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

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^*

^*Corresponding author for this work

Pharmacology

Research output: Contribution to journal › Article › peer-review

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

Original language	English (US)
Article number	102489
Journal	STAR Protocols
Volume	4
Issue number	3
DOIs	https://doi.org/10.1016/j.xpro.2023.102489
State	Published - Sep 15 2023

Funding

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

Cell Biology
Cell-based Assays
Molecular Biology
Molecular/Chemical Probes
Protein Biochemistry

ASJC Scopus subject areas

General Neuroscience
General Biochemistry, Genetics and Molecular Biology
General Immunology and Microbiology

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10.1016/j.xpro.2023.102489

Cite this

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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",

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AU - Fang, Shuai

AU - Chen, Geng

AU - Wang, Yiyang

AU - Ganti, Rakhee

AU - Chernova, Tatiana A.

AU - Zhou, Li

AU - Jacobs, Savannah E.

AU - Duong, Duc

AU - Kiyokawa, Hiroaki

AU - Chernoff, Yury O.

AU - Li, Ming

AU - Shcherbik, Natalia

AU - Zhao, Bo

AU - Yin, Jun

PY - 2023/9/15

Y1 - 2023/9/15

N2 - 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

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

Abstract

Funding

Keywords

ASJC Scopus subject areas

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