Identification of Opt3 as a putative ER glutathione disulfide exporter

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 Plasmamembran-Glutathiontransporters Opt1 ist. Ich stellte eine synthetisch negative genetische Interaktion zwischen OPT3 und GSH1 oder GSH2 fest, die für die Proteine der Glutathion-Biosynthese kodieren. Darüber hinaus führte die Überexpression von OPT3 zu einem starken Rückgang des zellulären Glutathion-Disulfid (GSSG)-Gehalts, der unabhängig von der Ycf1-vermittelten vakuolären GSSG-Speicherung war. Ich zeigte, dass ein genetisches Fusionskonstrukt zwischen OPT3 und dem ultrahellen grün fluoreszierenden Protein, ymNeonGreen, eine Lokalisierung im endoplasmatischen Retikulum (ER) aufweist. Außerdem war die Wirkung von OPT3-Deletion und -Überexpression auf den zellulären GSSG-Spiegel stark erhöht, wenn die Glutathion-Biosynthese in das ER verlagert wurde. Insgesamt denke ich, dass Opt3 ein im ER lokalisierter GSSG-Transporter ist, welcher GSSG aus dem ER in das Zytosol exportiert, wo es reduziert werden kann

Glutathione: subcellular distribution and membrane transport

Glutathione (Îł-glutamylcysteinylglycine) is a small tripeptide found at millimolar concentrations in nearly all eukaryotes as well as many prokaryotic cells. Glutathione synthesis is restricted to the cytosol in animals and fungi and to the cytosol and plastids in plants. Nonetheless, glutathione is found in virtually all subcellular compartments. This implies that transporters must exist, which facilitate glutathione transport into and out of the various subcellular compartments. Glutathione may also be exported and imported across the plasma membrane in many cells. However, in most cases, the molecular identity of these transporters remains unclear. Whilst glutathione transport is essential for the supply and replenishment of subcellular glutathione pools, recent evidence supports a more active role for glutathione transport in the regulation of subcellular glutathione redox homeostasis. However, our knowledge of glutathione redox homeostasis at the level of specific subcellular compartments remains remarkably limited and the role of glutathione transport remains largely unclear. In this review we discuss how new tools and techniques have begun to yield insights into subcellular glutathione distribution and glutathione redox homeostasis. In particular, we discuss the known and putative glutathione transporters and examine their contribution to the regulation of subcellular glutathione redox homeostasis.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author