Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast

Gupta, R. P.; Kus, Bart; Fladd, Christopher; Wasmuth, James D.; Tonikian, Raffi; Sidhu, Sachdev S.; Krogan, Nevan J.; Parkinson, John; Rotin, Daniela

doi:10.1038/msb4100159

Cited by 153 publications

(164 citation statements)

References 70 publications

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“…These data confirm the existence of ubiquitylation sites on Bul1 and Bul2 and suggest that these residues may play a role in Rsp5-dependent regulation of plasma membrane proteins. Notably, two other arrestins, Ecm21/Art2 and Art10, are also ubiquitylated by Rsp5 (54,55), and we detected decreased ubiquitylation of specific residues in tul1⌬ cells (Ecm21/Art2 K95, H/L ϭ 1.5; Art10 K118, H/L ϭ 1.6). However, analysis of ubiquitylation for all 10 Art proteins revealed that Tul1-dependent ubiquitylation of arrestins was limited to Ecm21/Art2 and Art10 (supplemental Table S5).…”

Section: Tul1 E3 Ligase Subunit Genes Show Genetic Interactions With mentioning

confidence: 69%

Identification of Candidate Substrates for the Golgi Tul1 E3 Ligase Using Quantitative diGly Proteomics in Yeast

Tong

Kim

Pandey

et al. 2014

Molecular & Cellular Proteomics

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Maintenance of protein homeostasis is essential for cellular survival. Central to this regulation are mechanisms of protein quality control in which misfolded proteins are recognized and degraded by the ubiquitin-proteasome system. One well-studied protein quality control pathway requires endoplasmic reticulum ( Control of protein homeostasis or proteostasis is key for cell function and survival (1). An important aspect of proteostasis is protein quality control in which misfolded proteins are recognized and degraded by the ubiquitin-proteasome pathway (2). Complex mechanisms regulate whether proteins are targeted for degradation, but ultimately misfolded proteins are recognized and ubiquitylated by specific E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases (3). One well-studied protein quality control pathway is ER-associated degradation (ERAD) 1 (4 -6). ER luminal and membrane proteins are targeted for cytosolic proteasomal degradation by a set of multisubunit E3 ligases integral to the ER membrane, such as Hrd1 and Doa10 in Saccharomyces cerevisiae and Hrd1 and gp78 in mammals. Key open questions in the protein quality control field are (i) what are the physiological substrates of protein quality control pathways and (ii) how do these E3 ligases recognize proteins for degradation.The sterol regulatory element-binding protein (SREBP) family of transcription factors regulates lipid homeostasis in mammals and fungi (7). These ER membrane-bound proteins are proteolytically activated in the Golgi to release the transcription factor domain from the membrane, allowing it to

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Section: Tul1 E3 Ligase Subunit Genes Show Genetic Interactions With mentioning

confidence: 69%

Identification of Candidate Substrates for the Golgi Tul1 E3 Ligase Using Quantitative diGly Proteomics in Yeast

Tong

Kim

Pandey

et al. 2014

Molecular & Cellular Proteomics

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“…3). Unlike known Nedd4-1 substrates, including LAPTM5, which typically contain PY motifs (27)(28)(29)(30)32), PTEN does not possess this motif, possibly explaining the observed lack of its interaction with Nedd4-1 ( Fig. 3 C and D) and unaffected PTEN ubiquitination in Nedd4-1-deficient cells (Fig.…”

Section: Discussionmentioning

confidence: 96%

“…The WW domain of Nedd4 proteins recognizes and binds a short amino acid sequence in substrate proteins, called the PY motif (L/PPxY) (26)(27)(28)(29)(30). By using two independent approaches, we recently generated distinct strains of mice with disrupted Nedd4-1 gene, leading to a complete loss of Nedd4-1 protein and embryonic lethality at mid to late gestation (F.F.…”

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confidence: 99%

The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability and localization

Fouladkou

Landry²,

Kawabe

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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PTEN is a tumor suppressor frequently mutated in cancer. Recent reports implicated Nedd4-1 as the E3 ubiquitin ligase for PTEN that regulates its stability and nuclear localization. We tested the physiological role of Nedd4-1 as a PTEN regulator by using cells and tissues derived from two independently generated strains of mice with their Nedd4-1 gene disrupted. PTEN stability and ubiquitination were indistinguishable between the wild-type and Nedd4-1-deficient cells, and an interaction between the two proteins could not be detected. Moreover, PTEN subcellular distribution, showing prominent cytoplasmic and nuclear staining, was independent of Nedd4-1 presence. Finally, activation of PKB/Akt, a major downstream target of cytoplasmic PTEN activity, and the ability of PTEN to transactivate the Rad51 promoter, a measure of its nuclear function, were unaffected by the loss of Nedd4-1. Taken together, our results fail to support a role for Nedd4-1 as the E3 ligase regulating PTEN stability and subcellular localization.E3 ligase ͉ tumor suppressor ͉ P13K signal ͉ WW domain P TEN is one of the most frequently mutated genes in human cancer (1-3). In the cytoplasm, PTEN functions as a lipid phosphatase by dephosphorylating the D3 position of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ], and directly antagonizing PI3K (4-6). Consistent with a negative regulatory role for PTEN in regulation of PI3 signaling, PTEN-deficient cells and tissues exhibit defects in cell proliferation, growth, survival, death, protein translation, metabolism, migration, and structural organization (2,3,7,8).One of the most intriguing features of PTEN is its subcellular localization. Although PTEN was originally found to be a cytoplasmic protein (9), its nuclear localization in many cell types has been reported by using various independently developed monoclonal and polyclonal anti-PTEN antibodies (10)(11)(12)(13)(14). A number of attempts have been made to uncouple the cytoplasmic and nuclear roles of PTEN, yielding incongruous results. For example, differing accounts of the respective levels of cytoplasmic and nuclear PTEN throughout the stages of the cell cycle have been documented (15, 16). Functionally, nuclear PTEN has been shown to induce cell cycle arrest in certain cell types (17), whereas in others, nuclear accumulation of PTEN increased upon stimulation with proapoptotic factors and correlated with induction of apoptosis (18). Finally, nuclear-targeted PTEN was shown to impede the growth of U251MG glioblastoma cells and down-regulate p70S6K in a PKB/Akt-independent manner but was unable to inhibit cell invasion (19).Recently, published work revealed a phosphatase activityindependent nuclear role for PTEN in the regulation of chromosomal stability and repair of DNA damage (20). Wang et al. (21) and Trotman et al. (22) reported that PTEN stability and nuclear translocation were regulated by ubiquitination mediated by the ubiquitin ligase Nedd4-1. Although the Nedd4-1-mediated polyubiquitination of PTEN in the cytosol caused ...

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“…To further characterize the structural determinants necessary for Gpa1 mono-ubiquitination, we examined the protein for potential Rsp5 docking sites. Rsp5 contains WW protein interaction domains that bind and recruit substrates or substrate adaptor proteins containing one or more short PY motifs (PPXY) within their primary structure (47,48). However, the PXY portion of the motif is the minimal FIGURE 5.…”

Section: Gpa1 Protein Folding Mutants Are Targeted For Poly-ubiquitinmentioning

confidence: 99%

“…requirement for establishing an interaction with Rsp5, whereas nonpolar hydrophobic residues can be tolerated at the first position of the motif (e.g. LPXY or APXY) (47,48). Gpa1 contains a single non-canonical PY motif (FPDY) that contains a nonpolar hydrophobic residue in position 1.…”

Section: Table 1 Relative Free Energies Of Gpa1 Protein Fold-destabilmentioning

confidence: 99%

G Protein Mono-ubiquitination by the Rsp5 Ubiquitin Ligase

Torres¹,

Lee²,

Ding

et al. 2009

Journal of Biological Chemistry

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Emerging evidence suggests that ubiquitination serves as a protein trafficking signal in addition to its well characterized role in promoting protein degradation. The yeast G protein ␣ subunit Gpa1 represents a rare example of a protein that undergoes both mono-and poly-ubiquitination. Whereas mono-ubiquitinated Gpa1 is targeted to the vacuole, poly-ubiquitinated Gpa1 is directed instead to the proteasome. Here we investigate the structural requirements for mono-and poly-ubiquitination of Gpa1. We find that variants of Gpa1 engineered to be unstable are more likely to be poly-ubiquitinated and less likely to be mono-ubiquitinated. In addition, mutants that cannot be myristoylated are no longer mono-ubiquitinated but are still polyubiquitinated. Finally, we show that the ubiquitin ligase Rsp5 is necessary for Gpa1 mono-ubiquitination in vivo and that the purified enzyme is sufficient to catalyze Gpa1 mono-ubiquitination in vitro. Taken together, these data indicate that mono-and poly-ubiquitination have distinct enzyme and substrate recognition requirements; whereas poly-ubiquitination targets misfolded protein for degradation, a distinct ubiquitination apparatus targets the fully mature, fully myristoylated G protein for mono-ubiquitination and delivery to the vacuole.

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Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast

Cited by 153 publications

References 70 publications

Identification of Candidate Substrates for the Golgi Tul1 E3 Ligase Using Quantitative diGly Proteomics in Yeast

Identification of Candidate Substrates for the Golgi Tul1 E3 Ligase Using Quantitative diGly Proteomics in Yeast

The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability and localization

G Protein Mono-ubiquitination by the Rsp5 Ubiquitin Ligase

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