Abstract
Ubiquitin-protein ligases (E3s) are responsible for target recognition and subsequent modification of selected substrates within the ubiquitin proteasomal system (UPS). Substrates of this pathway are covalently modified by the attachment of ubiquitin usually onto Lys residues. As a result, these modified proteins can be targeted for degradation, endocytosis, protein sorting, subnuclear trafficking, or other fates. Despite the advancements in understanding the underlying mechanisms of the ubiquitin system, the substrates of most E3 enzymes remain largely unknown. Here, we describe the development of a high-throughput method to identify in vitro substrates for E3 ligases on a global proteomic scale. The enzymatic activity (ubiquitylation) and binding of ubiquitin ligases to their substrates are performed using protein (proteome) microarrays as the experimental platform, and using Nedd4/Rsp5 family members as examples of HECT E3 ligases. The in vitro ubiquitylation and binding substrates identified in these screens can provide invaluable insight into the cellular pathways in which E3 ligases participate.
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Acknowledgments
The work described here was supported by the Canadian Institute of Health Research (CIHR) and the Canadian Cancer Society Research Institute (CCSRI/NCIC) to DR and by the CFI-funded SIDNET. AP is supported by RESTRACOMP and DR by a Canada Research Chair (CRC, Tier II).
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© 2011 Humana Press
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Persaud, A., Rotin, D. (2011). Use of Proteome Arrays to Globally Identify Substrates for E3 Ubiquitin Ligases. In: Castrillo, J., Oliver, S. (eds) Yeast Systems Biology. Methods in Molecular Biology, vol 759. Humana Press. https://doi.org/10.1007/978-1-61779-173-4_13
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DOI: https://doi.org/10.1007/978-1-61779-173-4_13
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