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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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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. 62 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).
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Figure 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. 62 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).

Mentions: In yeast, Rsp5 is the only HECT-domain-containing NEDD4 ubiquitin ligase. Rsp5 is an essential ubiquitin ligase that functions in many diverse processes, such as mRNA export, chromatin remodeling, and the regulation of transcription (60). However, the best-studied role of Rsp5 is in sorting membrane permeases and transporters into the vacuole for proteasome-independent protein degradation (61). Gupta and co-workers used protein microarrays to identify 150 potential in vitro targets of Rsp5 (62). In our dataset we quantified 158 ubiquitylation sites on 54 of these proteins and found that the putative Rsp5 targets identified by Gupta et al. were significantly more likely to harbor up-regulated ubiquitylation sites (Fig. 5A). Rsp5 contains a WW domain that binds to L/PPxY motifs and facilitates the recognition of target proteins (63). However, some proteins that undergo Rsp5-dependent degradation, such as Gap1, Pma1, and Smf1, do not have an L/PPxY recognition motif, and instead their Rsp5-dependent ubiquitylation is facilitated by adaptor proteins that recruit Rsp5 to its target proteins (27). Recently, it was shown that nitrogen permease reactivator 1, a direct target of TORC1, modulates the phosphorylation state of Art1 in a TORC1-dependent manner to modulate the interaction among Rsp5, Art1, and a target protein (26). The phosphorylation state of Rsp5 adaptor proteins often determines whether a protein is targeted for vacuolar degradation. In this study we quantified 58 class I phosphorylation sites (site localization probability > 0.75) and 34 class II phosphorylation sites (site localization probability < 0.75) on 11 Rsp5 adaptor proteins (supplemental Table S11). We found that Rsp5 adaptor proteins were significantly more likely to harbor up-regulated class I phosphorylation sites in rapamycin-treated cells (Fig. 5B). This bias was more pronounced, and more significant, when we included the poorly localized class II sites in our analysis (supplemental Fig. S4). In accordance with the known role of Rsp5 in the regulation of subcellular localization, trafficking, and degradation of transmembrane permeases and transporters, we found that GO terms related to transporters and permeases were enriched among proteins with down-regulated ubiquitylation sites (Fig. 4D, supplemental Figs. S3E and S3F). Consistent with the GO analysis, we found that down-regulated ubiquitylation occurred significantly more frequently on permeases and transporters (Fig. 5C). In addition, we found that permease and transporter protein abundance was significantly more frequently down-regulated, although a portion of these proteins were increased in abundance (Fig. 5D). These data indicate that the proteome, phosphoproteome, and ubiquitylome changes induced by rapamycin treatment converge on Rsp5, Rsp5 adaptor proteins, and Rsp5 targets (Fig. 6).


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

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

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. 62 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 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. 62 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).
Mentions: In yeast, Rsp5 is the only HECT-domain-containing NEDD4 ubiquitin ligase. Rsp5 is an essential ubiquitin ligase that functions in many diverse processes, such as mRNA export, chromatin remodeling, and the regulation of transcription (60). However, the best-studied role of Rsp5 is in sorting membrane permeases and transporters into the vacuole for proteasome-independent protein degradation (61). Gupta and co-workers used protein microarrays to identify 150 potential in vitro targets of Rsp5 (62). In our dataset we quantified 158 ubiquitylation sites on 54 of these proteins and found that the putative Rsp5 targets identified by Gupta et al. were significantly more likely to harbor up-regulated ubiquitylation sites (Fig. 5A). Rsp5 contains a WW domain that binds to L/PPxY motifs and facilitates the recognition of target proteins (63). However, some proteins that undergo Rsp5-dependent degradation, such as Gap1, Pma1, and Smf1, do not have an L/PPxY recognition motif, and instead their Rsp5-dependent ubiquitylation is facilitated by adaptor proteins that recruit Rsp5 to its target proteins (27). Recently, it was shown that nitrogen permease reactivator 1, a direct target of TORC1, modulates the phosphorylation state of Art1 in a TORC1-dependent manner to modulate the interaction among Rsp5, Art1, and a target protein (26). The phosphorylation state of Rsp5 adaptor proteins often determines whether a protein is targeted for vacuolar degradation. In this study we quantified 58 class I phosphorylation sites (site localization probability > 0.75) and 34 class II phosphorylation sites (site localization probability < 0.75) on 11 Rsp5 adaptor proteins (supplemental Table S11). We found that Rsp5 adaptor proteins were significantly more likely to harbor up-regulated class I phosphorylation sites in rapamycin-treated cells (Fig. 5B). This bias was more pronounced, and more significant, when we included the poorly localized class II sites in our analysis (supplemental Fig. S4). In accordance with the known role of Rsp5 in the regulation of subcellular localization, trafficking, and degradation of transmembrane permeases and transporters, we found that GO terms related to transporters and permeases were enriched among proteins with down-regulated ubiquitylation sites (Fig. 4D, supplemental Figs. S3E and S3F). Consistent with the GO analysis, we found that down-regulated ubiquitylation occurred significantly more frequently on permeases and transporters (Fig. 5C). In addition, we found that permease and transporter protein abundance was significantly more frequently down-regulated, although a portion of these proteins were increased in abundance (Fig. 5D). These data indicate that the proteome, phosphoproteome, and ubiquitylome changes induced by rapamycin treatment converge on Rsp5, Rsp5 adaptor proteins, and Rsp5 targets (Fig. 6).

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