L. A. Rutkevich et al. and somatostatin, as well as with unfolded RNase A, have been documented recently in NMR titration studies (Den- isov et al., 2009). On substrate oxidation, the catalytic sites of PDI are reduced and must be reoxidized for subsequent rounds of catalysis. This is accomplished by the FAD-bind- ing oxidase Ero1 that donates oxidizing equivalents to PDI in both yeast (Sevier and Kaiser, 2002) and mammals (Mezghrani et al., 2001; Wang et al., 2009). PDI has also been implicated in maintaining redox homeostasis as a feedback regulator of Ero1 (Appenzeller-Herzog et al., 2008) and by retaining Ero1 in the ER (Otsu et al., 2006). Although PDI carries out all the functions of a thiol oxidoreductase in the ER, many other enzymes in this family can catalyze these reactions in vitro (Alanen et al., 2006). Consequently, there has been great interest in determining the extent to which PDI family members exhibit functional overlap versus unique properties such as restricted substrate speciﬁcities. This issue has been addressed in S. cerevisiae that pos- sesses ﬁve PDI family members of which only PDI is essen- tial. In most instances, the ability of individual family mem- bers to restore viability to a PDI-deleted strain, when overexpressed, required low level expression of one of the other homologues. Furthermore, only PDI was capable of supporting native folding of the carboxypeptidase Y sub- strate (Norgaard et al., 2001). These ﬁndings indicate little functional interchangeability within yeast PDI homologues and underscore the predominant role of PDI in catalyzing oxidative folding of substrates. Similar studies in mammalian cells have been hampered by the plethora of homologues and by the difﬁculties in maintaining cell viability when PDI levels are constitutively depleted (Park et al., 2006). However, transient PDI knock- down experiments have implicated this enzyme in facilitat- ing cholera toxin retrotranslocation from ER to cytosol whereas the related family members ERp57 and ERp72 ei- ther had no effect or an opposing effect, respectively (Forster et al., 2006). Furthermore, PDI, but not ERp57, has been shown to affect the production of infectious rotavirus parti- cles (Maruri-Avidal et al., 2008) and the folding of human MHC class I heavy chains (Kang et al., 2009). Additional studies have focused on those members that most closely resemble PDI. Of these, the best characterized is ERp57 which shares a high degree of similarity with PDI, but its substrate-binding b domain has diverged such that it inter- acts with the ER lectin-chaperones calnexin (Cnx) and cal- reticulin (Crt) (Oliver et al., 1999; Kozlov et al., 2006). These chaperones are thought to present glycoprotein substrates to ERp57, and indeed, ERp57 has a well-established speciﬁcity for Asn-linked glycoproteins that is abrogated when its in- teractions with Cnx and Crt are disrupted (Zapun et al., 1998; Jessop et al., 2009a). Nevertheless, there are several reports of ERp57 acting directly on a few select substrates indepen- dently of a requirement for Cnx or Crt (Peaper et al., 2005; Schelhaas et al., 2007; Zhang et al., 2009). Studies in which ERp57 has been depleted through RNA interference or gene knockout have established that ERp57 is required for the efﬁcient oxidative folding of mouse MHC class I heavy chains (Zhang et al., 2006), inﬂuenza hemagglutinin (Solda et al., 2006), clusterin, and 1 integrin (Jessop et al., 2007). Another family member, ERp72, contains three catalytic and two noncatalytic domains (Mazzarella et al., 1990) and, although it shares high sequence similarity with ERp57, it is not able to bind to Cnx or Crt (Kozlov et al., 2009). Limited information is available concerning a functional relationship between ERp72 and ERp57. ERp72 was unable to substitute functionally for ERp57 in the oxidative folding of MHC class I molecules (Zhang et al., 2006) but it did replace ERp57 in 3094 mixed disulﬁde complexes with several glycoprotein sub- strates when ERp57 was either eliminated or its recruitment to substrates by Cnx/Crt impaired by treatment with cas- tanospermine (Solda et al., 2006; Jessop et al., 2009b). Addi- tionally, ERp72 has been implicated in the folding of thyro- globulin (Menon et al., 2007) and apolipoprotein B (Linnik and Herscovitz, 1998), but no consensus has been reached as to how ERp72 recognizes substrates. Finally, P5 is a poorly understood member of the PDI family with one noncatalytic and two catalytic domains (Rupp et al., 1994). Although found in a large multi-enzyme complex that also includes PDI, ERp72, and BiP (Meunier et al., 2002), only recently has it been implicated in a direct interaction with BiP, possibly mediating its substrate selection (Jessop et al., 2009b). Despite these ﬁndings, the question of why so many PDI family members exist in the ER remains enigmatic. The hypothesis of differences in substrate speciﬁcity has been impressively investigated using substrate-trapping mutants of PDI family members in which the second cysteine in the CXXC active site motif is mutated to alanine. This inactivates an escape pathway in which the second cysteine thiolate resolves mixed disulﬁde intermediates between the ﬁrst cys- teine and substrate (Walker and Gilbert, 1997). These studies revealed distinct substrate proﬁles for each PDI family mem- ber but with considerable overlap (Jessop et al., 2009a; Jessop et al., 2009b). Most notable was a strong preference for glycoprotein substrates by ERp57, a surprisingly wide range of substrates for P5 and an equally surprising dearth of identiﬁable substrates for PDI. As welcome as this informa- tion is, the approach has a number of limitations: substrate identiﬁcation is difﬁcult, trap mutants are overexpressed and must compete with endogenous enzymes for substrate, the functional role of individual family members cannot be assessed, and, because trap mutants do not permit the CXXC motif to become oxidized, substrates undergoing PDI mem- ber-catalyzed oxidative folding are not detected. To address some of these issues, we report the ﬁrst sys- tematic knockdown of multiple PDI family members in mammalian cells and its functional consequences on the oxidative folding and intracellular trafﬁcking of a number of endogenously expressed substrates, including albumin, -fetoprotein, transferrin, 2-HS-glycoprotein, and 1-antit- rypsin. By high efﬁciency depletion of PDI, ERp57, ERp72, and P5, either alone or in various combinations, we assess their relative importance in oxidative folding as well as their functional overlap when evaluated with a common group of substrates. MATERIALS AND METHODS Cell Lines and Antibodies HepG2 cells were maintained in high glucose DMEM containing 10% fetal bovine serum (FBS) and antibiotics. This was supplemented with either 1 g/ml puromycin (Bioshop, Toronto, ON, Canada) or 200 g/ml hygromy- cin B (Invitrogen, Carlsbad, CA) for HepG2 cells stably expressing ERp57 shRNAmir or mutant PDI, respectively. Antibodies used in this study were as follows: anti-PDI and -ERp72 (AssayDesigns, Plymouth Meeting, PA), anti-P5 (Afﬁnity Bioreagents, Cambridge, United Kingdom), anti-BiP (BD Biosciences, Franklin Lakes, NJ), anti-CHOP (Thermo, Rockford, IL), anti-actin (Chemicon, Temecula, CA), anti-albumin, -transferrin and - 1-antitrypsin (Sigma, St. Louis, MO) and anti- -fetoprotein and - 2-HS-glycoprotein (Abcam, Cam- bridge, MA). Rabbit antisera directed against calnexin, calreticulin, and ERp57 have been described previously (Danilczyk and Williams, 2001; Zhang et al., 2006; Ireland et al., 2008). The 12CA5 anti-HA mAb was provided by Dr. Tania Watts (University of Toronto). Human PDI cDNA Isolation and Mutagenesis mRNA was isolated from HepG2 cells and then cDNA was produced using the RevertAid H Minus kit (Fermentas, Burlington, ON, Canada). Using this as template, cDNA encoding PDI with an HA tag inserted after position D499, Molecular Biology of the Cell