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

作者：Shuai Fang, Geng Chen, Yiyang Wang, Rakhee Ganti, Tatiana A. Chernova, Li Zhou, Savannah E. Jacobs, Duc M. Duong, Hiroaki Kiyokawa, Yury O. Chernoff, Ming Li, Natalia Shcherbik, Bo Zhao, Jun Yin · 发表于：STAR Protocols · 年份：2023 · DOI：10.1016/j.xpro.2023.102489 · 被引用次数：2 · 研究领域：Ubiquitin and proteasome pathways、Protein Degradation and Inhibitors、Autophagy in Disease and Therapy

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