UCSF

In eukaryotic cells, secreted and membrane proteins fold within the endoplasmic reticulum (ER). Various physiological or pathophysiological conditions can disrupt ER protein folding homeostasis and cause unfolded proteins to accumulate within the ER. Unfolded proteins activate a conserved intracellular signaling pathway called the unfolded protein response (UPR) that increases the ER's protein-folding capacity in order to restore protein folding homeostasis. However, because it is infeasible to directly measure the concentration of unfolded proteins within the ER, any UPR-activated cell is generically described as experiencing "ER stress." To address this problem, I utilized an ER-targeted green fluorescent protein whose fluorescent output is responsive to its oxidation state (called eroGFP). I found that many stressors to ER protein folding homeostasis--both experimental and physiological--compromise oxidation of eroGFP in S. cerevisiae . By combining eroGFP with an additional fluorescent protein reporter to follow changes in UPR activity I was able to determine conditions in which the UPR is capable of promoting adaptation. Additionally, using high-throughput flow cytometry, I measured eroGFP oxidation in approximately 6000 yeast strains each with a deletion or hypomorphic allele of a single gene. Through this analysis, I was able to identify genes important for maintaining oxidative protein folding during normal growth conditions and during protein folding stress. The strategy of utilizing eroGFP as a proximal reporter for ER stress proved to be complimentary to UPR-based metrics to provide a more comprehensive understanding of ER protein folding. The tools and concepts developed here should be broadly applicable to other biological processes and should aid investigations of how ER stress affects human disease.