The family of ubiquitin C-terminal hydrolases (UCHs(releases ε-linked amide bonds positioned at the C-terminus of ubiquitin. UCHL3 is a highly conserved and dual functional member of this family, recognizing C-terminal extensions of two paralogous modifiers: ubiquitin and NEDD8. The Saccharomyces cerevisiae orthologue of UCHL3, namely, Yuh1, is the only UCH family member in this organism. Like UCHL3, Yuh1 recognizes ubiquitin as well as Rub1, the direct orthologue of NEDD8 in S. cerevisiae. We describe here a method for examining the activity of bacteria and yeast expressed Yuh1 by monitoring the C-terminal trimming of UBB + 1 and Rub1 + 1 through immunoblotting and the increased AMC fluorescence readout detected through a plate reader.
|Title of host publication||Methods in Molecular Biology|
|Publisher||Humana Press Inc.|
|Number of pages||16|
|State||Published - 2023|
|Name||Methods in molecular biology (Clifton, N.J.)|
Bibliographical noteFunding Information:
We thank Michael Glickman for providing us with plasmids. Studies at the Pick lab are supported by the Israel Science Foundation (ISF) grant no. 192/20 and Bet Margolin fellowships for E. B. and S. S. The authors would like to acknowledge the networking support by the ProteoCure COST action (CA20113).
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
- Saccharomyces cerevisiae Proteins
- Saccharomyces cerevisiae/genetics
- Ubiquitin Thiolesterase