Endoplasmic reticulum: reduced and oxidized glutathione revisited

The reducing power of glutathione, expressed by its reduction potential E-GSH, is an accepted measure for redox conditions in a given cell compartment. In the endoplasmic reticulum (ER), E-GSH is less reducing than elsewhere in the cell. However, attempts to determine E-GSH(ER) have been inconsistent and based on ineligible assumptions. Using a codon-optimized and evidently glutathione-specific glutaredoxin-coupled redox-sensitive green fluorescent protein (roGFP) variant, we determined E-GSH(ER) in HeLa cells as -208 +/- 4 mV (at pH 7.0). At variance with existing models, this is not oxidizing enough to maintain the known redox state of protein disulfide isomerase family enzymes. Live-cell microscopy confirmed ER hypo-oxidation upon inhibition of ER Ca2+ import. Conversely, stressing the ER with a glycosylation inhibitor did not lead to more reducing conditions, as reported for yeast. These results, which for the first time establish the oxidative capacity of glutathione in the ER, illustrate a context-dependent interplay between ER stress and E-GSH(ER). The reported development of ER-localized E-GSH sensors will enable more targeted in vivo redox analyses in ER-related disorders.