Table 1. Summary of oxidative folding defects accompanying single and combined PDI family member knockdown MW (kDa) Glycosylation sites (N-linked) PDI ERp57 ERp72 P5 66 66 77 44 52 0 1 2 2 3 ERp57/PDI ERp72/PDI PDI/ERp57/ERp72 ERp57/ERp72 ERp57/P5 PDI/P5 L. A. Rutkevich et al. Substrate Albumin -Fetoprotein Transferrin 2-HS glycoprotein 1-Antitrypsin Substrate Albumin -Fetoprotein Transferrin 2-HS glycoprotein 1-Antitrypsin The degree of delay in formation of the fully oxidized protein substrate and/or its ER to Golgi trafﬁcking rate in response to the indicated individual or combined PDI family member knockdowns is scored from , signifying no effect, to , signifying a profound delay. DISCUSSION Our current understanding of the relationships among mammalian PDI family members comes mainly from the use of substrate trap mutants to examine the spectrum of client proteins acted on by individual family members (Jessop et al., 2007; Jessop et al., 2009b). However, this approach does not detect substrates undergoing oxidative folding nor does it permit an assessment of the functional impact of each family member. Some functional studies on select substrates have been undertaken by depletion of individual family members but comparative assessment of the impact of de- pleting different PDI members on oxidative folding has been very limited (Solda et al., 2006; Kang et al., 2009; Zhang et al., 2009). The present study represents the ﬁrst systematic com- parison of the functions of mammalian PDI, ERp57, ERp72, and P5 in the oxidative folding of multiple endogenous substrates. One of our more surprising ﬁndings is that depletion of PDI in HepG2 cells had a relatively modest impact on the oxidative folding capacity of the ER. For all substrates tested, oxidative folding was delayed rather than arrested, with the half-time for acquisition of the fully oxidized state being extended from less than twofold for the 2HS and TF substrates to approximately eightfold for Alb and FP. A trivial explanation is that low levels of PDI remaining after knockdown can still catalyze substantial disulﬁde forma- tion. However, this possibility is unlikely given that esti- mates of PDI activity required to maintain yeast viability range from 10 to 20% (Hatahet et al., 2009), far higher than the trace levels detectable in our experiments. Rather, the modest phenotypes we observe are consistent with a sub- stantial degree of redundancy among mammalian PDI fam- ily members, although they were not able to fully comple- ment PDI deﬁciency. Interestingly, less redundancy is observed in yeast where PDI is absolutely required for efﬁ- cient oxidative folding of carboxypeptidase Y; none of the remaining family members could support carboxypeptidase Y folding and maturation to even 50% of the efﬁciency observed with PDI (Norgaard et al., 2001). We also noted that PDI depletion resulted in a delay in ER to Golgi trans- port of FP, 2HS, and TF. This occurred after the acquisi- tion of an apparent fully oxidized state, suggesting the pres- ence of nonnative disulﬁdes and hence a role for PDI in catalyzing disulﬁde isomerization. This is consistent with the ﬁnding that the isomerase activity of yeast PDI is re- quired for efﬁcient carboxypeptidase folding in vivo (Xiao et al., 2004). Despite the relative subtlety of the folding defects observed upon loss of PDI, it is noteworthy that PDI silenc- ing affected the oxidation of all substrates examined, irre- spective of their glycosylation status. This observation is in agreement with the generally held view that PDI is the predominant ER thiol oxidoreductase (Hatahet et al., 2009), directly binding to most folding proteins via hydrophobic interactions mediated largely through its b domain (Pirnes- koski et al., 2004; Tian et al., 2006). The modest delay in oxidative folding upon PDI depletion is also surprising given that oxidizing equivalents within the ER are thought to pass from Ero1 through PDI to substrate protein (Sevier and Kaiser, 2008). Although controversial (Mezghrani et al., 2001), there is mounting evidence that other 3102 Figure 7. Effect of combined PDI and ERp72 knockdown on oxidative folding of albumin. HepG2 cells were depleted of PDI and ERp72 by siRNA or treated with control siRNA for 6 d and then subjected to pulse-chase radio- labeling and immunoisolation of Alb as de- scribed for Figure 3. (A) Representative gel of Alb oxidation from three independent knock- down experiments. (B) Quantiﬁcation of gel shown in panel A. Solid lines, control condi- tions; dashed lines, knockdown conditions. Error bars represent the average of three in- dependent experiments one SD. Molecular Biology of the Cell