Molecular Biology of the Cell Vol. 21, 3093–3105, September 15, 2010 Functional Relationship between Protein Disulﬁde Isomerase Family Members during the Oxidative Folding of Human Secretory Proteins Lori A. Rutkevich, Myrna F. Cohen-Doyle, Ulf Brockmeier,* and David B. Williams Department of Biochemistry, University of Toronto, Toronto, Canada M5S 1A8 Submitted April 26, 2010; Accepted July 8, 2010 Monitoring Editor: Benjamin S. Glick To examine the relationship between protein disulﬁde isomerase family members within the mammalian endoplasmic reticulum, PDI, ERp57, ERp72, and P5 were depleted with high efﬁciency in human hepatoma cells, either singly or in combination. The impact was assessed on the oxidative folding of several well-characterized secretory proteins. We show that PDI plays a predominant role in oxidative folding because its depletion delayed disulﬁde formation in all secretory proteins tested. However, the phenotype was surprisingly modest suggesting that other family members are able to compensate for PDI depletion, albeit with reduced efﬁcacy. ERp57 also exhibited broad speciﬁcity, overlapping with that of PDI, but with preference for glycosylated substrates. Depletion of both PDI and ERp57 revealed that some substrates require both enzymes for optimal folding and, furthermore, led to generalized protein misfolding, impaired export from the ER, and degradation. In contrast, depletion of ERp72 or P5, either alone or in combination with PDI or ERp57 had minimal impact, revealing a narrow substrate speciﬁcity for ERp72 and no detectable role for P5 in oxidative protein folding. INTRODUCTION Proteins entering the secretory pathway are initially targeted to the endoplasmic reticulum (ER) where protein folding and posttranslational modiﬁcations take place in a special- ized environment. Critical among these modiﬁcations is the formation, isomerization, and reduction of disulﬁde bonds catalyzed by thiol oxidoreductases of the protein disulﬁde isomerase (PDI) family. Assignment of as many as 20 pro- teins to the mammalian PDI family is based on the presence of at least one thioredoxin-like domain, with catalytic activ- ity determined by the presence of a pair of cysteine residues within a CXXC motif that is able to alternate between disul- ﬁde and dithiol forms (Appenzeller-Herzog and Ellgaard, 2008; Hatahet et al., 2009). To act as an oxidase and form disulﬁde bonds, the active site must itself be oxidized, whereas reduction and isomerization events are carried out This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E10 – 04 – 0356) on July 21, 2010. * Present address: Institut fu¨ r Physiologie, Universita¨t Duisburg- Essen, Hufelandstrasse 55, 45122 Essen, Germany. Address correspondence to: David B. Williams (david.williams@ utoronto.ca). Abbreviations used: 1AT, 1-antitrypsin; FP, -fetoprotein; 2HS, 2HS glycoprotein; Alb, albumin; Cnx, calnexin; Crt, calreticulin; DTT, dithiothreitol; ERAD, ER-associated degradation; NEM, N- ethylmaleimide; PDI, protein disulﬁde isomerase; TF, transferrin; UPR, unfolded protein response. © 2010 L. A. Rutkevich et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution– Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). when the active site is reduced. Although relatively few substrates have been examined in detail, it appears that disulﬁde isomerization is a slow process that may be rate- limiting in the folding of certain proteins. In vitro studies on the oxidative folding of bovine pancreatic trypsin inhibitor revealed that partially disulﬁde-bonded intermediates be- come kinetically trapped during folding and that isomeriza- tion of disulﬁdes was the main process responsible for the 1000-fold enhancement in folding rate observed in the presence of PDI (Weissman and Kim, 1993). Furthermore, in vivo studies have shown that both the HIV-1 envelope gly- coprotein (Land et al., 2003) and low-density lipoprotein receptor (Jansens et al., 2002) form a number of incorrect disulﬁde bonds early in folding and that posttranslational isomerization events are then required for reshufﬂing and native folding. PDI was the ﬁrst ER thiol oxidoreductase to be character- ized (Goldberger et al., 1964), and its oxidase and isomerase activities have been validated in vitro (Lyles and Gilbert, 1991), in yeast (Laboissiere et al., 1995; Kulp et al., 2006), and in mammalian systems (Bulleid and Freedman, 1988). The structure of yeast PDI reveals a U-shaped orientation of its four thioredoxin-like domains, termed a, b, b , and a , where a and a denote the catalytically active domains containing the CXXC motif and b and b are noncatalytic domains (Kemmink et al., 1997; Tian et al., 2006). The b domain appears to be the main site for substrate binding and for the reported chaperone functions of PDI (Klappa et al., 1998; Horibe et al., 2004). There is a hydrophobic surface within the interior of the U structure that is provided by the b domain as well as the other domains that appears well suited for interaction with nonnative folding intermediates (Tian et al., 2006). Indeed, direct interactions between a hydrophobic pocket in the b domain of PDI spanning resi- dues 240 –320 and the amphipathic peptides mastoparan 3093