Evaluation of activation of HSF and UPR protein folding pathways by chemical stressors using a yeast reporter system for a hypothesis driven laboratory exercise

Abstract

Heat shock factor (HSF) is a transcription factor that is activated in response to an accumulation of misfolded proteins in the cytoplasm. In contrast, the unfolded protein response (UPR) is a transcriptional response pathway that responds to the accumulation of misfolded proteins in the endoplasmic reticulum. Both pathways, when activated by misfolded proteins, result in a transcriptional response that helps the cell to survive by making factors to help the cell sequester and destroy the misfolded proteins. Using aseptic techniques, Saccharomyces cerevisiae with UPR or HSF Beta‐galactosidase reporter plasmids were grown and tested with various doses of 4 different chemical stressors (Dithiothreitol, Diamide, Hydrogen Peroxide, Beta‐Mercaptoethanol) that may induce protein misfolding. Beta‐galactosidase assays were completed to determine the transcriptional response of the HSF and UPR pathways to the chemical stressors. Importantly, this activity emphasizes the mechanism of action of two different cell signaling pathway and demonstrates the use of a marker gene to determine transcriptional activity of a pathway. This laboratory exercises engages the student in critical thinking and data analysis skills as they evaluate data to determine if their chemical stressor activated both pathways or only one pathway.