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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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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.
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Figure 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.

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).


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

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

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 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.
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).

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