Glutathione (L-γ-glutamyl-L-cysteinylglycine) (GSH) is the most abundant low molecular weight thiol in eukaryotic cells. Its ability to serve as a reducing equivalent in several biological reactions offers protection against reactive oxygen species, xenobiotics, and heavy metals. In addition, GSH serves as an iron ligand for some Fe-S binding proteins involved in both iron regulation and the synthesis and maturation of Fe-S proteins. Hence, GSH is important for iron homeostasis. The biosynthesis of GSH occurs via a two-step ATP-dependent enzymatic process. In the first reaction, γ-glutamylcysteine is formed by γ-glutamylcysteine synthetase (Gsh1). In the second step, glycine is added to the peptide via GSH synthetase (Gsh2). Deletion of the γ-glutamylcysteine synthase gene (GSH1) in yeast leads to growth arrest without GSH supplementation.1 In Saccharomyces cerevisiae, extracellular GSH can be imported via a high affinity GSH transporter, Hgt1.2 The physiological role of GSH depletion on cellular function and subcellular redox status has been well characterized. However, it has not been determined how cells depleted of GSH (∆gsh1) respond to increased GSH uptake. To study this, our lab employed a ∆gsh1 yeast strain engineered to overexpress Hgt1. Yeast strains where Hgt1 is overexpressed have been shown to overaccumulate GSH and glutathione disulfide (GSSG) when GSH is added to the media.3 Our data suggests that the overexpression of Hgt1 alone can partially rescue cells devoid of GSH in a redox-dependent manner. To future elucidate this observation, we will express subcellular targeted redox sensors developed in our lab that specifically equilibrates with GSH:GSSG redox changes.