Modular and integrative activity reporters enhance biochemical studies in the yeast ER

Abstract The yeast endoplasmic reticulum sequestration and screening (YESS) system is a broadly applicable platform to perform high-throughput biochemical studies of post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substr...

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Bibliographic Details
Published in:	Protein engineering, design and selection Vol. 37
Main Authors:	Martinusen, Samantha G, Slaton, Ethan W, Nelson, Sage E, Pulgar, Marian A, Besu, Julia T, Simas, Cassidy F, Denard, Carl A
Format:	Journal Article
Language:	English
Published:	England Oxford University Press 29-01-2024
Subjects:	Endopeptidases - genetics Endopeptidases - metabolism Endoplasmic Reticulum - genetics Endoplasmic Reticulum - metabolism Genes, Reporter Plasmids - genetics Plasmids - metabolism Protein Processing, Post-Translational Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae - metabolism Protease engineering enzyme activity reporters yeast surface display high-throughput screening and selection yeast endoplasmic reticulum sequestration and screening
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Summary:	Abstract The yeast endoplasmic reticulum sequestration and screening (YESS) system is a broadly applicable platform to perform high-throughput biochemical studies of post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substrate specificity of PTM-enzymes and to discover inhibitor-resistant enzyme mutants. In this study, we expand the capabilities of YESS by transferring its functional components to integrative plasmids. The YESS integrative system yields uniform protein expression and protease activities in various configurations, allows one to integrate activity reporters at two independent loci and to split the system between integrative and centromeric plasmids. We characterize these integrative reporters with two viral proteases, Tobacco etch virus (TEVp) and 3-chymotrypsin like protease (3CLpro), in terms of coefficient of variance, signal-to-noise ratio and fold-activation. Overall, we provide a framework for chromosomal-based studies that is modular, enabling rigorous high-throughput assays of PTM-enzymes in yeast.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1741-0126 1741-0134
DOI:	10.1093/protein/gzae008