Reductive stress and compartmental glutathione homeostasis

Abstract

While reduced glutathione (GSH) is associated with critical functions in the endoplasmic reticulum (ER) including protein folding and prevention of hyperoxidising conditions, GSH also constitutes a reductive burden on certain processes in the cell including those performed by the ER disulfide bond forming machinery whereby GSH may compete with unfolded proteins for oxidising equivalents derived from Ero1p. As part of this study, erodoxin was used to inhibit Ero1p, in an attempt to make cells more susceptible to GSH-associated reductive stress, with the aim of making GSH-associated reductive stress biased towards effects mediated through the ER. This study aimed to improve understanding of how glutathione-associated reductive stress impacts the cell and the ER, and how cells maintain GSH homeostasis. This study also sought to understand how compartmental movement of GSH is regulated, as well as identify genes involved in countering GSH-associated reductive stress. In this study, genome-wide screening revealed 279 mutants that exhibited resistance to erodoxin and GSH treatment and 482 mutants that exhibited sensitivity to erodoxin and GSH treatment. This study identified 188 mutants that exhibited altered tolerance to erodoxin, in a manner dependent on GSH synthesis, and 339 mutants that exhibited altered tolerance to erodoxin, in a manner independent of GSH synthesis. These mutants are hypothesised to be affected in compartmentalisation or degradation of GSH, altered GSH interaction with the ER protein folding machinery, or altered tolerance to erodoxin independent of GSH. This study revealed numerous mutants that exhibited erodoxin resistance dependent on GSH synthesis suggesting that the deleted genes would normally encode proteins that influence GSH synthesis. These included genes/mutants affecting the Rpd3L histone deacetylase complex, and mutants affected in ribosomal function. This study also revealed that endoplasmic reticulum-associated degradation (ERAD) may play an important role in modulating GSH-associated reductive stress. Furthermore, this study identified that Yml082wp may act as a novel regulator of GSH homeostasis. Numerous neurodegenerative diseases including Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), Huntington’s disease, and Parkinson’s disease have been associated with dysfunction of the ER and altered glutathione homeostasis. Data from this study may have broader implications on future research of neurodegenerative diseases.