Profiling and verifying the substrates of E3 ubiquitin ligase Rsp5 in yeast cells

• Type of Material: Article
• Authors: Shuai Fang, Shanghai Jiao Tong University; Geng Chen, Georgia State University; Yiyang Wang, Georgia State University; Rakhee Ganti, Georgia Institute of Technology; Tatiana Chernova, Emory University; Li Zhou, Georgia State University; Savannah E. Jacobs, Georgia State University; Duc Duong, Emory University; Hiroaki Kiyokawa, Northwestern University; Yury O. Chernoff, Georgia Institute of Technology; Ming Li, University of Michigan Ann Arbor; Natalia Shcherbik, Rowan University; Bo Zhao, Shanghai Jiao Tong University; Jun Yin, Georgia State University
• Language: English
• Date: 2023-09-15
• Publisher: CellPress
• Publication Version: Final Published Version
• Copyright Statement: © 2023 The Author(s)
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1016/j.xpro.2023.102489
• Title of Journal or Parent Work: STAR Protocols
• Volume: 4
• Issue: 3
• Start Page: 102489
• End Page: 102489
• Grant/Funding Information: This work was supported by grants from the National Institutes of Health (NIH) of the United States (R01GM104498 to J.Y. and H.K., R01GM114308 to N.S., and R01GM133873 to M.L.) and the National Science Foundation of the United States (NSF) (1817976 to Y.O.C. and 2109051 to J.Y.). B.Z. was supported by grants from the Natural Science Foundation of China (31770921 and 31971187).
• Keywords: Protein Biochemistry; Cell Biology; Molecular/Chemical Probes; Cell-based Assays; Molecular Biology
• Research Categories: Biology, Cell; Biology, Molecular; Chemistry, Biochemistry

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