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

Gupta R, Kus B, Fladd C, Wasmuth J, Tonikian R, Sidhu S, Krogan NJ, Parkinson J, Rotin D - Mol. Syst. Biol. (2007)

Bottom Line: Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase.Our enzymatic approach was complemented by a parallel protein microarray protein interaction study.Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
Ubiquitin-protein ligases (E3s) are responsible for target recognition and regulate stability, localization or function of their substrates. However, the substrates of most E3 enzymes remain unknown. Here, we describe the development of a novel proteomic in vitro ubiquitination screen using a protein microarray platform that can be utilized for the discovery of substrates for E3 ligases on a global scale. Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase. Our enzymatic approach was complemented by a parallel protein microarray protein interaction study. Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5. The development of a platform for global discovery of E3 substrates is invaluable for understanding the cellular pathways in which they participate, and could be utilized for the identification of drug targets.

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Validation of substrate ubiquitination in vitro and in vivo. (A, B) In vitro ubiquitination: (A) 15 proteins identified as ‘high-confidence' Rsp5 substrates using protein microarrays were expressed (fused to GST) in yeast, purified and incubated in ubiquitination reactions containing Rsp5. (B) Six randomly selected proteins that were not identified as Rsp5 substrates in the protein microarray experiments were used as negative controls. All 15 of the ‘high-confidence' Rsp5 substrates and none of the negative control proteins were visibly ubiquitinated in the Western blots with anti-GST antibodies (arrows indicate the original size of the protein in the absence of ubiquitination (i.e. without ATP)). (C) In vivo ubiquitination: example of three Rsp5 substrates from the protein microarray exhibiting ubiquitination in vivo. The three proteins (HA tagged) were expressed in RSP5 (WT) or rsp5-1 mutant yeast cells. Following a shift to the non-permissive temperature (37°C), proteins were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-ubiquitin antibodies. Note ubiquitination in the RSP5-WT but not the rsp5-1 cells.
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f2: Validation of substrate ubiquitination in vitro and in vivo. (A, B) In vitro ubiquitination: (A) 15 proteins identified as ‘high-confidence' Rsp5 substrates using protein microarrays were expressed (fused to GST) in yeast, purified and incubated in ubiquitination reactions containing Rsp5. (B) Six randomly selected proteins that were not identified as Rsp5 substrates in the protein microarray experiments were used as negative controls. All 15 of the ‘high-confidence' Rsp5 substrates and none of the negative control proteins were visibly ubiquitinated in the Western blots with anti-GST antibodies (arrows indicate the original size of the protein in the absence of ubiquitination (i.e. without ATP)). (C) In vivo ubiquitination: example of three Rsp5 substrates from the protein microarray exhibiting ubiquitination in vivo. The three proteins (HA tagged) were expressed in RSP5 (WT) or rsp5-1 mutant yeast cells. Following a shift to the non-permissive temperature (37°C), proteins were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-ubiquitin antibodies. Note ubiquitination in the RSP5-WT but not the rsp5-1 cells.

Mentions: Fifteen proteins from the Rsp5 high-confidence substrate set, and six proteins that were not identified as substrates of Rsp5, were purified from yeast using glutathione affinity purification, incubated in ubiquitination reactions containing Rsp5 and the above described E1 and E2 (Ubc4), and assayed for ubiquitination using anti-ubiquitin antibodies and Western blots. All of the proteins whose ubiquitination was detected on the protein microarray were verified to be ubiquitinated by Western blot analysis (Figure 2A; Table I). Most of the proteins were efficiently polyubiquitinated or ubiquitinated on multiple lysines. In contrast, the six proteins tested whose ubiquitination was not detected on the protein microarray did not appear to be ubiquitinated after Western blot analysis (Figure 2B), confirming that the enzymatic activity detected is specific and that the data generated by the protein microarray approach are consistent with established methods of detecting ubiquitination.


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

Gupta R, Kus B, Fladd C, Wasmuth J, Tonikian R, Sidhu S, Krogan NJ, Parkinson J, Rotin D - Mol. Syst. Biol. (2007)

Validation of substrate ubiquitination in vitro and in vivo. (A, B) In vitro ubiquitination: (A) 15 proteins identified as ‘high-confidence' Rsp5 substrates using protein microarrays were expressed (fused to GST) in yeast, purified and incubated in ubiquitination reactions containing Rsp5. (B) Six randomly selected proteins that were not identified as Rsp5 substrates in the protein microarray experiments were used as negative controls. All 15 of the ‘high-confidence' Rsp5 substrates and none of the negative control proteins were visibly ubiquitinated in the Western blots with anti-GST antibodies (arrows indicate the original size of the protein in the absence of ubiquitination (i.e. without ATP)). (C) In vivo ubiquitination: example of three Rsp5 substrates from the protein microarray exhibiting ubiquitination in vivo. The three proteins (HA tagged) were expressed in RSP5 (WT) or rsp5-1 mutant yeast cells. Following a shift to the non-permissive temperature (37°C), proteins were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-ubiquitin antibodies. Note ubiquitination in the RSP5-WT but not the rsp5-1 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1911201&req=5

f2: Validation of substrate ubiquitination in vitro and in vivo. (A, B) In vitro ubiquitination: (A) 15 proteins identified as ‘high-confidence' Rsp5 substrates using protein microarrays were expressed (fused to GST) in yeast, purified and incubated in ubiquitination reactions containing Rsp5. (B) Six randomly selected proteins that were not identified as Rsp5 substrates in the protein microarray experiments were used as negative controls. All 15 of the ‘high-confidence' Rsp5 substrates and none of the negative control proteins were visibly ubiquitinated in the Western blots with anti-GST antibodies (arrows indicate the original size of the protein in the absence of ubiquitination (i.e. without ATP)). (C) In vivo ubiquitination: example of three Rsp5 substrates from the protein microarray exhibiting ubiquitination in vivo. The three proteins (HA tagged) were expressed in RSP5 (WT) or rsp5-1 mutant yeast cells. Following a shift to the non-permissive temperature (37°C), proteins were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-ubiquitin antibodies. Note ubiquitination in the RSP5-WT but not the rsp5-1 cells.
Mentions: Fifteen proteins from the Rsp5 high-confidence substrate set, and six proteins that were not identified as substrates of Rsp5, were purified from yeast using glutathione affinity purification, incubated in ubiquitination reactions containing Rsp5 and the above described E1 and E2 (Ubc4), and assayed for ubiquitination using anti-ubiquitin antibodies and Western blots. All of the proteins whose ubiquitination was detected on the protein microarray were verified to be ubiquitinated by Western blot analysis (Figure 2A; Table I). Most of the proteins were efficiently polyubiquitinated or ubiquitinated on multiple lysines. In contrast, the six proteins tested whose ubiquitination was not detected on the protein microarray did not appear to be ubiquitinated after Western blot analysis (Figure 2B), confirming that the enzymatic activity detected is specific and that the data generated by the protein microarray approach are consistent with established methods of detecting ubiquitination.

Bottom Line: Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase.Our enzymatic approach was complemented by a parallel protein microarray protein interaction study.Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
Ubiquitin-protein ligases (E3s) are responsible for target recognition and regulate stability, localization or function of their substrates. However, the substrates of most E3 enzymes remain unknown. Here, we describe the development of a novel proteomic in vitro ubiquitination screen using a protein microarray platform that can be utilized for the discovery of substrates for E3 ligases on a global scale. Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase. Our enzymatic approach was complemented by a parallel protein microarray protein interaction study. Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5. The development of a platform for global discovery of E3 substrates is invaluable for understanding the cellular pathways in which they participate, and could be utilized for the identification of drug targets.

Show MeSH
Related in: MedlinePlus