title: Functional in vitro analysis of the ERO1 protein and protein-disulfide isomerase pathway.
abstract: Oxidative protein folding in the endoplasmic reticulum is supported by efficient electron relays driven by enzymatic reactions centering on the ERO1-protein-disulfide isomerase (PDI) pathway. A controlled in vitro oxygen consumption assay was carried out to analyze the ERO1-PDI reaction. The results showed the pH-dependent oxidation of PDI by ERO1α. Among several possible disulfide bonds regulating ERO1α activity, Cys(94)-Cys(131) and Cys(99)-Cys(104) disulfide bonds are dominant regulators by excluding the involvement of the Cys(85)-Cys(391) disulfide in the regulation. The fine-tuned species specificity of the ERO1-PDI pathway was demonstrated by functional in vitro complementation assays using yeast and mammalian oxidoreductases. Finally, the results provide experimental evidence for the intramolecular electron transfer from the a domain to the a' domain within PDI during its oxidation by ERO1α.
authors.affiliation: Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Araki
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Nagata
mesh_terms: D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D006023:Glycoproteins; D008562:Membrane Glycoproteins; D029701:Saccharomyces cerevisiae Proteins; C405327:ERO1A protein, human; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; C113186:ERO1 protein, S cerevisiae; D019704:Protein Disulfide-Isomerases; D003545:Cysteine