When CT reaches the ER from the cell surface, it coopts the ERAD quality control machinery, gains access to the cytosol, and evades proteasomal degradation. How these distinct steps are achieved remains under intense investigation. In this study, we identify new components of the ERAD machinery that mediate retro-translocation of CTA1. We demonstrated previously that the ER membrane protein, Derlin-1, facilitates retro-translocation of CTA1 (Bernardi et al., 2008
), similar to a subsequent report (Dixit et al., 2008
). As E3 ubiquitin ligases Hrd1 and gp78 form complexes with Derlin-1 (Ye et al., 2005
, Lilley and Ploegh, 2005
) and are responsible for the degradation of a variety of misfolded substrates (Hirsch et al., 2009
), we tested the possibility that this ubiquitination machinery may similarly regulate toxin retro-translocation, despite the observation that the CTA1 chain is neither ubiquitinated on its two lysines nor at its N-terminus when the toxin reaches the cytosol (Rodighiero et al., 2002
Using siRNA knockdown and dominant-negative approaches, we demonstrate that Hrd1 is involved in the ER-to-cytosol transport of CTA1. Specifically, down-regulation of Hrd1, as well as expression of the enzymatically inactive C291A Hrd1 and the truncated TM1-6 Hrd1 mutants, inhibited CTA1 retro-translocation. In the case of the dominant-negative approach, a trivial explanation to account for this block in toxin retro-translocation is that expression of the Hrd1 mutants causes the buildup of endogenous misfolded substrates at the retro-translocation sites, potentially inducing ER stress and thereby preventing CT from engaging this machinery to reach the cytosol. However, we found that expressing the Hrd1 mutants did not induce massive ER stress, and CTA bound more strongly with C291A Hrd1 than with WT Hrd1.
These findings therefore indicate that expression of the Hrd1 mutants interferes with a specific step in the retro-translocation of CTA1. Although the ligase activity of this E3 is not required to bind initially the CTA substrate, it appears to be necessary for the subsequent transport and release of the toxin into the cytosol. Similar to CTA, the Hrd1-dependent substrate CD3δ displayed a more stable interaction with C291A Hrd1 than with WT Hrd1 (Kikkert et al., 2004
), suggesting that the mechanistic basis of Hrd1's role in CD3δ degradation and CTA1 retro-translocation may be similar.
Expression of TM1-6 Hrd1 decreased CTA1 retro-translocation. This finding demonstrates that the cytosolic domain of Hrd1 is essential for transport. That CTB binds more strongly to TM1-6 Hrd1 than WT Hrd1 suggests that CTB normally undergoes cycles of binding and release from Hrd1 that is disrupted in the absence of its cytosolic domain. This observation supports the view that active communication between the Hrd1 transmembrane and cytosolic domains plays a crucial role in transferring a substrate from the ER into the cytosol.
What might be the functions of the Hrd1 transmembrane and cytosolic domains in the toxin translocation process? The Hrd1 cytosolic domain contains the active site responsible for ubiquitination. Recent findings indicate that the Hrd1 transmembrane domain transfers a membrane substrate from the ER into the cytosol (Omura et al., 2008
) and senses the misfolded state of a membrane protein (Sato et al., 2009
). Thus, the different Hrd1 domains have distinct roles. A potential scenario of how Hrd1 might eject the toxin into the cytosol could be envisioned as follows: the Hrd1 lumenal domain first engages CT, followed by the Hrd1 transmembrane domain assisting in the transfer of CTA1 into the cytosol. Finally, the Hrd1 cytosolic domain promotes ubiquitination of CTA1 (on nonlysine residues, see below) to allow the toxin to be extracted. Alternatively, the Hrd1 cytosolic domain ubiquitinates a cellular factor that releases the toxin from the ER membrane. Expression of the Hrd1 cytosolic domain did not affect CTA1 retro-translocation (not shown), perhaps because this domain neither binds to CT nor disrupts any cytosolic components required for extracting the toxin into the cytosol.
The repertoire of E3 ligases involved in ERAD continues to expand (Vembar and Brodsky, 2008
; Hirsch et al., 2009
). We therefore asked whether CT utilizes other E3 ligases in addition to Hrd1. As Hrd1 hetero-dimerizes with the E3 ligase gp78 (Ye et al., 2005
), we tested whether gp78 plays any role in the ER-to-cytosol transport of CTA1. Our functional and interaction analyses demonstrate that the gp78 E3 ligase regulates CTA1 retro-translocation, suggesting that there is conservation in function among the E3 ligases to enable CT to utilize these proteins interchangeably. This finding is similar to the retro-translocation of certain misfolded substrates such as TCRα and CD3δ (Fang et al., 2001
; Kikkert et al., 2004
), but different from that of other substrates (such as CFTR and HMG CoA reductase) that show preference for a specific E3 ligase (Song et al., 2005
; Chen et al., 2006
; Morito et al., 2008
). Thus the selection of an E3 ligase during retro-translocation appears to be substrate-dependent.
We found that in contrast to the enzymatic-inactive Hrd1, the inactive gp78 mutant binds less efficiently to CT than WT gp78. Because expression of this mutant gp78 did not induce significant ER stress, we suspect that the mutant gp78's decreased affinity for the toxin is unlikely due to a build-up of misfolded proteins that would preclude its interaction with CT. Instead, potential structural changes imparted on the mutant gp78 might prevent its interaction with CT directly or disrupt the E3 ligase's interaction with other cellular components used to recruit CT to gp78. Further experiments are required to clarify these possibilities.
How a particular substrate engages sequentially the various ERAD components is not clear. In the case of CT, we found that dominant-negative Derlin-1 (i.e., Derlin-1-YFP) blocks the CT-Hrd1 and -gp78 interactions, suggesting that the toxin is first recruited to Derlin-1 and then transferred to Hrd1/gp78. Derlin-1-YFP's inhibitory effect is likely imparted within the ER lumen because CTB never reaches the cytosol. Moreover, we showed previously that Derlin-1-YFP exerts its dominant-negative effect by titrating CT away from endogenous Derlin-1 (Bernardi et al., 2008
). Thus, by preventing endogenous Derlin-1 from engaging the toxin, Derlin-1-YFP is also expected to block interaction between CT and cellular components that normally interacts with the toxin after Derlin-1, such as Hrd1 and gp78. This sequential transfer mechanism depicted for CT has also been proposed in the Der1/Hrd1-dependent degradation of the yeast ERAD substrate CPY* (Gauss et al., 2006
). Clearly, elucidating the fate of the toxin after it is released from Hrd1 will be important to understand the entire CTA1 retro-translocation pathway.
Our previous results showed that the ER lumenal factor PDI unfolds CTA1 to prime the toxin for transport across the ER membrane (Tsai et al., 2001
; Forster et al., 2006
). We then determined that PDI interacts with the Derlin-1 membrane protein (Bernardi et al., 2008
), suggesting that events within the ER lumen and on the membrane that control toxin retro-translocation are coupled. That PDI also associates with the membrane proteins Hrd1 and gp78 further supports this view. As the holotoxin is transferred from Derlin-1 to Hrd1, we believe that the Hrd1-bound PDI unfolds CTA1 once the toxin reaches Hrd1.
The observations that expression of two enzymatic defective E3 ligases and a catalytic-inactive E2 enzyme dedicated to ERAD decreased the ER-to-cytosol transport of CTA1 led us to conclude that a functional ubiquitin system is required for toxin retro-translocation. This possibility was surprising because CT has been considered to be a nonubiquitinated ERAD substrate (Rodighiero et al., 2002
; Kothe et al., 2005
), a conclusion based on the finding that a CTA1 variant in which the two lysines are mutated, and where the N-terminus was blocked chemically, displayed an activity similar to WT toxin. However, as recent studies have identified nonlysine ubiquitination sites on substrates, including serine, threonine, and cysteine residues (Cadwell and Coscoy, 2005
; Wang et al., 2007
), ubiquitination of CTA1 (which contains numerous serines/threonines and a single cysteine) remains a formal possibility. In this context, we have not observed any higher molecular weight CTA1 in our experiments that might correspond to ubiquitinated CTA1. This may be because ubiquitinated CTA1 is deubiquitinated rapidly or that CTA1 is not ubiquitinated. Interestingly, although degradation of TCRα requires a functional ubiquitination pathway (Yu and Kopito, 1999
), ubiquitination of TCRα on lysine residues is not required for its degradation (Yu et al., 1997
If CTA1 is not ubiquitinated, ubiquitination of a cellular factor may be required for toxin transport. For example, it has been postulated that ubiquitination of adapter proteins may be required to recruit cellular components to the retro-translocation complex to properly execute the US11-mediated retro-translocation of MHC class I molecules, as retro-translocation does not require ubiquitination of the substrate itself (Hassink et al., 2006
), but an intact ubiquitination system is necessary (Shamu et al., 1999
; Kikkert et al., 2001
). These findings further support the contention that ubiquitination of cellular components other than the substrate regulates retro-translocation. We suspect that a similar mechanism is operating in the ubiquitin-dependent ER-to-cytosol transport of CTA1. Perhaps auto-ubiquitination of Hrd1/gp78 (Fang et al., 2001
; Kaneko et al., 2002
; Nadav et al., 2003
; Kikkert et al., 2004
) recruits adaptor proteins that facilitate CTA1 release into the cytosol. Alternatively, it is also conceivable that CTA1 “shuttles” on a misfolded substrate that is ubiquitinated upon entry into the cytosol. Clearly, understanding how CT utilizes the ubiquitination machinery during retro-translocation will illuminate a critical step in CT's intoxication process as well as the fundamental mechanics of ERAD.