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

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...

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Detalles Bibliográficos
Autores principales: Fang, Shuai, Chen, Geng, Wang, Yiyang, Ganti, Rakhee, Chernova, Tatiana A., Zhou, Li, Jacobs, Savannah E., Duong, Duc, Kiyokawa, Hiroaki, Chernoff, Yury O., Li, Ming, Shcherbik, Natalia, Zhao, Bo, Yin, Jun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10440593/
https://www.ncbi.nlm.nih.gov/pubmed/37561636
http://dx.doi.org/10.1016/j.xpro.2023.102489
Descripción
Sumario: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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