Regulation of Ero1p by Pdi1p. (A) Reoxidation (ox) of Ero1pc-C100A-C105A, which is impaired for autonomous oxidation, can be stimulated by oxidized Pdi1p but not by oxidized Trx1. Ero1pc-C100A-C105A was reduced (red) by incubation with reduced Trx1 followed by Trx1 depletion. Reoxidation of Ero1pc-C100A-C105A was followed after addition of a 20-fold molar excess of either oxidized Trx1 (ox-Trx1), oxidized Pdi1p (ox-PDI), or oxidized Pdi1p variants (a′ mutant–ox-CCSS, a mutant–ox-SSCC, and both the a and a′ mutant–SSSS). (B) Reduced Pdi1p is capable of reducing Ero1pc-C100A-C105A albeit at a slower rate than reduction by reduced Trx1. Ero1pc-C100A-C105A was incubated with a 20-fold excess of reduced Trx1 (red-Trx1), reduced Pdi1p (red-Pdi1p), or reduced Pdi1p variants (the red-Pdi1p panel is presented again in Fig. 4 C). (C) Oxidized Ero1pc-C100A-C105A was mixed with different admixtures of reduced and oxidized Pdi1p for 1 h (the time required for 100% reduced Pdi1p to reduce Ero1pc-C100A-C105A). The relative amounts of reduced, partially reduced, and oxidized Ero1pc-C100A-C105A were quantified by band intensity.