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

Open Access
Publisher Website
Google Scholar
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. ### 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][1]). Therefore, the yeast proteome can be readily exploited using traditional biochemical approaches. Recent 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. Mol Syst Biol. 3: 116 [1]: #F1