Profiling and verifying the substrates of E3 ubiquitin ligase Rsp5 in yeast cells.
Profiling and verifying the substrates of E3 ubiquitin ligase Rsp5 in yeast cells.
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DOI：
10.1016/j.xpro.2023.102489
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发表时间：
2023-09-15
期刊：
影响因子：
--
通讯作者：
Yin, Jun
中科院分区：
文献类型：
--
作者：
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
酵母是研究蛋白质泛素化途径的重要模式生物;然而，确定细胞中E3的直接底物是一个挑战。在这里，我们提出了一个协议，使用正交泛素转移（OUT）级联来分析酵母E3 Rsp 5的底物特异性。我们描述了OUT分析、蛋白质组学分析、体外和细胞内泛素化以及稳定性测定的步骤。该方案可适用于鉴定和验证酵母中其他E3的泛素化靶标。有关本方案使用和执行的完整详细信息，请参见Wang et al.通过酵母中的正交泛素转移（OUT）级联识别E3底物在酵母中表达Rsp 5的OUT级联并制备蛋白质组学样品通过体外和酵母中的泛素化测定来识别Rsp 5的底物测量Rsp 5底物在酵母细胞中的稳定性出版商说明：进行任何实验方案都需要遵守当地机构的实验室安全和伦理指南。酵母是研究蛋白质泛素化途径的重要模式生物;然而，确定细胞中E3的直接底物是一个挑战。在这里，我们提出了一个协议，使用正交泛素转移（OUT）级联来分析酵母E3 Rsp 5的底物特异性。我们描述了OUT分析、蛋白质组学分析、体外和细胞内泛素化以及稳定性测定的步骤。该方案可适用于鉴定和验证酵母中其他E3的泛素化靶标。
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. Identifying E3 substrates by orthogonal ubiquitin transfer (OUT) cascade in yeast Expressing the OUT cascade of Rsp5 in yeast and preparing samples for proteomics Verifying the substrates of Rsp5 by ubiquitination assays in vitro and in yeast Measuring the stability of Rsp5 substrates in yeast cells Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.
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影响因子：
6.6
作者：
Sane S;Hafner A;Srinivasan R;Masood D;Slunecka JL;Noldner CJ;Hanson AD;Kruisselbrink T;Wang X;Wang Y;Yin J;Rezvani K
通讯作者：
Rezvani K
影响因子：
--
作者：
Zhao B;Bhuripanyo K;Zhang K;Kiyokawa H;Schindelin H;Yin J
通讯作者：
Yin J
影响因子：
4.5
作者：
Liu, Jingxuan;Sitaram, Anand;Burd, Christopher G.
通讯作者：
Burd, Christopher G.
影响因子：
64.8
作者：
GHISLAIN, M;UDVARDY, A;MANN, C
通讯作者：
MANN, C
DOI：
10.1007/978-1-4939-7493-1_7
发表时间：
2018-01-01
期刊：
CANCER SYSTEMS BIOLOGY: METHODS AND PROTOCOLS
影响因子：
--
作者：
Tyanova, Stefka;Cox, Juergen
通讯作者：
Cox, Juergen