Limits...
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.

Show MeSH

Related in: MedlinePlus

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


getmorefigures.php?uid=PMC4125731&req=5

Figure 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.

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)

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

Show All Figures
getmorefigures.php?uid=PMC4125731&req=5

Figure 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.
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