1. A high-throughput genetic screening protocol to measure lipid bilayer stress-induced unfolded protein response in Saccharomyces cerevisiae
- Author
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Nurulain Ho, Wei Sheng Yap, Guillaume Thibault, School of Biological Sciences, and Institute of Molecular and Cell Biology, A*STAR
- Subjects
Saccharomyces cerevisiae Proteins ,Science (General) ,Lipid Bilayers ,Saccharomyces cerevisiae ,Gene Expression ,General Biochemistry, Genetics and Molecular Biology ,Cell membrane ,Q1-390 ,Model Organisms ,Gene expression ,Protocol ,medicine ,Genetics ,Flow Cytometry/Mass Cytometry ,Lipid bilayer ,General Immunology and Microbiology ,biology ,Chemistry ,General Neuroscience ,Endoplasmic reticulum ,Cell Membrane ,Biological sciences [Science] ,Endoplasmic Reticulum Stress ,biology.organism_classification ,Synthetic genetic array ,High Throughput Screening ,High-Throughput Screening Assays ,Cell biology ,medicine.anatomical_structure ,Genetic Techniques ,Unfolded Protein Response ,Unfolded protein response ,Cell-based Assays ,Signal transduction ,Signal Transduction - Abstract
Summary The endoplasmic reticulum (ER) stress is defined by the accumulation of unfolded proteins at the ER and perturbation at the ER membrane, known as lipid bilayer stress (LBS). In turn, ER stress triggers the unfolded protein response (UPR) to restore ER homeostasis. Here, we provide a modified protocol based on the synthetic genetic array analysis in Saccharomyces cerevisiae to identify genetic perturbations that induce the UPR by LBS. This method is adaptable to other canonical stress pathways. For complete details on the use and execution of this protocol, please refer to Ho et al. (2020), Jonikas et al. (2009) and Baryshnikova et al. (2010)., Graphical abstract, Highlights • Generation and validation of IRE1 and IRE1ΔLD query strains with a UPR reporter • Detailed protocol of query strains mated to the yeast deletion library using SGA • High-throughput measurement of reporter fluorescence levels by flow cytometry • Data analysis to identify gene deletions activating the UPR by lipid bilayer stress, The endoplasmic reticulum (ER) stress is defined by the accumulation of unfolded proteins at the ER and perturbation at the ER membrane, known as lipid bilayer stress (LBS). In turn, ER stress triggers the unfolded protein response (UPR) to restore ER homeostasis. Here, we provide a modified protocol based on the synthetic genetic array analysis in Saccharomyces cerevisiae to identify genetic perturbations that induce the UPR by LBS. This method is adaptable to other canonical stress pathways.
- Published
- 2021