Glutathione is a small thiol-containing tripeptide, which is found in all eukaryotes, and has numerous essential functions. In mammals and yeast, glutathione is synthesized exclusively in the cytosol, yet is found in nearly all subcellular compartments. This implies the existence of glutathione transporters in most intracellular membranes, although the identity of these proteins is largely unknown. In this study, I characterized the protein product of the Saccharomyces cerevisiae open reading frame, YGL114W, hereinafter referred to as Opt3, which is a homolog of the plasma membrane glutathione transporter, Opt1. I observed a strong synthetic negative genetic interaction between OPT3 and either GSH1 or GSH2, which encode the proteins responsible for glutathione biosynthesis. Furthermore, overexpression of OPT3 led to a strong decrease in cellular glutathione disulfide (GSSG) levels, which was independent of Ycf1-mediated vacuolar GSSG storage. I found that a genetic fusion construct between OPT3 and the ultra-bright green fluorescent protein, ymNeonGreen, showed an exclusive endoplasmic reticulum (ER) localization. I observed that the effect of OPT3 deletion and overexpression on cellular GSSG levels was strongly increased when glutathione biosynthesis was re-localized to the ER. Taken together, I propose that Opt3 is an ER-localized GSSG transporter, which functions to export GSSG from the ER to the cytosol, where it can be efficiently reduced. ; Glutathion ist ein kleines Thiol haltiges Tripeptid, das in allen Eukaryonten vorkommt und diverse wichtige Funktionen hat. In Säugern und Hefen wird Glutathion ausschließlich im Zytosol synthetisiert, ist aber in fast allen Zellkompartimenten zu finden. Dies impliziert eine Existenz von Glutathiontransportern in intrazellulären Membranen, obwohl deren Identität weitgehend unbekannt ist. In dieser Studie habe ich das Proteinprodukt des offenen Leserahmens von Saccharomyces cerevisiae, YGL114W, im Folgenden als Opt3 bezeichnet, charakterisiert,welches ein Homolog des ...