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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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Related in: MedlinePlus

Rsp5-mediated ubiquitination of the yeast proteome. (A) Assay development. To optimize ubiquitination conditions using protein microarrays, known substrates of Rsp5 (CTD and Ydl203c) and proteins not ubiquitinated by Rsp5 in vitro (Yer036c and GST alone) were robotically printed on slides and incubated in ubiquitination reactions containing Rsp5 and FITC-labeled ubiquitin. The fluorescent signal demonstrates CTD and Ydl203c ubiquitination in the presence of ATP (right panel), while negative control proteins are not ubiquitinated (left panel). Blue color represents ubiquitination (detected with FITC-Ub). GFP was used as a positive control, as it has the same excitation wavelength as FITC. The colors associated with the protein microarray spots indicate of the intensity of the signal (with light blue<bright blue<white). (B) Image of a scanned ubiquitinated protein microarray with an enlargement of one grid. All proteins are printed in duplicate and arrows indicate proteins that were identified as substrates after quantitative data analysis. Alexa dyes are spotted as controls in the left-hand corners of each grid. (C) Reproducibility. Two protein microarrays were ubiquitinated in separate experiments and the same grid from each array is shown. Arrows point to ubiquitinated proteins that were identified as substrates. Most spots producing significantly higher signal than background can be seen on both arrays, suggesting high reproducibility between slides.
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f1: Rsp5-mediated ubiquitination of the yeast proteome. (A) Assay development. To optimize ubiquitination conditions using protein microarrays, known substrates of Rsp5 (CTD and Ydl203c) and proteins not ubiquitinated by Rsp5 in vitro (Yer036c and GST alone) were robotically printed on slides and incubated in ubiquitination reactions containing Rsp5 and FITC-labeled ubiquitin. The fluorescent signal demonstrates CTD and Ydl203c ubiquitination in the presence of ATP (right panel), while negative control proteins are not ubiquitinated (left panel). Blue color represents ubiquitination (detected with FITC-Ub). GFP was used as a positive control, as it has the same excitation wavelength as FITC. The colors associated with the protein microarray spots indicate of the intensity of the signal (with light blue<bright blue<white). (B) Image of a scanned ubiquitinated protein microarray with an enlargement of one grid. All proteins are printed in duplicate and arrows indicate proteins that were identified as substrates after quantitative data analysis. Alexa dyes are spotted as controls in the left-hand corners of each grid. (C) Reproducibility. Two protein microarrays were ubiquitinated in separate experiments and the same grid from each array is shown. Arrows point to ubiquitinated proteins that were identified as substrates. Most spots producing significantly higher signal than background can be seen on both arrays, suggesting high reproducibility between slides.

Mentions: Before assaying for ubiquitinated proteins on the protein microarray, we developed conditions in which Rsp5 could ubiquitinate one of its known substrates, the C-terminal domain of Rpb1 (CTD) (Beaudenon et al, 1999). The ubiquitination of CTD was dependent on the budding yeast E1 enzyme, an E2 enzyme (Ubc4), ubiquitin, Rsp5 and ATP, and was visualized by Western blotting. This control reaction was used to optimize conditions for Rsp5-dependent ubiquitination on nitrocellulose-coated glass slides. In these experiments, the CTD and other proteins were robotically spotted onto slides and incubated with a reaction mixture containing Rsp5 and FITC-labeled ubiquitin. The proteome array was then assayed for Rsp5-dependent ubiquitination using the optimized conditions (Figure 1A).


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)

Rsp5-mediated ubiquitination of the yeast proteome. (A) Assay development. To optimize ubiquitination conditions using protein microarrays, known substrates of Rsp5 (CTD and Ydl203c) and proteins not ubiquitinated by Rsp5 in vitro (Yer036c and GST alone) were robotically printed on slides and incubated in ubiquitination reactions containing Rsp5 and FITC-labeled ubiquitin. The fluorescent signal demonstrates CTD and Ydl203c ubiquitination in the presence of ATP (right panel), while negative control proteins are not ubiquitinated (left panel). Blue color represents ubiquitination (detected with FITC-Ub). GFP was used as a positive control, as it has the same excitation wavelength as FITC. The colors associated with the protein microarray spots indicate of the intensity of the signal (with light blue<bright blue<white). (B) Image of a scanned ubiquitinated protein microarray with an enlargement of one grid. All proteins are printed in duplicate and arrows indicate proteins that were identified as substrates after quantitative data analysis. Alexa dyes are spotted as controls in the left-hand corners of each grid. (C) Reproducibility. Two protein microarrays were ubiquitinated in separate experiments and the same grid from each array is shown. Arrows point to ubiquitinated proteins that were identified as substrates. Most spots producing significantly higher signal than background can be seen on both arrays, suggesting high reproducibility between slides.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Rsp5-mediated ubiquitination of the yeast proteome. (A) Assay development. To optimize ubiquitination conditions using protein microarrays, known substrates of Rsp5 (CTD and Ydl203c) and proteins not ubiquitinated by Rsp5 in vitro (Yer036c and GST alone) were robotically printed on slides and incubated in ubiquitination reactions containing Rsp5 and FITC-labeled ubiquitin. The fluorescent signal demonstrates CTD and Ydl203c ubiquitination in the presence of ATP (right panel), while negative control proteins are not ubiquitinated (left panel). Blue color represents ubiquitination (detected with FITC-Ub). GFP was used as a positive control, as it has the same excitation wavelength as FITC. The colors associated with the protein microarray spots indicate of the intensity of the signal (with light blue<bright blue<white). (B) Image of a scanned ubiquitinated protein microarray with an enlargement of one grid. All proteins are printed in duplicate and arrows indicate proteins that were identified as substrates after quantitative data analysis. Alexa dyes are spotted as controls in the left-hand corners of each grid. (C) Reproducibility. Two protein microarrays were ubiquitinated in separate experiments and the same grid from each array is shown. Arrows point to ubiquitinated proteins that were identified as substrates. Most spots producing significantly higher signal than background can be seen on both arrays, suggesting high reproducibility between slides.
Mentions: Before assaying for ubiquitinated proteins on the protein microarray, we developed conditions in which Rsp5 could ubiquitinate one of its known substrates, the C-terminal domain of Rpb1 (CTD) (Beaudenon et al, 1999). The ubiquitination of CTD was dependent on the budding yeast E1 enzyme, an E2 enzyme (Ubc4), ubiquitin, Rsp5 and ATP, and was visualized by Western blotting. This control reaction was used to optimize conditions for Rsp5-dependent ubiquitination on nitrocellulose-coated glass slides. In these experiments, the CTD and other proteins were robotically spotted onto slides and incubated with a reaction mixture containing Rsp5 and FITC-labeled ubiquitin. The proteome array was then assayed for Rsp5-dependent ubiquitination using the optimized conditions (Figure 1A).

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