Strategies for Monitoring “Ubiquitin C-Terminal Hydrolase 1” (Yuh1) Activity

Shahaf Saad; Eden Berda; Yuval Klein; Suha Issa; Elah Pick

doi:10.1007/978-1-0716-2859-1_8

Strategies for Monitoring “Ubiquitin C-Terminal Hydrolase 1” (Yuh1) Activity

Shahaf Saad, Eden Berda, Yuval Klein, Suha Issa, Elah Pick

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

Abstract

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.

Original language	English
Title of host publication	Methods in Molecular Biology
Publisher	Humana Press Inc.
Pages	107-122
Number of pages	16
Volume	2602
DOIs	https://doi.org/10.1007/978-1-0716-2859-1_8
State	Published - 2023

Publication series

Name	Methods in molecular biology (Clifton, N.J.)
ISSN (Print)	1064-3745

Bibliographical note

Funding 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).

Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

Amides
Proteolysis
Saccharomyces cerevisiae Proteins
Saccharomyces cerevisiae/genetics
Ubiquitin
Ubiquitin Thiolesterase
Ubiquitins

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10.1007/978-1-0716-2859-1_8

Cite this

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abstract = "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.",

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Strategies for Monitoring “Ubiquitin C-Terminal Hydrolase 1” (Yuh1) Activity

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Keywords

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