title: Two conserved cysteine triads in human Ero1alpha cooperate for efficient disulfide bond formation in the endoplasmic reticulum.
abstract: Human Ero1alpha is an endoplasmic reticulum (ER)-resident protein responsible for protein disulfide isomerase (PDI) oxidation. To clarify the molecular mechanisms underlying its function, we generated a panel of cysteine replacement mutants and analyzed their capability of: 1) complementing a temperature-sensitive yeast Ero1 mutant, 2) favoring oxidative folding in mammalian cells, 3) forming mixed disulfides with PDI and ERp44, and 4) adopting characteristic redox-dependent conformations. Our results reveal that two essential cysteine triads (Cys85-Cys94-Cys99 and Cys391-Cys394-Cys397) cooperate in electron transfer, with Cys94 likely forming mixed disulfides with PDI. Dominant negative phenotypes arise when critical residues within the triads are mutated (Cys394, Cys397, and to a lesser extent Cys99). Replacing the first cysteine in either triad (Cys85 or Cys391) generates mutants with weaker activity. In addition, mutating either Cys85 or Cys391, but not Cys397, reverts the dominant negative phenotype of the C394A mutant. These findings suggest that interactions between the two triads, dependent on Cys85 and Cys391, are important for Ero1alpha function, possibly stabilizing a platform for efficient PDI oxidation.
authors.affiliation: Department of Biology and Technology (DiBiT), San Raffaele Scientific Institute, Milano, Italy.
authors.forename: Gloria
authors.initials: G
authors.lastname: Bertoli
authors.forename: Thomas
authors.initials: T
authors.lastname: Simmen
authors.forename: Tiziana
authors.initials: T
authors.lastname: Anelli
authors.forename: Silvia Nerini
authors.initials: SN
authors.lastname: Molteni
authors.forename: Riccardo
authors.initials: R
authors.lastname: Fesce
authors.forename: Roberto
authors.initials: R
authors.lastname: Sitia
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D003553:Cystine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004583:Electrons; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017353:Gene Deletion; D005799:Genes, Dominant; D005816:Genetic Complementation Test; D006023:Glycoproteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D013997:Time Factors; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D004220:Disulfides; C451899:ERP44 protein, human; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; D003553:Cystine; C405327:ERO1A protein, human; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; C113186:ERO1 protein, S cerevisiae; D019704:Protein Disulfide-Isomerases; D003545:Cysteine; D010100:Oxygen