PDI dominantly alters the activity of endogenous Ero1-α and increases conformational flexibility in a shuttle cysteine, Cys99, of Ero1-α. (A and B) HEK293T cells in which the series of oxidoreductases was overexpressed for 24 h (A) or knocked down for 72 h (B) were trapped by alkylation with NEM and solubilized in lysis buffer. The supernatant was subjected to precipitation with Con A–Sepharose, and the glycoprotein fraction was analyzed by immunoblot analysis with the anti–Ero1-α antibody under nonreducing condition. Black lines indicate the removal of intervening lanes for presentation purposes. As controls, cells were transfected with mock vector or two different siRNAs (L, low GC content; M, medium GC content). The activated states of Ero1-α (O_x1/ (O_x1 + O_x2)) were quantified as shown in the bottom graphs of Fig. 2 (A and B). Mutants of full-length PDI containing only the intact a or intact a′ catalytic thioredoxin domain are shown as PDI(a) and PDI(a′), respectively (see also Fig. 2 C). Data represent means ± SDs from three independent experiments (see also Fig. S2 A). Although we have reported that ERdj5 works as a reductase in the ER-associated degradation process, it had almost no significant effect on the redox states of Ero1-α (Ushioda et al., 2008). (C) Schematic representation of human PDI proteins with the CGHC active sites and the mutated AGHA sites indicated. x denotes a linker region between the b′ and the a′ domain. (D) Schematic and simplified model of Ero1-α and its intramolecular electron flow (Araki and Inaba, 2012). Four spheres show the catalytically essential cysteines, two of which form the shuttle disulfide (light gray, Cys94-Cys99) on the flexible loop. The other cysteines form active site disulfides (dark gray, Cys394-Cys397) located proximally to the cofactor (flavin adenine dinucleotide [FAD]). Dashed arrows indicate the electron transfer pathway. The four-helix core is shown as a cylinder. (E) ¹³C NMR spectra of the constitutively active Ero1-α(C104A/C131A) selectively labeled with ¹³C at the carbonyl carbons of cysteine residues (Ero1*). Spectra were measured in the absence (top) and presence of equimolar amounts of WT PDI (top middle), PDI(AA) mutant (bottom middle), or WT PDI together with 4.25 mM somatostatin (bottom). The spectrum of the unlabeled protein has been subtracted. The asterisk indicates the peak originating from Cys99. A.U., arbitrary unit.