PDI family members such as ERp57, ERp46, and ERp18 form mixed disulﬁdes with Ero1 and hence may accept oxidizing equivalents in a similar manner (Jessop et al., 2009b). Alterna- tively, because more than half of ER-localized glutathione has been shown to be in the form of mixed disulﬁdes with protein (Bass et al., 2004), this may serve as an alternative source of oxidative equivalents in the absence of PDI. Our studies suggest that ERp57 is a leading candidate among PDI family members for activities that partially com- plement PDI deﬁciency. ERp57 had the broadest substrate speciﬁcity of the remaining PDI family members examined in this study, contributing to the efﬁcient oxidative folding of FP, 2HS, and TF but not of Alb. Furthermore, when ERp57 was depleted in combination with PDI, a greater impairment in oxidative folding rates was observed for both TF and 2HS compared with either single knockdown alone and slower export of all substrates from the ER was apparent along with evidence of misfolding and disposal by ERAD. Such extensive effects on protein folding and trafﬁcking were not observed when either ERp72 or P5 was depleted in combination with PDI, although a greater delay in the oxi- dation of Alb was noted with combined PDI and ERp72 knockdown. These ﬁndings indicate overlapping substrate speciﬁcities for PDI and ERp57 as well as illustrating that both enzymes are required for the efﬁcient folding of certain substrates, most notably TF. The generalized impairment of protein export from the ER and disposal by ERAD upon depletion of PDI and ERp57, but not with either enzyme individually, suggests that both enzymes are required for the folding of many other substrates as well. Our ﬁndings indicate that, of the four PDI family members examined, PDI and ERp57 contribute most substantially to oxidative pro- tein folding within the ER. It will be of considerable interest in the future to test the generality of this observation with an expanded set of glycosylated and nonglycosylated sub- strates as well as other PDI family members. Why might an individual substrate require the activities of both PDI and ERp57 during oxidative folding? One pos- sibility is that individual substrate thiols exhibit substantial variation in redox potential that require different PDI family members for thiol-disulﬁde exchange reactions. However, both PDI and ERp57 possess similar reduction potentials that are substantially more oxidizing than those of substrate thiols (reviewed in (Hatahet et al., 2009), rendering such a requirement unlikely. Another possibility is that one en- zyme acts primarily to catalyze disulﬁde formation whereas the other is more involved in isomerization reactions. How- ever, current evidence supports the ability of PDI and ERp57 to catalyze both types of reactions in vivo (Xiao et al., 2004 and see below) and further work would be required to determine whether this varies from one substrate to another. Perhaps a more likely possibility is that PDI and ERp57 recognize distinct substrate conformers during the folding process. This may occur either through direct interaction with hydrophobic patches on the substrate in the case of PDI or indirectly through association with different chaperones. As discussed below, ERp57 is thought to be recruited to folding glycoproteins primarily through its association with Cnx and Crt which recognize nonnative folding intermedi- ates by virtue of a lectin site speciﬁc for monoglucosylated oligosaccharides as well as through a hydrophobic polypep- tide binding site (Williams, 2006). PDI has also been detected in a large complex that contains BiP and Grp94, both of which may inﬂuence PDI-substrate interactions (Meunier et al., 2002). Such distinct modes of substrate recognition may dictate the utilization of PDI or ERp57 at different stages of the substrate folding pathway. Vol. 21, September 15, 2010 Oxidative Folding by PDI Family Members Previous studies using substrate-trapping mutants of ERp57 (Jessop et al., 2007) or that interfered with the forma- tion of ERp57-Cnx/Crt (Jessop et al., 2009a) or Cnx/Crt- glycoprotein complexes (Oliver et al., 1997; Zapun et al., 1998; Solda et al., 2006) have concluded that ERp57 has glycoprotein speciﬁcity mediated through its association with these lectin-chaperones. Our ﬁnding that Alb, the only nonglycosylated substrate examined in this study, was con- spicuously excluded from those substrates affected by the loss of ERp57 is in good agreement with this view. Interest- ingly, the impact of ERp57 depletion varied among the glycoproteins examined. Whereas the effect was mild for FP and 2HS, the impact on TF was substantial. Molinari and coworkers recently examined the impact of ERp57 de- pletion on different glycoproteins and found that the sever- ity of the defect in oxidative folding correlated with the dependency of the individual glycoprotein on the Cnx/Crt chaperone system for folding (Solda et al., 2006). Although the dependence on Cnx and Crt of the various secretory glycoproteins in the present study has not been rigorously examined, TF has been established as an obligate client of these chaperones (Wada et al., 1997), consistent with the strong impairment we observe upon ERp57 depletion. It is noteworthy that delays in oxidative folding were even de- tectable for FP, 2HS, and TF upon ERp57 depletion, given that all three glycoproteins were demonstrated to be sub- strates for PDI and that PDI was fully present when ERp57 was depleted. Recent studies from Jessop and coworkers have shed light on this issue (Jessop et al., 2009a). They specu- lated that entry of glycoproteins into the Cnx/Crt cycle might impede access of other PDI family members, even when ERp57 was depleted. Consistent with this view, they showed that the impaired oxidative folding of inﬂuenza HA observed upon ERp57 depletion could be largely bypassed if HA was pre- vented from associating with these chaperones. There is some controversy as to whether ERp57 functions primarily as an isomerase or oxidase during protein folding. Previous studies by Jessop and coworkers, in which the disulﬁde content of HA was assessed through chemical al- kylation, revealed that the HA produced in ERp57-deﬁcient cells was fully oxidized but contained nonnative disulﬁdes (Jessop et al., 2009a). This led to the conclusion that ERp57 functions primarily as an isomerase. Our current ﬁndings, as well as previous work showing that ERp57 deﬁciency slows the formation of fully oxidized class I heavy chains by as much as 10-fold (Zhang et al., 2006), point to a role for ERp57 in catalyzing disulﬁde formation but do not preclude the possibility of an additional role in isomerization. Future experiments using the chemical alkylation approach on se- cretory proteins in ERp57-depleted HepG2 cells will help to clarify this issue. ERp72 is expected to possess a different substrate speci- ﬁcity compared with PDI or ERp57. The crystal structure of the ERp72 bb domain fragment revealed that it lacks the hydrophobic face that is thought to mediate PDI interactions with nonnative substrates and, on the opposite face, lacks the positively charged patch that mediates ERp57 binding to Cnx and Crt (Kozlov et al., 2009). Consistent with a limited substrate speciﬁcity, a substrate trap mutant of ERp72 failed to stabilize a signiﬁcant level of mixed disulﬁde complexes with substrates (Jessop et al., 2009b). Thus, our inability to detect any impact of ERp72 depletion is perhaps not sur- prising. Only when PDI was depleted in combination with ERp72 was a role for ERp72 revealed in the oxidative folding of Alb, a role previously masked by PDI in the single knock- down experiments. The lack of effect of this double knock- down on the three glycoprotein substrates further under- 3103