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.
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.
STAR Protoc. 2023 Sep 15;4(3):102489. doi: 10.1016/j.xpro.2023.102489. Epub 2023 Aug 9.
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..
酵母是研究蛋白质泛素化途径的重要模式生物；然而，鉴定细胞中 E3 的直接底物是一项挑战。在这里，我们提出了一种使用正交泛素转移 (OUT) 级联来分析酵母 E3 Rsp5 底物特异性的方案。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。该方案可用于鉴定和验证酵母中其他 E3 的泛素化靶标。有关此方案的使用和执行的完整详细信息，请参阅 Wang 等人的文章。
