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.
Highlights
IntroductionPhosphorylation and Ubiquitylation Dynamics in target of rapamycin (TOR) Signaling quantification of several posttranslational modifications (PTMs) on a global scale [9, 10]
It should be mentioned that the di-Gly remnant is not absolutely specific for proteins modified by ubiquitin; proteins modified by NEDD8 generate an identical di-Gly remnant, and it is not possible to distinguish between these posttranslational modifications (PTMs) using this approach
We found that Ser100 and 101 were ϳ70% phosphorylated in untreated cells, and phosphorylation was decreased to ϳ45% and ϳ23% after 1 and 3 h of rapamycin treatment, respectively, indicating that phosphorylation at these positions might be important for the activity of Pol12 in initiating DNA replication, which is presumably inhibited in rapamycin-treated cells
Summary
Phosphorylation and Ubiquitylation Dynamics in TOR Signaling quantification of several PTMs on a global scale [9, 10]. Proteome-wide mapping of ubiquitylation sites via mass spectrometry relies on the identification of the di-glycine (di-Gly) remnant that is derived from trypsin digestion of ubiquitylated proteins and remains conjugated to modified lysines [15, 16]. We previously optimized a single-step, immunoaffinity purification method for large-scale analysis of ubiquitylated peptides [17, 18]. This approach has been used successfully to identify thousands of endogenous ubiquitylation sites [17, 18] and to quantify site-specific changes in ubiquitylation in response to different cellular perturbations [19, 20]. A great majority of di-Gly modified sites originate from ubiquitylated peptides [21]
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