Oxidative Folding by PDI Family Members Figure 4. ERp57 depletion delays oxidative folding of glycosylated substrates. HepG2 cells were depleted of ERp57 or treated with control siRNA and pulse-chase experiments were conducted and analyzed as described in Figure 3. (A) Kinetics of disulﬁde formation. (B) Secretion kinetics. Black and gray bars represent control and knockdown conditions, respectively. (C) Quantiﬁcation of oxidative folding. Solid lines, control conditions; dashed lines, knockdown conditions. (D) ER to Golgi transport kinetics. (E) Quantiﬁcation of the gels in panel D. Error bars represent the average of three independent experiments one SD, except in the case of TF which was examined in a single experiment. We next knocked down ERp57 and monitored the same substrates for changes in oxidative folding and intracellular transport. For Alb there was no observed difference in oxi- dative folding in cells with or without ERp57 and no changes in secretion efﬁciency (Figure 4, A–C). In contrast, FP and 2HS, exhibited modest delays in oxidative folding similar to those observed upon depletion of PDI. However, unlike PDI depletion, there was little impact on rates of ER to Golgi transport, suggesting that native disulﬁdes were being formed (Figure 4, D and E). For TF, ERp57 depletion resulted in a marked delay in oxidative folding that was considerably more pronounced than that observed with PDI depletion (Figure 4, A–C). This occurred through 60 min of chase, at which time only 45% of transferrin had reached a fully oxidized state in the absence of ERp57 compared with 85% fully oxidized transferrin in PDI-depleted cells (com- pare Figures 3C and 4C). By 2h, almost all of TF in the cells and medium was fully oxidized (Figure 4, A–C, TF panel). Although secretion of TF was not signiﬁcantly delayed upon ERp57 depletion, ER to Golgi transport was substantially impaired (Figure 4, D and E). This was observed throughout 30 –90 min of chase, reﬂecting the slower formation of disul- Vol. 21, September 15, 2010 ﬁde bonds. These data indicate that TF is a substrate highly speciﬁc to ERp57 and that other family members, such as PDI, are acting on this substrate with reduced efﬁcacy. Even the modest phenotypes observed upon ERp57 depletion for FP and 2HS suggest that PDI, despite being able to act on these substrates, is not able to fully complement the loss of ERp57 for these client proteins. The lack of impact of ERp57 depletion on the folding of Alb stands in contrast to the other substrates. Alb is the only protein in this set that is not glycosylated and hence is less likely to be targeted via Cnx or Crt to ERp57 (Jessop et al., 2009a). This is in agreement with previous work suggesting that the action of ERp57 is largely speciﬁc for glycoproteins (Oliver et al., 1999; Solda et al., 2006; Jessop et al., 2009a). Individual knockdowns of ERp72 and P5 were also car- ried out. However, there was no change in the oxidative folding of any of the substrates tested and no changes were observed in their rates of ER to Golgi transport or secretion (data summarized in Table 1). Because the preceding data indicated that the secretory proteins under study are sub- strates for PDI and/or ERp57, it is possible that any contri- bution of ERp72 or P5 to the oxidative folding of these 3099