L. A. Rutkevich et al. Figure 3. Effect of PDI knockdown on oxidative folding of secretory proteins. HepG2 cells were depleted of PDI or treated with control siRNA for 6 d, radiolabeled with [35S]Met for 3 min, then chased with unlabeled Met for the indicated times. The medium was removed, and then cells were treated on ice with 20 mM NEM in PBS and lysed in RIPA buffer containing 20 mM NEM. Both cell lysates and media were immunoisolated with antisera directed against the indicated proteins. (A) Kinetics of disulﬁde formation. Immune complexes from cell lysates were subjected to SDS-PAGE under reducing (ﬁrst lane) or nonreducing conditions and proteins were visualized and quantiﬁed by phosphorimaging. Shown are representative gels of three independent experiments for substrates albumin (Alb), -fetoprotein ( FP), transferrin (TF), and 2-HS-glycoprotein ( 2HS). Reduced (R), partially oxidized (PO), oxidized (O), and Golgi (G, G1) forms of each protein are indicated. (B) Secretion kinetics. Immune complexes recovered from media samples at the indicated chase times were analyzed by SDS-PAGE and visualized and quantiﬁed by phosphorimaging. Histograms indicate the amount of protein observed in the medium as a percentage of the combined intra- and extracellular signal at each chase time. Black and gray bars represent control (C) and knockdown (KD) conditions, respectively. (C) Quantiﬁcation of gels shown in panels A and B was carried out by expressing the amount of fully oxidized protein in lysate and media samples as a percentage of the total combined amount of reduced, partially oxidized, and oxidized forms at a given time point. Solid lines represent control conditions; dashed lines represent knockdown conditions. (D) ER to Golgi transport kinetics. The indicated proteins were immunoisolated and either digested or not with endo H as indicated and analyzed by reducing SDS-PAGE. Endo HS and Endo HR represent endo H–sensitive and -resistant species, respectively. G and ER represent Golgi-processed and ER forms which could be resolved without endo H digestion. (E) Quantiﬁcation of the gels in panel D. Histograms represent the amount of protein remaining in the ER as a percentage of the total protein recovered from cells and medium at each time point. Black bars, control; gray bars, knockdown. Error bars represent the average of three independent experiments one SD, except in the case of TF which was examined in a single experiment. (Figure 3B). The ﬁnal secretory protein tested, 2HS, yielded clearly distinguishable fully reduced, partially oxidized, and fully oxidized forms, but only a slight delay in oxidative folding was observed at the 10- and 20-min chase times in PDI-depleted cells (Figure 3, A and C). By 30 min of chase, 2HS was fully oxidized (species O, G, G1) in both control and PDI-depleted cells. A modest delay in ER to Golgi transport was also observed in PDI-depleted cells at 20 and 30 min of chase (Figure 3, D and E). It is noteworthy that slower ER to Golgi transport was manifested for both TF and 2HS at time 3098 points when they were maximally oxidized, again suggestive of nonnative disulﬁdes in the absence of PDI. Overall, PDI appears to be a versatile and prominent oxidoreductase in that its depletion affected all substrates tested and other PDI family members could not fully com- plement its loss. However, the phenotypes observed were more modest than expected given the essential role of PDI in yeast, raising the question of whether other PDI family members are able to substitute to some degree for the func- tions of PDI. Molecular Biology of the Cell