Figure 6. Misfolded transferrin is degraded by the proteasome in PDI/ERp57-depleted cells. (A) Pulse-chase data from Figures 3–5 was analyzed for changes in the total amount of radiolabeled Alb, FP, TF, 2HS, and 1AT recovered from the combined intracellular and medium fractions. Total protein, as measured by densitometry, was deﬁned as 100% at the 10 min chase time. Error bars represent the average of three independent experiments one SD. (B, C) HepG2 cells were treated with control siRNA or depleted of PDI and ERp57 for 6 d and then incubated in the absence or presence of 5 g/ml lactacystin for 5 h. Cells were either plated on Cell-Tak- treated coverslips (BD Biosciences), ﬁxed in paraformaldehyde, and permeabilized for confocal microscopy (B) or lysed in RIPA buffer for Western blot analysis (C). Immunoblot or immunoﬂuorescence was carried out using the indicated antibodies (Scale bar 10 m). transport were strongly impaired for all proteins tested (Fig- ure 5, B, D, and E). On comparison to the 1AT control, it was further observed that even a secretory protein lacking disulﬁde bonds was delayed both in its trafﬁcking from ER to Golgi and its secretion. This suggests that combined de- pletion of ERp57 and PDI leads to a generalized impairment of ER function. The observed degradation of substrate pro- teins (Figure 6A) was investigated further in the case of TF by treating control and PDI/ERp57-depleted cells with the proteasome inhibitor lactacystin and examining both the subcellular distribution of TF by immunoﬂuorescence mi- croscopy and its steady state levels by immunoblotting. As shown in Figure 6B, ER morphology as assessed by Cnx staining was not grossly altered upon combined PDI and ERp57 depletion either alone or after treatment with lacta- cystin. TF exhibited a reticular ER staining pattern in both Vol. 21, September 15, 2010 Oxidative Folding by PDI Family Members control and PDI/ERp57-depleted cells. However, staining was more intense in the depleted cells consistent with ER accumulation and the intensity increased substantially upon inhibition of proteasomal degradation. This was conﬁrmed by immunoblotting wherein cellular levels of TF increased 2.2-fold upon combined PDI and ERp57 depletion relative to control cells and threefold following lactacystin treatment (Figure 6C). These data are consistent with a substantial degree of protein misfolding accompanying the combined loss of PDI and ERp57 and the subsequent disposal of mis- folded proteins by ERAD. Such efﬁcient disposal of mis- folded proteins by HepG2 cells may explain the absence of an observable UPR upon depletion of PDI and ERp57 (Fig- ure 1, A and B). Collectively, the stronger phenotypes observed in the double versus single knockdown studies support the view that both PDI and ERp57 are involved in the oxidative folding of the glycoprotein substrates and that neither can fully replace the functions of the other. Furthermore, in the context of the mild phenotypes observed with the other combinatorial knockdowns (see below and Table 1), these experiments underscore the prominent role that these thiol oxidoreductases play among the four PDI family members examined. We next tested whether depletion of ERp72 or P5 in combination with PDI would yield enhanced defects in fold- ing. The combination of PDI P5 showed no differences for any of the four substrates tested compared with data ob- tained for PDI knockdown alone (Table 1). For the PDI ERp72 knockdown combination, oxidative folding proﬁles were comparable to PDI-depleted cells for the three glyco- protein substrates. In contrast, there was a more pronounced delay in the oxidative folding of Alb compared with PDI- depletion alone suggesting that both enzymes can act on this substrate (compare Figure 3, A and C and Figure 7). Unlike the PDI ERp57 combination, these cells did not display a generalized delay in ER to Golgi trafﬁcking or in secretion rate; indeed, the rate of Alb secretion was the same in both control and PDI ERp72 knockdown cells (data not shown). Therefore, ERp72 likely recognizes a more limited set of substrates compared with PDI or ERp57. Its effect on Alb was likely masked in the individual ERp72 knockdown by the presence of PDI. To assess whether an effect of ERp72 or P5 knockdown could be assigned in the absence of ERp57, combination knockdowns of ERp57 ERp72 as well as ERp57 P5 were carried out. There was no change observed in the oxidative folding of the four substrates beyond what had been noted for ERp57 knockdown alone (Table 1). Because the presence of PDI in these cells may have yet been inﬂuencing our ability to observe the impact of these family members on glycoprotein folding, a triple knockdown was carried out, depleting cells of PDI, ERp57, and ERp72. Remarkably, the cells grew and remained viable and behaved as previously observed for the PDI ERp57 double knockdown. Alb oxidation was delayed in a manner similar to what had been observed in the PDI ERp72 combination, but there was no additional change in the oxidative folding of the glycopro- teins due to ERp72 depletion (Table 1). This again suggests that ERp72 does not act on these substrates but is speciﬁc for others such as Alb. The inability to detect any effect of P5 depletion may indicate speciﬁcity for a narrow range of substrates, none of which were included in this study, or that it may be involved in other processes such as substrate reduction. 3101