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Updated: Jul 19, 2025
Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
Published on: May 10, 2022
Profiling and verifying the substrates of E3 ubiquitin ligase Rsp5 in yeast cells
Shuai Fang1, Geng Chen2, Yiyang Wang3
1Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China; Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA.
Abstract:
Yeast is an essential model organism for studying protein ubiquitination pathways; however, identifying the direct substrates of E3 in the cell presents a challenge. Here, we present a protocol for using the orthogonal ubiquitin transfer (OUT) cascade to profile the substrate specificity of yeast E3 Rsp5. We describe steps for OUT profiling, proteomics analysis, in vitro and in cell ubiquitination, and stability assay. The protocol can be adapted for identifying and verifying the ubiquitination targets of other E3s in yeast. For complete details on the use and execution of this protocol, please refer to Wang et al.1.
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