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

Author:

Martinusen Samantha G1,Slaton Ethan W1,Nelson Sage E1,Pulgar Marian A1,Besu Julia T2,Simas Cassidy F3,Denard Carl A14ORCID

Affiliation:

1. Department of Chemical Engineering, University of Florida , Gainesville, 32611 , USA

2. Department of Biology, University of Florida , Gainesville, 32611 , USA

3. J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida , Gainesville, 32611 , USA

4. UF Health Cancer Center, University of Florida , Gainesville, 32611 , USA

Abstract

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.

Funder

National Institute of General Medical Sciences

National Institute of Health

Publisher

Oxford University Press (OUP)

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