Different Interaction Modes for Protein-disulfide Isomerase (PDI) as an Efficient Regulator and a Specific Substrate of Endoplasmic Reticulum Oxidoreductin-1α (Ero1α)

Lihui Zhang,Yingbo Niu,Li Zhu,Jingqi Fang,Xi'E Wang,Lei Wang,Chih-Chen Wang

doi:10.1074/jbc.m114.602961

Abstract

Protein-disulfide isomerase (PDI) and sulfhydryl oxidase endoplasmic reticulum oxidoreductin-1α (Ero1α) constitute the pivotal pathway for oxidative protein folding in the mammalian endoplasmic reticulum (ER). Ero1α oxidizes PDI to introduce disulfides into substrates, and PDI can feedback-regulate Ero1α activity. Here, we show the regulatory disulfide of Ero1α responds to the redox fluctuation in ER very sensitively, relying on the availability of redox active PDI. The regulation of Ero1α is rapidly facilitated by either a or a' catalytic domain of PDI, independent of the substrate binding domain. On the other hand, activated Ero1α specifically binds to PDI via hydrophobic interactions and preferentially catalyzes the oxidation of domain a'. This asymmetry ensures PDI to function simultaneously as an oxidoreductase and an isomerase. In addition, several PDI family members are also characterized to be potent regulators of Ero1α. The novel modes for PDI as a competent regulator and a specific substrate of Ero1α govern efficient and faithful oxidative protein folding and maintain the ER redox homeostasis.

Highlights

ER oxidoreductin-1 (Ero1)␣ and Protein-disulfide isomerase (PDI) constitute the pivotal oxidative protein folding pathway in mammalian endoplasmic reticulum (ER)
At 10 mM GSH, the majority of Ero1␣ shifted to Ox1 state but no band at the mobility of C85/104/131/391A was observed, indicating that Cys85-Cys391 was still intact after the reduction of Cys94-Cys131
Role of Hydrophobic Interaction in Ero1␣-PDI Interplay—We further explored the mechanisms underlying the intriguing phenomenon that both catalytic domains of PDI can react with the regulatory disulfides of Ero1␣ without discrimination but only the aЈ domain can mediate efficient electron transfer to the outer active site of Ero1␣

Summary

Background

Ero1␣ and PDI constitute the pivotal oxidative protein folding pathway in mammalian ER. To reveal the molecular mechanism underlying the interplay between Ero1␣ and PDIs is central and crucial for understanding how efficient oxidative folding and redox balance in the ER are maintained in mammalian cells. We report that (i) Cys85-Cys391 disulfide in Ero1␣ is stable and remains intact during the physiological activation of the enzyme; (ii) Cys94-Cys131 regulatory disulfide responds to the redox fluctuation in ER very sensitively, and its reduction/oxidation can be facilitated by PDI and some other PDIs; (iii) either catalytic domain of PDI is able to facilitate the regulation of Ero1␣, and the substrate binding domain bЈ of PDI is not essential for activation/inactivation of Ero1␣; (iv) the functional oxidation of PDI catalyzed by Ero1␣ is asymmetric to make the aЈ domain act primarily as an oxidase and the a domain as an isomerase. The above findings shed great light on the mechanism underlying the interplay between Ero1␣ and PDI proteins, which ensures the efficiency and fidelity of oxidative protein folding and maintains the thiol-disulfide redox homeostasis in the ER

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION