns were generated, and the match between the profile of the cluster and ubiquitylation change is described by the membership value. D, the heatmap shows the clustering of GO terms associated with the temporal clusters from C. A more detailed description of the enriched GO terms is provided in supplemental Fig. S3F. E, sequence motifs for distinct clusters were generated using IceLogo and show the percent difference in amino acid frequency relative to unregulated sites at a p value cutoff of 0.05.

Fig. 5.

Regulation of the Rsp5 system by rapamycin. Significantly regulated sites after 1 and 3h (see legend) were determined based on a cutoff of two standard deviations from the median for unmodified peptides. All p values were calculated using Fisher's exact test. A, the column graph compares the frequency of regulated ubiquitylation sites occurring on putative Rsp5 target proteins (Rsp5 targets) identified in Ref. to all other proteins (not Rsp5 targets). B, the column graph compares the frequency of regulated class I phosphorylation sites occurring on the Rsp5 adaptor proteins (adaptors) Aly1, Aly2, Art5, Bul1, Bul2, Ecm21, Ldb19, Rod1, and Rog3 to all other proteins (not adaptors). C, the column graph compares the frequency of regulated ubiquitylation sites occurring on permeases and transporters (transporters) to all other proteins (not transporters). D, the column graph compares the frequency of regulated protein abundance between permeases and transporters (transporters) and all other proteins (not transporters).

Fig. 6.

Co-regulation of permeases and transporters by ubiquitylation and phosphorylation. The figure shows permeases, transporters, and adaptors in which ubiquitylation or phosphorylation changed significantly after 3h of rapamycin treatment. Proteins are decorated with circles and squares, which represent the number of quantified phosphorylation and ubiquitylation sites, as well as their regulation in rapamycin-treated cells as indicated in the provided color-code key. Significantly up- or down-regulated sites are indicated in red or blue, respectively. Significantly regulated proteins, phosphorylation sites, and ubiquitylation sites were identified as described in Figs. 2A, 3A, and 4A, respectively.