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 quantified 2299 di-Gly-modified lysines, providing an in-depth analysis of the ubiquitylation changes in rapamycin-treated yeast (Fig. 1B and supplemental Table S5). A vast majority (∼93%) of the quantified sites were corrected for differences in protein abundance, and as with phosphorylation, only a small fraction of the observed changes in ubiquitylation could be attributed to changes in protein abundance (supplemental Fig. S3A). SILAC ratio changes were well correlated between experimental replicates (supplemental Fig. S3B). The cutoff for identifying significantly changed ubiquitylation sites was calculated based on the distribution of unmodified peptides (Fig. 4A and supplemental Fig. S3C). 204 and 377 sites were significantly up-regulated, and 69 and 198 sites were significantly down-regulated, after 1 h and 3 h of rapamycin treatment, respectively (supplemental Fig. S3D and supplemental Table S5), indicating that the fraction of up-regulated sites was 2- to 3-fold larger than that of down-regulated sites at both time points. We compared GO term enrichment among proteins that showed up- or down-regulated ubiquitylation at both time points (supplemental Fig. S3E). The most significantly enriched terms associated with up-regulated ubiquitylation were “ribosome” and “posttranscriptional regulation of gene expression,” suggesting a role for ubiquitylation in regulating protein translation or ribophagy. A majority of the down-regulated ubiquitylation sites were present on proteins that were highly significantly associated with the term “intrinsic to membrane,” with smaller fractions of down-regulated ubiquitylation sites occurring on proteins associated with the terms “vacuole,” “ion transport,” and “amino acid transport.” These data indicate globally reduced ubiquitylation of membrane proteins, possibly linked to the known role of ubiquitylation in regulating membrane protein trafficking (56).
Affiliation: From the ‡Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.