Thioredoxin domain–containing ER proteins function in a wide variety of processes, both physiological and pathological. The normal processes of protein folding, ER quality control, and secretion rely on PDI-like proteins that distinguish between correctly folded, secretion-competent substrates and misfolded substrates that need to be retained and then refolded or degraded. Moreover, to cause disease, pathogens such as Py and cholera toxin traffic from the plasma membrane to the ER where they hijack PDI-like proteins (Magnuson et al., 2005
; Forster et al., 2006
; Gilbert et al., 2006
) in order to gain access to the cytosol. The regulatory mechanisms that control the various functions of a given PDI-like protein have not been well characterized.
To assess whether dimerization could regulate the activities of a specific PDI-like protein, we elucidated the role of dimerization of ERp29, a PDI-like protein shown to have at least two distinct activities, one in mediating the entry of a viral pathogen and the other in the escorting of secretory proteins. Our studies revealed that homodimerization of ERp29 is essential for both its protein unfolding and escort functions. A single amino acid mutation in the N-terminal domain of ERp29 (D42A) abrogates dimerization, disrupting its ability to unfold Py. Why dimerization of ERp29 is required to stimulate Py unfolding is unclear. Because we have not observed physical binding between Py and ERp29, their interaction is assumed to be transient. Whether dimeric ERp29 is required for this transient interaction is unknown. After NTD-mediated dimerization, the two C-terminal domains of ERp29 are brought together and may become active only in close proximity to one another. This idea is supported by the observation that ERp29 NTD can block unfolding and infection. In this scenario, NTD acts in a dominant-negative manner by titration of ERp29 monomers, preventing the two CTD domains from joining together.
Through a rational design approach based on the crystal structure of the Drosophila
ERp29 orthologue, we added a compensatory mutation to the D42A ERp29 mutant (G37D/D42A) that partially restored its ability to form dimers and found the unfolding activity to be rescued partially. Surprisingly, the G37D/D42A mutant is able to stimulate Py infection to a similar extent as wild-type ERp29. It is known that only a small fraction of virus taken up by cells during infection reaches the ER (Gilbert and Benjamin, 2004
), and even fewer viral particles are transported out of the ER and into the nucleus to cause infection (Greber and Kasamatsu, 1996
). Therefore, only a small percentage of G37D/D42A ERp29 overexpressed in the cell is needed to dimerize in order to provide the unfolding activity necessary to stimulate infection by one or a few viral particles. By contrast, in the in vitro unfolding assay, the virus:G37D/D42A ERp29 ratio is higher by necessity due to the detection limit of the immunoblot analysis. As a consequence, the higher virus:ERp29 ratio results in the unfolding of only a fraction of the viral particles.
In addition to the role of the ERp29 dimer in viral infection, our findings also revealed that dimerization of ERp29 is essential during its normal cellular function. ERp29 was shown previously to bind to the secretory protein Tg in the ER (Sargsyan et al., 2002
) and facilitate its secretion (Baryshev et al., 2006
). However, the detailed mechanism underlying this function remains unclear. We found that ERp29 maintains its interaction with Tg once Tg is secreted, suggesting that ERp29 aids Tg secretion by serving as an escort factor. This finding is consistent with the previously shown cosecretion of ERp29 with Tg despite the presence of the ER-retrieval signal in its C-terminus that normally facilitates recycling of ER-resident proteins back to the ER (Sargsyan et al., 2002
). Other ER proteins, such as SCAP (Brown et al., 2002
) or RAP (Bu, 2001
), have also been shown to act as escort factors in guiding the transport of proteins further along the secretory pathway. Importantly, the dimer of ERp29 was found to be crucial for this activity as overexpression of wild-type ERp29, but not the D42A mutant, stimulated Tg secretion. In fact, D42A overexpression led to a decrease in Tg secretion. Our evidence suggests that the inhibitory action of the mutant ERp29 is attributed to its inability to escort the bound Tg out of the ER. This is consistent with the notion that the ER contains quality control machineries that can recognize misassembled proteins (Ellgaard and Helenius, 2003
), preventing them from exiting this compartment. In this context, we observed a ~25-kDa protein that coimmunoprecipitated with mutant but not wild-type ERp29 (B). This protein may represent either an additional secretory substrate similar to Tg trapped by the defective D42A ERp29 in the ER or another helper protein involved along with ERp29 in the escort of Tg.
It was shown previously, using a gel filtration approach, that a His-tagged, purified mutant Wind protein, in which the aspartic acid residue at position 31 was changed to asparagine (D31N), is essentially monomeric (Ma et al., 2003
). The D31N Wind protein was shown to be active, as assessed by its ability to transport Pipe, a substrate of Wind, from the ER to the Golgi in COS cells (Ma et al., 2003
). In a subsequent chemical crosslinking study, the D31N mutant expressed in COS cells was impaired in dimerization when compared to the wild-type protein, although a low level of dimer can be observed (Barnewitz et al., 2004
). In this report, the D31N mutant displayed activity similar to the wild-type protein, consistent with the authors' previous observations (Ma et al., 2003
). As suggested by the authors, the residual level of dimer in the D31N protein may be sufficient for its activity. However, we found that the D42A ERp29 single mutant (corresponding to D31N Wind) failed to dimerize and was functionally compromised in both the Py infection and Tg secretion processes. The reason for this apparent discrepancy remains unclear, but may be due to differences in the chosen mutations (i.e. D mutated to A versus N), subtle structural differences in the ERp29 and Wind dimers, or differences in substrate concentrations in the ER (i.e. Py and Tg versus Pipe).
When a second site mutation (R41S) was introduced to the D31N Wind single mutant, the D31N/R41S double mutant was reported to exhibit an even more severe dimerization defect than the D31N mutant (Barnewitz et al., 2004
). Importantly, this double mutant was shown to be inactive in transporting Pipe to the Golgi. The authors reported that a high expression level of the Wind double mutant in the ER caused the defect in Pipe transport. Although this raises the possibility that the high concentration of Wind mutant, but not the absence of Wind dimerization per se
, prevented Pipe transport, these findings suggest that dimerization of Wind plays a role in regulating its function, consistent with our finding that dimerization regulates the activities of ERp29.
PDI and ERp29 have been shown to oligomerize under certain conditions (Pace and Dixon, 1979
; Yu et al., 1994
; Mkrtchian et al., 1998b
). In contrast, other studies indicated that PDI (Solovyov and Gilbert, 2004
; Li et al., 2006
) and ERp57 (Frickel et al., 2004
) exist as monomers. The reported discrepancy in PDI's dimerization state may reflect a difference in the purification procedures. It is also possible that the high-concentration storage of PDI induced oligomerization in vitro. Further studies aimed at determining the oligomerization state of PDI (if any), as well as other PDI-family members, in live cells may help to elucidate the oligomerization properties of these proteins. Of note, as PDI was shown previously to participate in Py infection (Gilbert et al., 2006
) and in cholera toxin intoxication (Tsai et al., 2001
; Forster et al., 2006
), it is unclear whether oligomerization of PDI, should it occur, regulates any of these processes.
How do PDI-like proteins possess such tremendous flexibility in selecting a wide myriad of substrates, ranging from endogenous secretory proteins to foreign pathogens? Perhaps the process of oligomerization, which increases the number of conformations that can be adopted by these proteins, provides a mechanism by which they may engage their numerous substrates. Alternatively, interactions with other ER-resident factors may enable the PDI-like proteins to act on a specific substrate. In this regard, identifying the binding partners of the PDI-like proteins would help to clarify whether this interaction regulates the substrate selection process.