A decisive step in the intoxication of CT is the transfer of the toxic CTA1 subunit from the ER lumen into the cytosol. How CTA1 is prepared in the ER lumen before its arrival in the cytosol is not fully understood. Based on an in vitro approach, we determined previously that the ER lumenal proteins PDI and Ero1α likely represent two central players in this process. Specifically, we found that the reduced form of PDI binds and unfolds CTA1 (Tsai et al., 2001
), whereas subsequent oxidation of PDI by Ero1α releases the toxin from PDI (Tsai and Rapoport, 2002
). We postulated that these events initiate retro-translocation of CTA1 in cells. Using a siRNA-mediated approach, we observed that PDI is essential for CTA1 retro-translocation (Forster et al., 2006
). However, the role of Ero1α in controlling the toxin retro-translocation process remained to be clarified.
Using loss-of-function and gain-of-function approaches, we have demonstrated in this study that Ero1α plays a central role in facilitating retro-translocation of CTA1. We found that down-regulation of Ero1α decreases toxin retro-translocation in a specific manner. It is not due to a general induction in ER stress as knockdown of Ero1α neither up-regulates several UPR markers, induces XBP1 splicing, nor affects the retro-translocation and degradation of the established ERAD substrates TCRα and CD3δ. Instead, we showed that down-regulation of Ero1α leads to a decrease in PDI oxidation that precludes the toxin from being released from PDI efficiently, thereby blocking toxin transport.
Likewise, overexpression of the catalytically active Ero1α also blocks CTA1 retro-translocation. In this case, increased PDI oxidation due to Ero1α overexpression prevents the toxin from engaging PDI effectively. These two findings not only pinpoint Ero1α as a critical player in toxin retro-translocation, but also support the redox-dependent model of CTA1 retro-translocation described in vitro. Furthermore, our analyses demonstrate that reduced PDI displays an increased affinity for its binding partner Derlin-1, a key component of the retro-translocon. Overall, it appears that the ability of PDI to engage a binding partner, as well as a substrate, is redox-dependent (G). Specifically, reduced PDI engages both CTA1 and Derlin-1; oxidation of PDI by Ero1α releases the unfolded toxin from PDI as well as PDI from Derlin-1.
Our findings implicate that, under normal conditions, a cell maintains a fine balance of Ero1α and PDI levels. This balance enables sufficient amounts of reduced PDI to bind and unfold the toxin while simultaneously maintaining enough oxidation equivalents to subsequently oxidize PDI and induce toxin release. The observation that simultaneous overexpression of PDI and Ero1α rescues toxin transport further supports this idea. Although there is a much higher cellular concentration of PDI in comparison to Ero1α, only a small fraction of PDI is likely to be dedicated to retro-translocation. Consistent with this idea, we previously observed only a small fraction of PDI binds to Derlin-1 (Bernardi et al., 2008
); this pool of PDI is expected to be involved in retro-translocation.
The Ero1α-PDI redox cycle described in this study is not designed primarily for pathogen entry. Instead, this system is likely geared to drive the retro-translocation of misfolded substrates during ERAD (Vembar and Brodsky, 2008
). For example, PDI displays redox-dependent binding to the ERAD substrates such as BACE (Molinari et al., 2002
) and the nonglycosylated pro-α factor (Wahlman et al., 2007
), suggesting Ero1α may act in the retro-translocation of these substrates. Moreover, although the specific ER factors have not yet been identified, the cellular redox state appears to control the degradation of several ER proteins (Young et al., 1993
; Wainwright and Field, 1997
; Courageot et al., 1999
), signifying that the Ero1α-PDI complex may be generally involved. In addition to our finding that PDI acts as a chaperone to unfold CTA1 and initiate toxin retro-translocation, it is important to note that PDI is recognized classically as an enzyme that catalyzes the formation, breakage, and rearrangement of disulfide bonds during the protein folding process (Ellgaard and Ruddock, 2005
We note that down-regulation of Ero1α does not block toxin retro-translocation completely. This result may be due to the incomplete knockdown of Ero1α or to the complementary activity of other undiscovered PDI oxidases. In this context, there is another isoform of Ero1 called Ero1β (Pagani et al., 2000
). However, our previous in vitro analysis suggested that Ero1β does not oxidize PDI to release CTA1 (Tsai and Rapoport, 2002
). This result is supported by our finding that overexpression of Ero1β does not affect toxin retro-translocation (data not shown). Furthermore, Ero1β is found to be expressed at low levels in 293T cells (Pagani et al., 2000
), implying that Ero1β does not contribute significantly to the toxin transport process.
Structurally, the observation that PDI engages a substrate and a binding partner in a redox-dependent manner suggests that the PDI redox state may regulate the conformation of multiple binding sites. In this context, we have demonstrated previously that reduced and oxidized PDI exist in different conformations (Tsai et al., 2001
). Pinpointing the specific sites on PDI that bind to CTA1 (Forster et al., 2009
) and Derlin-1 will be crucial in assessing whether these binding sites are altered in response to the redox state of PDI.
The fact that reduced PDI binds to Derlin-1 with increased affinity has major implications for the mechanism by which the unfolding process is coupled to events on the ER membrane. Preferential targeting of reduced PDI to the retro-translocation machinery permits efficient temporal and spatial interactions with CTA1. Subsequent oxidation of PDI by Ero1α, which is itself tethered to the ER membrane by a poorly described mechanism (Otsu et al., 2006
), would thus allow direct presentation of unfolded toxin to the retro-translocon. Oxidized PDI, which is no longer capable of binding and unfolding toxin, is then released from the membrane and replaced by reduced PDI. This binding–release cycle is perpetuated by regeneration of reduced PDI by unknown reductase activity. Clearly, addressing how Ero1α is coupled physically to the retro-translocation machinery, as well as how reduced PDI is regenerated, will elucidate the precise mechanism by which CTA1 is primed for retro-translocation across the ER membrane.