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

doi:10.1016/j.xpro.2023.102489

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

STAR Protoc. 2023 Sep 15;4(3):102489. doi: 10.1016/j.xpro.2023.102489. Epub 2023 Aug 9.

Authors

Shuai Fang¹, Geng Chen², Yiyang Wang³, Rakhee Ganti⁴, Tatiana A Chernova⁵, Li Zhou⁶, Savannah E Jacobs⁶, Duc Duong⁷, Hiroaki Kiyokawa⁸, Yury O Chernoff⁴, Ming Li⁹, Natalia Shcherbik¹⁰, Bo Zhao¹¹, Jun Yin¹²

Affiliations

¹ 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.
² 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.
³ 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.
⁴ School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USA.
⁵ Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
⁶ Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA.
⁷ Integrated Proteomics Core, Emory University, Atlanta, GA 30322, USA.
⁸ Department of Pharmacology, Northwestern University, Chicago, IL 60611, USA.
⁹ Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48019, USA.
¹⁰ Department of Cell Biology and Neuroscience, Rowan University School of Osteopathic Medicine, Stratford, NJ 08084, USA. Electronic address: shcherna@rowan.edu.
¹¹ Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China. Electronic address: bozhao@sjtu.edu.cn.
¹² Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA. Electronic address: junyin@gsu.edu.

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.

Keywords: Cell Biology; Cell-based Assays; Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry.

MeSH terms

Saccharomyces cerevisiae / metabolism
Saccharomyces cerevisiae Proteins* / metabolism
Ubiquitin / metabolism
Ubiquitin-Protein Ligases* / metabolism
Ubiquitination

Substances

Ubiquitin-Protein Ligases
Ubiquitin
Saccharomyces cerevisiae Proteins

Grants and funding

R01 GM114308/GM/NIGMS NIH HHS/United States