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

Shuai Fang; Geng Chen; Yiyang Wang; Rakhee Ganti; Tatiana A. Chernova; Li Zhou; Savannah E. Jacobs; Duc Duong; Hiroaki Kiyokawa; Yury O. Chernoff; Ming Li; Natalia Shcherbik; Bo Zhao; Jun Yin

STAR Protocols (Sep 2023)

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

Shuai Fang,
Geng Chen,
Yiyang Wang,
Rakhee Ganti,
Tatiana A. Chernova,
Li Zhou,
Savannah E. Jacobs,
Duc Duong,
Hiroaki Kiyokawa,
Yury O. Chernoff,
Ming Li,
Natalia Shcherbik,
Bo Zhao,
Jun Yin

Affiliations

Shuai Fang: Engineering 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
Geng Chen: Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA; Kobilka Institute of Innovative Drug Discovery, School of Life and Health Sciences, The Chinese University of Hong Kong, Shenzhen, Guangdong 518172, China
Yiyang Wang: Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA; Department of Pathophysiology, School of Medicine, Jinan University, Guangzhou, Guangdong 510632, China
Rakhee Ganti: School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USA
Tatiana A. Chernova: Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA
Li Zhou: Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA
Savannah E. Jacobs: Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA
Duc Duong: Integrated Proteomics Core, Emory University, Atlanta, GA 30322, USA
Hiroaki Kiyokawa: Department of Pharmacology, Northwestern University, Chicago, IL 60611, USA
Yury O. Chernoff: School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USA
Ming Li: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48019, USA
Natalia Shcherbik: Department of Cell Biology and Neuroscience, Rowan University School of Osteopathic Medicine, Stratford, NJ 08084, USA; Corresponding author
Bo Zhao: Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China; Corresponding author
Jun Yin: Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA; Corresponding author

Journal volume & issue: Vol. 4, no. 3
p. 102489

Abstract

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Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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