FIGURE 2.

Fully reduced Pdi1p contributes to the reduction of all regulatory disulfides in Ero1p. A, the redox states of Pdi1p oxidized by 10 mm GSSG (oxidized), reduced by 7 mm GSH and 0.2 mm GSSG (GSH-reduced), or reduced by 20 mm DTT (DTT-reduced) were monitored by mPEG-5k modification and Coomassie staining. B, oxidized Ero1p C100A/C105A-FLAG at 0.5 μm was incubated with 10 μm reduced Pdi1p or Trx1 as indicated. Aliquots were taken at the indicated times, and the redox states of NEM-blocked Ero1p were analyzed under non-reducing conditions by Western blotting using αFLAG. C, the fraction of reduced Ero1p doublet in each lane in B was quantified by densitometry and plotted against time (mean ± S.D., n = 3 independent experiments). D, experiments were carried out as in B except that AMS was used for the alkylation of Ero1p. Ero1p C100A/C105A-FLAG, reduced by excess DTT, precipitated by TCA and blocked with AMS, was loaded as a marker for fully reduced Ero1p. The asterisk (*) indicated Ero1p C100A/C105A with only the Cys¹⁴³-Cys¹⁶⁶ regulatory disulfide reduced (34).