Figure 2. Identiﬁcation of PDI substrates. (A) Lysates of HepG2 cells stably expressing either the pQCXIH expression vector (vector) or HA-tagged PDI carrying C56A and C400A mutations (PDICXXA- HA) were immunoblotted with anti-HA antibody. (B) HepG2 cells expressing either vector or PDICXXA-HA were radiolabeled with [35S]Met for 15 min, and treated with 20 mM NEM in PBS. Cell lysates were immunoisolated with anti-HA antibody, immune com- plexes were disrupted and then immunoisolated a second time with the indicated antibodies. Proteins were separated by reducing SDS- PAGE and visualized by ﬂuorography. Alb, albumin; FP, -feto- protein; TF, transferrin. various secretory proteins. As shown in Figure 2B, Alb, FP, and TF could all be recovered as complexes with the PDI trap mutant, conﬁrming them as PDI substrates. Other bands observed in complex with the PDI trap mutant were not identiﬁed but presumably correspond to additional PDI substrates (Figure 2B, lane 2). In addition to Alb, FP, and TF, 2-HS-glycoprotein ( 2HS) was selected for study based on high disulﬁde content and available reagents. As a neg- ative control to detect any pleiotropic effects of PDI family member knockdown, 1-antitrypsin ( 1AT) was selected be- cause it is a glycoprotein that lacks disulﬁde bonds. Individual Knockdown of PDI Family Members To determine the contributions of PDI, ERp57, ERp72, and P5 to the oxidative folding of secretory proteins, pulse-chase experiments were carried out under control and knockdown conditions. Free thiols were alkylated with membrane-per- meable NEM before cell lysis to prevent air oxidation and disulﬁde isomerization. As shown for PDI knockdown in Figure 3A, nonreducing SDS-PAGE was used to monitor the progression of substrate proteins from the fully reduced (R) form of slow mobility to partially oxidized (PO) and fully oxidized forms (O) of higher mobilities. For FP and 2HS, the fully oxidized form was further converted to one or two slower-mobility species (G, G1), reﬂecting processing of their oligosaccharide chains to larger complex structures in the Golgi. A reduction in signal was also apparent at late chase times due to secretion, and fully oxidized species could be recovered from the culture medium (Figure 3B). These were included when calculating percentages of proteins oxidized or Vol. 21, September 15, 2010 Oxidative Folding by PDI Family Members secreted (Figure 3, B and C). In addition, the region near the top of the gel as well as the stacking gel were monitored for disulﬁde-linked aggregates, because previous PDI and ERp57 depletion studies involving overexpressed exogenous sub- strates had reported signiﬁcant quantities of such aggregates (Solda et al., 2006; Lee et al., 2010). However, we did not observe disulﬁde-linked aggregates upon PDI family member deple- tion, either individually or in combination, for any of our endogenous substrates (shown only for Alb in Figures 3– 6). During the oxidative folding of Alb and FP, formation of disulﬁde bonds was very rapid and a substantial shift in gel mobility was seen even in the 3 min pulse period (Figure 3A, control; compare lane P -/ DTT). Only one distinct par- tially oxidized species was observed under control condi- tions that slowly progressed to fully oxidized forms (species O and G) over the course of 30 – 60 min. On depletion of PDI, the impact on oxidative folding was remarkably modest for both proteins. The initial rapid formation of the partially oxidized species during the 3-min pulse period was un- changed, but progression to the fully oxidized species dur- ing the subsequent 10 min of chase was delayed (Figure 3A, PDI lanes and Figure 3C, dashed lines). No other partially oxidized forms were observed. By 20 –30 min of chase, the proportion of fully oxidized Alb and FP (O G species) in PDI-depleted cells became similar to that in control cells, although it was noted that in the PDI-depleted cells the conversion of the FP O species to the Golgi-processed G species was delayed. Because nonnative protein conformers are normally subject to quality control and retained in the ER, we also monitored secretion rates and, in the case of glycoproteins, ER to Golgi transport rates as an indirect measure of folding efﬁciency. This method has been used previously to detect incorrectly disulﬁde-bonded conform- ers in proteins that exhibit an otherwise normal extent of oxidation (Chakravarthi and Bulleid, 2004; Solda et al., 2006). Both Alb and FP appeared in the culture medium at similar rates in the absence or presence of PDI (Figure 3B). ER to Golgi transport of FP was measured by the rate at which its oligosaccharides were processed from an endo H-sensitive ER form to a complex endo H–resistant form in the medial- Golgi (Figure 3D, quantiﬁed in Figure 3E). In the absence of PDI, ER to Golgi transport of FP was notably delayed at the 30 min chase time, consistent with the delay noted above in the conversion of the FP O species to the G species at 30 min in Figure 3A. Because control experiments revealed no delay in the ER to Golgi transport of nondisulﬁde-bonded 1-antitrypsin ( 1AT), the delay observed for FP was spe- ciﬁc to this protein. Given that FP exhibited a similar degree of disulﬁde formation at the 30 min chase point in the absence or presence of PDI (Figure 3C), these data are con- sistent with the presence of nonnative folding intermediates that may require a disulﬁde isomerization event, delayed in the absence of PDI, to be released from the ER quality control machinery. The oxidative folding of TF occurred at a slower pace than Alb or FP, and the fully oxidized form was not detectable until 10 min of chase under control conditions (Figure 3, A and C). Multiple partially oxidized folding intermediates were visible as a smear between the fully reduced and fully oxidized forms. On depletion of PDI, the formation of the various partially oxidized species appeared unaffected but the rate of their subsequent conversion to the fully oxidized species was delayed. However, by 60 min of chase, TF was maximally oxidized in both control and PDI-depleted cells. PDI-depletion also resulted in slower ER to Golgi transport of TF, most apparent by 60 min of chase (Figure 3, D and E), although no difference in its rate of secretion was detected 3097