title: Glutathione limits Ero1-dependent oxidation in the endoplasmic reticulum.
abstract: Many proteins of the secretory pathway contain disulfide bonds that are essential for structure and function. In the endoplasmic reticulum (ER), Ero1 alpha and Ero1 beta oxidize protein disulfide isomerase (PDI), which in turn transfers oxidative equivalents to newly synthesized cargo proteins. However, oxidation must be limited, as some reduced PDI is necessary for disulfide isomerization and ER-associated degradation. Here we show that in semipermeable cells, PDI is more oxidized, disulfide bonds are formed faster, and high molecular mass covalent protein aggregates accumulate in the absence of cytosol. Addition of reduced glutathione (GSH) reduces PDI and restores normal disulfide formation rates. A higher GSH concentration is needed to balance oxidative folding in semipermeable cells overexpressing Ero1 alpha, indicating that cytosolic GSH and lumenal Ero1 alpha play antagonistic roles in controlling the ER redox. Moreover, the overexpression of Ero1 alpha significantly increases the GSH content in HeLa cells. Our data demonstrate tight connections between ER and cytosol to guarantee redox exchange across compartments: a reducing cytosol is important to ensure disulfide isomerization in secretory proteins.
authors.affiliation: Department of Biological and Technological Research, San Raffaele Scientific Institute, 20132 Milan, Italy.
authors.forename: Silvia Nerini
authors.initials: SN
authors.lastname: Molteni
authors.forename: Anna
authors.initials: A
authors.lastname: Fassio
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Ciriolo
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Filomeni
authors.forename: Elena
authors.initials: E
authors.lastname: Pasqualetto
authors.forename: Claudio
authors.initials: C
authors.lastname: Fagioli
authors.forename: Roberto
authors.initials: R
authors.lastname: Sitia
mesh_terms: D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005822:Genetic Vectors; D005978:Glutathione / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D010100:Oxygen / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017510:Protein Folding; D020033:Protein Isoforms; D013696:Temperature; D013997:Time Factors; D014162:Transfection
chemical_list: D004220:Disulfides; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D020033:Protein Isoforms; C405327:ERO1A protein, human; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D004072:Digitonin; D010100:Oxygen