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

Cell Biology

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1 Scopus citations

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

All Science Journal Classification (ASJC) codes

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

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title = "Profiling and verifying the substrates of E3 ubiquitin ligase Rsp5 in yeast cells",

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",

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

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doi = "10.1016/j.xpro.2023.102489",

language = "English (US)",

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T1 - Profiling and verifying the substrates of E3 ubiquitin ligase Rsp5 in yeast cells

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

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