Functional in vitro analysis of the ERO1 protein and protein-disulfide isomerase pathway |
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Authors: | Araki Kazutaka Nagata Kazuhiro |
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Affiliation: | Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan. |
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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. |
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Keywords: | Disulfide Electron Transfer Endoplasmic Reticulum Oxidase Protein Isomerase |
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