Through a series of genetic and molecular studies, we demonstrate here the inhibition of UPR by ricin. Specifically, we showed that expression of RTA with its own signal sequence resulted in its translocation to the ER and subsequent glycosylation. Interestingly, expression of the inactive form that could translocate to the ER-induced UPR, but not the wild type RTA, providing evidence that the active site of ricin, played a role in perturbing the ER stress response. The inhibition of UPR was not unique to ER stress induced by the wild type protein but also by global effectors with different mechanisms of action, such as tunicamycin and DTT. Analysis of the nontoxic RTA mutants provided a causal link between the ability of ricin to inhibit ER stress induced UPR and its cytotoxicity.
For ricin to be successful in reaching its cytosolic target, the ribosome, it must be retrotranslocated from the ER to the cytosol. Considering RTA must unfold within the ER, it would seem likely that activation of UPR would occur. Induction of UPR would lead to activation of transcription of ERAD components (21
). Because ERAD is normally used to retrotranslocate misfolded proteins from the ER to the cytosol for destruction by the ubiquitin proteasome pathway, ricin must evade complete degradation by this pathway. Evidence for this was provided from recent studies, which showed that overexpression of an ERAD-related protein, the α
-mannosidase I-like protein (EDEM) strongly protected against the cytotoxicity of ricin by decreasing its retrotranslocation to the cytosol (28
). However, when EDEM was available for ricin and the interaction between EDEM and misfolded proteins was inhibited, it promoted retrotranslocation of ricin from the ER to the cytosol (28
). Therefore, for RTA to retrotranslocate to the cytosol, this ER stress response must either be inhibited greatly or abolished altogether. Otherwise, induction of UPR might successfully disable the toxin.
Two well characterized activities of ricin are to inhibit translation and subsequently reduce cell viability. It is important to verify that the inhibition of UPR observed is not confounded by inhibition of translation by ricin. To confirm that translation inhibition was not responsible for inhibition of lacZ
expression from the UPRE promoter, we demonstrated that expression of lacZ
from a PGK promoter was unaffected in the presence of RTA. Additionally, RTA did not inhibit UPR in cells expressing the spliced form of HAC1
, indicating that it does not completely inhibit Hac1p translation (). Immunoblot analysis confirmed that Hac1p accumulated in cells expressing RTA (). Third, we presented evidence that the nontoxic RTA mutants that depurinated ribosomes and inhibited translation were not able to inhibit activation of UPR in response to ER stress, separating the inhibitory activity of RTA on translation from its activity on UPR. Last, a nontoxic RTA mutant that did not depurinate ribosomes reduced the viability of cells that could not induce UPR, indicating that Ire1p function was critical for survival. The northern data provided the most definitive evidence of a defect at the level of splicing, independent of translation. It has recently been shown that the pro-apoptotic protein Bax is required for efficient UPR in mammalian cells (29
). We have observed that overexpression of Bax can also induce UPR in yeast.4
Using this finding as a guide, we speculate that the decrease in viability seen in cells expressing RTA, if due to apoptosis, would not be expected to promote inhibition of UPR.
RTA expressed in yeast with its signal sequence as well as the mature RTA without the signal sequence that reduced the viability of yeast cells were both associated with the membrane fraction. However, only RTA with the signal sequence was translocated into the ER. In cells expressing either protein we did not observe induction of β
-galactosidase expression or splicing of HAC1
mRNA after treatment with ER stress inducers, indicating that inhibition of UPR occurred on the cytosolic face of the ER. In contrast, UPR was induced in cells expressing the active site mutant with the signal sequence, but not the mature E177K, demonstrating that the inactive RTA could modulate UPR only when it could translocate into the ER. All nontoxic RTA mutants that did not depurinate ribosomes induced UPR, possibly by accumulating in the ER, since activation of UPR would lead to overexpression of ERAD components, which would inhibit retrotranslocation of these mutants to the cytosol (28
). In contrast, all toxic mutants that depurinated ribosomes, inhibited activation of UPR. These results provided evidence that an intact active site was needed for inhibition of UPR by the wild type protein, possibly to allow the protein to retrotranslocate to the cytosol. We have previously shown that ribosome depurination and translation inhibition are not sufficient for ricin-mediated cell death (5
), suggesting that the protein must attenuate another target to enhance its cytotoxicity. The results presented here demonstrate that the active site of RTA appears to be critical not only for ribosome depurination but also for inhibition of UPR.
Only the precursor form of the HAC1
mRNA was detected in cells expressing RTA after ER stress, indicating that ricin inhibits UPR by preventing splicing of the HAC1
mRNA. The unspliced HAC1
mRNA is localized to the cytoplasm and is associated with functional polyribosomes, which are stalled on the mRNA due to a direct 16-nucleotide-long base pairing interaction between the HAC1
5′-untranslated region and the intron (16
). Because inhibition of UPR occurred on the cytosolic face of the ER and required an intact active site, RTA may inhibit UPR by targeting the unspliced HAC1
mRNA. This is consistent with evidence indicating that ribosome-inactivating proteins target other RNAs besides the rRNA (32
). A single chain ribosome-inactivating protein, pokeweed antiviral protein, binds to the cap structure on mRNA and depurinates the mRNA downstream of the cap structure in vitro
). Pokeweed antiviral protein autoregulates its own mRNA (34
) and can target other RNAs in vivo
). By analogy with pokeweed antiviral protein, RTA may bind to the unspliced form of HAC1
mRNA and depurinate it, preventing long distance base-pairing between the 5′-untranslated region and the intron. Disruption of the interaction between HAC1
5′-untranslated region and intron has been shown to lead to accumulation of the unspliced HAC1
mRNA and reduce the efficiency of splicing (31
). Alternatively, RTA may attenuate the enzymatic activity of Ire1p either through a direct interaction with Ire1p or through interactions with lumenal proteins.
We present evidence here that ricin inhibits adaptation responses to ER stress by preventing HAC1
mRNA splicing and Ire1p signaling to downstream mediators of UPR, such as KAR2
. If cells are subjected to continuous ER stress and cannot respond by inducing UPR, they commit to apoptosis (20
). Viability of yeast cells expressing the precursor or the mature form of RTA is decreased significantly (5
). A nontoxic RTA mutant that could not depurinate ribosomes reduced the viability of Δire1
cells, providing evidence that Ire1p function was critical for survival in the presence of RTA. The Δire1
cells expressing RTA were slightly more viable than the isogenic BY4743 cells expressing RTA (), suggesting that the Ire1p function was critical for the cytotoxicity of RTA. Taken together, these results suggest that there is an ideal balance between the presence of Ire1p, so that some of the toxin is allowed into the cytosol, and its complete absence, which causes so much ER stress that the cell inevitably dies. The RTA mutants that depurinated ribosomes but did not kill cells were not able to inhibit activation of UPR after ER stress, providing further evidence that the inability to activate UPR in response to ER stress contributes to ricin-mediated cell death. Consistent with these results, recent evidence in mammalian cells suggested a link between UPR signaling and cell survival after ER stress by demonstrating that termination of IRE1 activity is an important factor in allowing cell death after UPR activation (37
). Other ribosome-inactivating toxins that need to enter the cytosol from the ER to carry out their enzymatic action may also inhibit UPR to avoid destruction by the ubiquitin proteasome pathway. Therefore, the results described here for ricin may be broadly applicable to other toxins. Several viruses have been shown to induce ER stress and activate UPR (38
). Cancer cells need UPR for their survival (42
). Modulation of UPR by ricin may represent a unique opportunity to inhibit a pathway required by viruses to replicate and cancer cells to grow. Additionally, activation of UPR is required for effective antibody production (43
). Through the knowledge gained by studying the effect of RTA on UPR, protection strategies against ricin to counter the threat of its use as a potential bioweapon can be developed so that the beneficial immune response is not dampened.