Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER.
Proteins are basically strings of amino acids that have folded into a specific three-dimensional shape, and this shape is often important for the protein's function. Some proteins have bonds between pairs of cysteines—an amino acid that contains sulfur—in different parts of the protein to maintain its correct shape.
In eukaryotes, such as plants and animals, these so-called ‘disulfide bonds’ are formed inside a structure within each cell called the endoplasmic reticulum, which is where many proteins are folded. Occasionally, disulfide bonds form in the wrong place in a protein, so they need to be broken and re-positioned—a process sometimes called editing—for the protein to fold correctly. It was widely assumed that a chemical called ‘reduced glutathione’ fuels the breaking of disulfide bonds in the endoplasmic reticulum, but to date few researchers have tried to test this assumption.
Tsunoda et al. have now taken an enzyme that degrades glutathione elsewhere in the cell and modified it in a way that allows it to work inside the endoplasmic reticulum. When this modified enzyme was produced in human cells grown in the laboratory, it purged the endoplasmic reticulum of glutathione. However, the lack of glutathione had no effect on the folding of a large protein with 30 disulfide bonds, many of which need to be edited at one time or another for the protein to fold correctly. The destruction of a poorly folded protein, via a process that also needs this protein's disulfide bonds to be broken down, was also not affected by a lack of reduced glutathione in the endoplasmic reticulum.
Furthermore, decreasing these levels of glutathione did not affect the unfolded protein response: a stress response in cells that are experiencing a build-up of unfolded or poorly folded proteins within the endoplasmic reticulum.
As such, the findings of Tsunoda et al. challenge the importance of reduced glutathione in the endoplasmic reticulum and suggest that other chemical processes might be involved in editing disulfide bonds. Further work is now needed to investigate the other known processes that might complete this task instead to see which, if any, are involved.