Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast
Ronish Gupta1,2,a, Bart Kus1,2,a, Christopher Fladd1,2, James Wasmuth3, Raffi Tonikian4,5, Sachdev Sidhu6, Nevan J Krogan7, John Parkinson2,3,5 & Daniela Rotin1,2
- Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada
- Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
- Program in Molecular Structure and Function, The Hospital for Sick Children, Toronto, Ontario, Canada
- Banting & Best Department of Medical Research, University of Toronto, Canada
- Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada
- Department of Protein Engineering, Genentech, South San Francisco, CA, USA
- Department of Cellular and Molecular Pharmacology, University of California-San Francisco, San Francisco, CA, USA
Correspondence to: Daniela Rotin1,2 Program in Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Tel.: +1 416-813-5098; Fax: +1 416-813-8456; Email: drotin@sickkids.ca
Received 14 March 2007; Accepted 24 April 2007; Published online 5 June 2007
aThese authors contributed equally to this work
Top of pageArticle highlights
- A high throughput ubiquitination screen using protein microarrays was performed to identify in one experiment all (or most) of the substrates of the ubiquitin ligase Rsp5 in yeast (S. cerevisae).
- Most of the top hits (substrates) of the screens (~40 proteins) were proteins that contain a PY motif (mainly PPxY), the known motif that binds Rsp5-WW domains.
- The screen identified several known and numerous novel substrates for Rsp5.
- A parallel screen was performed to identify Rsp5 interacting proteins on parallel protein microarrays. The results of the ubiquitination screen were compared to those of the interaction screen, as well as to other recent biochemical and genetic screens, to generate an Rsp5 interaction map using bioinformatic analysis.
Synopsis
Post-translational modification of proteins by the ubiquitin pathway has been implicated in numerous cellular processes. Substrates of this pathway are covalently modified by the attachment of a small protein called ubiquitin and as a result are targeted for degradation, endocytosis, protein sorting or subnuclear trafficking. An enzyme called E3, or ubiquitin ligase, is responsible for the specificity of the reaction, and associates with specific substrates in order to ensure their ubiquitination. Defects in the ability of the E3 to interact with substrates have been implicated in numerous diseases, including neurodegeneration, immunological disorders, hypertension and cancers.
A significant fraction of the proteome is regulated by the ubiquitin pathway and eukaryotic genomes express hundreds of E3 enzymes to coordinate the ubiquitination of cellular proteins. Currently, most E3 enzymes have not been linked to any specific substrates despite advances in understanding the mechanics of the ubiquitin system and its role in the cell. We have developed a platform that allows systematic and high-throughput discovery of ubiquitinated E3 substrates. We expect that this application will be tremendously useful for gaining insights into cellular systems and will likely be exploited by the biomedical industry.
In order to develop this platform, we used Rsp5, a yeast E3, as a model system. E3 enzymes from this family have been implicated in numerous cellular functions including protein degradation, endocytosis, sorting and trafficking. For example, Rsp5 regulates mitochondrial inheritance, drug resistance, intracellular pH, fatty acid biosynthesis, protein sorting at the trans-Golgi network and transcriptional regulation. Nedd4 (or Nedd4-2), the human Rsp5 homologue, prevents hypertension by ubiquitinating and regulating endocytosis of ENaC in the kidney.
Our aim was to identify substrates of Rsp5 in the yeast proteome using the protein microrarray technology as our experimental platform. The arrays used in this study contain purified proteins immobilized at a high spatial density on standard sized slides and contain the majority of yeast proteins (Figure 1). Therefore, the yeast proteome can be readily exploited using traditional biochemical approaches.
Figure 1
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.
Full figure and legend (307K)Figures & Tables indexRecent studies have employed protein microarrays containing full-length proteins to discover calmodulin interacting proteins and to probe for a variety of other protein–protein interactions, as well as antibody–antigen, protein–small molecule, protein–lipid and protein–nucleotide associations. Protein microarrays are expected to provide excellent platforms for identifying post-translational modifications, but to date, only a few studies have assayed an enzymatic activity using this technology.
In the current study, we have successfully used yeast (S. cerevisae) protein microarrays, covering most of the yeast proteome, to assay the enzymatic (ubiquitination) activity and binding ability of Rsp5 to substrate proteins, and we have identified previously reported and novel ubiquitinated substrates and interacting partners of this E3 ligase. The data generated in this study were integrated with published data from large-scale physical and genetic interaction studies to generate Rsp5 interaction networks.
Our results demonstrate the feasibility of identifying substrates of E3 ligases and possibly other enzymes using a proteome microarray approach, and demonstrate how this approach can yield informative data regarding the binding mechanisms and substrate specificity of an E3 ligase enzyme.
Acknowledgements
We thank R Kardish for protein printing on array slides and C Boone for helpful comments on the manuscript. Computational analyses were performed at the Center for Computational Biology, Hospital for Sick Children, Toronto. This work was supported by grants from the Canadian Institute of Health Research (to DR), the Canadian Foundation for Innovation (SIDNET) to DR and the Natural Sciences and Engineering Research Council of Canada to JP. JW and BK were supported by the Hospital for Sick Children (Toronto, Ontario, Canada) Research Training Centre. JP is supported by a New Investigators award from the Canadian Institute of Health Research. DR is a recipient of a CRC chair (Tier I). NJK is Sandler Family Fellow.
