L. A. Rutkevich et al. scores the narrow substrate speciﬁcity of ERp72. It has been noted that when ERp57 is depleted (Solda et al., 2006) or the binding of Cnx or Crt to substrates is impeded through treatment with castanospermine (Jessop et al., 2009b) several glycoprotein substrates of ERp57 can shift to an association with ERp72, suggesting overlapping substrate speciﬁcities. However, we were unable to detect any involvement of ERp72 in the oxidative folding of FP, 2HS, or TF when ERp57 was depleted along with ERp72 or even in triple knockdowns lacking PDI, ERp57, and ERp72. It is possible that the engagement of these glycoproteins within the Cnx/ Crt cycle precludes interactions with ERp72 or that ERp72 simply has a narrow substrate range not sampled in the present study. Finally, we did not observe any impact on oxidative fold- ing of depleting P5 alone or in combination with other PDI family members. These data suggest either a minor role for P5 or one speciﬁc for substrates not included in this study. P5 has previously been reported to be part of a multi- enzyme complex containing several ER chaperones includ- ing BiP (Meunier et al., 2002). In the substrate-trapping study by Jessop et al. (2009b), the authors conﬁrmed a noncovalent interaction between P5 and BiP and reported mixed disul- ﬁde complexes between P5 and a broad range of substrates tested, although such complexes could only be observed when substrates were selectively translated in a semiperme- abilized cell system. The authors speculated that in a manner analogous to ERp57 and Cnx/Crt, P5 is targeted to sub- strates via its interaction with BiP. Given that BiP interacts with many newly synthesized proteins in the ER including those in hepatoma cells (Molinari and Helenius, 2000; Schmidt and Perlmutter, 2005), the discrepancy between our respective ﬁndings may reﬂect differences in the methods used or may indicate that P5, although reported to have oxidase activity in vitro (Lappi et al., 2004), may act predom- inantly as a reductase to remove disulﬁde bonds in mis- folded proteins targeted to BiP and degraded by ERAD (Jessop et al., 2009b). The existence of a multi-enzyme complex containing PDI, ERp72, P5, BiP, and Grp94, but lacking Cnx, Crt, and ERp57, has led to the suggestion that the folding of various proteins may be segregated either spatially or temporally between different chaperone networks (Meunier et al., 2002) and that this may partially explain differences in PDI family member substrate speciﬁcity (Hatahet et al., 2009; Jessop et al., 2009b). Our data highlighting the apparent functional overlap of PDI and ERp72 in Alb oxidation as well as the lack of substrate sharing for ERp57 and ERp72 are in good agree- ment with such physical separation of function. However, our observation that both PDI and ERp57 are involved in the oxidative folding of most substrates tested is consistent with a more homogenous distribution of PDI family members in the ER. We also do not observe any impact of combined depletion of PDI and P5, which might have been expected if substrate selection was based on physical proximity of the oxidoreductases within a chaperone complex. In summary, using a functional approach we have begun to deﬁne the relative roles of different PDI family members in oxidative protein folding within the ER. 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