Cell survival under ER-stress conditions is largely dependent on the UPR pathway (Patil and Walter, 2001
; Schroder and Kaufman, 2005
; Marciniak and Ron, 2006
; Zhao and Ackerman, 2006
; Lai et al., 2007
; Ron and Walter, 2007
). However, if the overload caused by ER stressors cannot be counteracted, the UPR response switches from its prosurvival function to the signaling of apoptotic death. A major current question regarding the cell's response to ER stress is how the switch from prosurvival to prodeath is mechanistically decided. According to currents models, the mammalian UPR senses intralumenal stress through the three upstream ER transducers: IRE1, PERK, and ATF6; signaling via these stress receptors is regulated by the ER molecular chaperone BiP (Lee et al., 1999
; Li and Lee, 2006
; Ni and Lee, 2007
). Nevertheless, it is unclear whether other upstream regulators of the mechanism ER-stress apoptosis do exist, and much remains to be elucidated about the downstream factors and interactions involved in the transduction of the cell-death signal.
Recent work showed that mammalian calnexin is required for the cleavage of Bap31 and thus for the generation of the proapoptotic p20 fragment under tunicamycin stress; these authors proposed that calnexin acts as a scaffold for Bap31 processing by caspase 8 (Zuppini et al., 2002
; Delom et al.
, 2006; Groenendyk et al., 2006
; Delom et al., 2007
). These observations raise the possibility that calnexin could be involved in the early steps relaying the signal toward apoptotic death initiated by overwhelming ER stress.
Here, we demonstrate that the overexpression of calnexin in S. pombe induces cell death with typical apoptotic features including early death, phosphatidyl serine exposure, metacaspase activation, ROS production, nuclear fragmentation, and DNA breakage. This effect is specific to calnexin and is not due to overloading of the ER capacity, because overproduction of the ER proteins PDI or Sec61β did not induce the apoptotic-death phenotypes and is not due to the loss of ER membrane integrity, as confirmed by microsome analysis.
It has been previously reported that the expression of mammalian Bak in the fission yeast is lethal and that Bak requires interactions with S. pombe
calnexin to mediate this cell-death phenotype (Torgler et al., 1997
). The authors proposed that the interaction between Bak and calnexin results in a dominant lethal effect due to the propagation of a death signal or the recruitment of additional interactors to the Bak–calnexin complex (Torgler et al., 1997
). Our results show that when overexpressed, calnexin induces apoptosis without exogenous factors or ER-stress conditions. In general, overexpression causes a bypass in the regulation of the pathway studied (Ramer et al., 1992
). This suggests that calnexin overexpression may disturb the control of an apoptotic pathway in which calnexin takes part. A possibility is that the overexpression of calnexin mimics conditions of ER stress in otherwise resting cells.
Truncation of the TM and cytosolic tail of calnexin abolished the apoptotic death caused by overexpression. Using various deletion mutants, we demonstrated that the anchoring of calnexin to the ER membrane is required to induce apoptosis by overexpression. Although the overexpression of the cytosolic tail anchored to the membrane induced cell death, the strongest apoptotic effect was observed by overproducing a calnexin mutant spanning the lumenal domain with the TM. These results may imply that residues on both the intralumenal portion and the cytosolic tail of calnexin could play roles in the induction of apoptosis and that the sequences on either side of the TM may participate in different prodeath interactions. Supporting the importance of the cytosolic tail and the TM, Torgler et al. (1997)
reported that expression of human Bak was lethal in an S. pombe
strain expressing wild-type calnexin but not in a strain expressing a mutant calnexin lacking both the TM and the cytosolic tail. Importantly, our results demonstrate that sequences within the lumenal domain of calnexin are also involved in proapoptotic interactions. The requirement of the TM to elicit apoptotic death by overexpression suggests that the anchoring of calnexin to the ER membrane may be required for its interaction with some key partner(s) for the assembly of a lethal complex.
Our observations provide first direct evidence that altering the levels of calnexin causes death with apoptotic features. That its overexpression induces programmed cell death is indicative that calnexin could play a role in apoptotic processes induced by “natural” ER stresses. An increase in the calnexin levels due to ER stress may constitute a part of a branch in the mechanism of induction of apoptotic death. Tunicamycin is a potent inhibitor of N-g
lycosylation that is currently used as an elicitor of ER stress in the study of the apoptosis mechanisms in mammalian cells and in the budding yeast (Perez-Sala and Mollinedo, 1995
; Hacki et al., 2000
; Hauptmann et al., 2006
; Hauptmann and Lehle, 2008
). We have previously reported that tunicamycin induces ER stress in S. pombe
and increases the expression of cnx1+
, the gene encoding calnexin (Jannatipour and Rokeach, 1995
). In this work, we show that also in the case of S. pombe
, tunicamycin provokes cell death with typical apoptosis markers.
Using S. pombe
strains expressing calnexin mutants at basal levels, we demonstrated that apoptotic cell death induced by tunicamycin is significantly less efficient in cells expressing calnexin without its TM. Cells expressing only the lumenal portion of calnexin exhibited a 50% reduction in apoptotic cell death due to exposure to tunicamycin. Our observations implicate calnexin in apoptosis caused by ER stress. In agreement with our results, calnexin-deficient rodent cells are relatively resistant to apoptosis induced by ER stress (Zuppini et al., 2002
; Groenendyk et al., 2006
). However, it has been reported that siRNA inhibition of calnexin expression increases the sensitivity to tunicamycin of apoptosis-resistant human-breast carcinoma MCF-7 cells (Delom et al.
, 2006). This disparity in the results could be due to differences in the cell lines used in these studies. Nevertheless, transfection of a ΔE calnexin mutant lacking most of the lumenal domain into MCF-7 cells restored the sensitivity to tunicamycin-induced apoptosis (Delom et al.
, 2006). Together, these observations clearly implicate calnexin in ER-stress–induced apoptosis, in mammalian and in S. pombe
encodes several homologues of proteins characterized for their implication in apoptosis including the thus far only metacaspase identified Pca1p (geneID: SPCC1840.04) and a Bap31 homologue (geneID: SPAC9E9.04). Our results demonstrate that the knockout of these genes does not affect the final level of apoptosis provoked by calnexin overexpression; however, we observed a slower kinetics of induction. Likewise, induction of apoptosis by tunicamycin was not blocked in the Δpca1
strains (data not shown). Importantly, our results clearly demonstrate that another, yet uncharacterized, caspase-like activity is involved in this apoptotic process. Similarly, the homologue of Pca1p in S. cerevisiae
, Yca1p, is not required for apoptotic death induced by numerous conditions (Madeo et al., 2002
; Bettiga et al., 2004
; Fannjiang et al., 2004
; Herker et al., 2004
; Wadskog et al., 2004
; Ivanovska and Hardwick, 2005
; Reiter et al., 2005
; Liang et al., 2008
; Mazzoni and Falcone, 2008
). More recently, it was reported that Yca1p is not required for apoptosis induced by tunicamycin in S. cerevisiae
(Hauptmann and Lehle, 2008
). These observations suggest the existence of uncharacterized caspase-like activities in both, S. pombe
and S. cerevisiae
. That cell death occurs by calnexin overexpression in the Δdma1/bap31
background does not exclude the possibility that Bap31 could be part of a complex involving calnexin, but it is indicative that the role of Bap31 is not essential for apoptosis. In this vein, it was shown in mammalian cells that an uncleavable mutant of Bap31 does not abrogate the capacity to the cells to enter FAS-mediated apoptosis but it delays some cytoplasmic apoptotic events (Nguyen et al., 2000
The significant reduction in the levels of tunicamycin-induced apoptosis observed with the lumenal_cnx1
mutant could be due to a diminution or a loss of effective interactions with lethal partners as observed for mammalian cells (Zuppini et al., 2002
; Delom et al.
, 2006; Groenendyk et al., 2006
; Delom et al., 2007
). Ire1p could be taking part of this interaction since its presence is required to attain maximum apoptosis induction by calnexin overexpression. Further experiments are required to dissect the molecular interaction of calnexin with Ire1p and other lethal partners. Such interactions probably require the anchoring of calnexin to the ER membrane to be optimal in transducing the death signal. Interestingly, the lumenalTM_cnx1
strain exhibited the highest levels of apoptosis induced by tunicamycin. These, and our results obtained by overexpression experiments implicate lumenal sequences of calnexin in apoptosis. However, it appears that the chaperone function of calnexin is distinct from its role in apoptosis since the mutant lumenal_Cnx1p is a very effective chaperone (Marechal et al., 2004
) but a poor effector of tunicamycin-induced apoptosis. Further supporting this point, mini_Cnx1p does not exhibit chaperone activity (Marechal et al., 2004
) but is highly efficient in inducing apoptosis by overexpression, and mini_cnx1
cells display levels of tunicamycin-induced apoptosis as the wild-type strain.
BiP anchoring to the ER membrane was reported in mammalian cells subjected to ER stress (Rao et al., 2002
; Reddy et al., 2003
). We have previously reported that the last 52 residues of the lumenal portion of calnexin are required for viability and do interact with ER chaperone BiP (Jannatipour et al., 1998
; Beaulieu et al., 1999
; Elagoz et al., 1999
; Marechal et al., 2004
). Because BiP regulates the ER stress receptors IRE1, PERK, and ATF6, it is therefore tempting to speculate that BiP could also play a regulatory role in the proapoptotic function of calnexin (Patil and Walter, 2001
; Szegezdi et al., 2006
; Ni and Lee, 2007
; Ron and Walter, 2007
). Our coimmunoprecipitation experiments showed that tunicamycin treatment enhances Cnx1p-BiP interaction in both the wild-type calnexin and the mutant lumenal_Cnx1p. However, apoptosis is significantly reduced in the case of the lumenal_Cnx1p mutant. These observations raise the possibility that stronger interaction between Cnx1p and BiP is required for ER-stress–induced apoptosis and that this complex needs to be anchored to the ER membrane to efficiently transduce the apoptotic signal. In such scenario, the diminution of tunicamycin-induced apoptosis in lumenal_Cnx1p cells could be due to a mislocalization of the Cnx1p-BiP complex, because this complex is not localized to the ER membrane in the case of this mutant.
In a genetic screen designed to isolate suppressors of lethality caused by calnexin overexpression we identified Hmg1/2p, the S. pombe
homologue of HMGB1. The HMGB1 protein is part of the HMG family that interacts with DNA for replication, transcription, and DNA repair and is a protein overexpressed in human breast carcinoma (Brezniceanu et al., 2003
; Ulloa and Messmer, 2006
). Importantly, HMGB1 is an antiapoptotic protein that was identified as an inhibitor of Bak-mediated cell death in mammalian cells and in S. pombe
(Brezniceanu et al., 2003
). In this work we showed that like its mammalian counterpart, Hmg1/2p inhibits the ER-derived apoptotic pathway involving calnexin. Because Bak-induced apoptotic death in S. pombe
is dependent on calnexin (Torgler et al., 1997
) and is inhibited by HMGB1 and because Hmg1/2 represses death by overexpression of calnexin in S. pombe
, we hypothesize that the apoptotic pathway involving calnexin are similar in S. pombe
and in mammalian cells. Although no sequence homologue of Bak was described in the fission yeast, it is likely that some S. pombe
protein plays an analogous function to that of mammalian Bak. The discovery of this lethal partner will be important for the elucidation of the apoptotic processes caused by ER stress in S. pombe
and are likely to contribute to our understanding of the mechanism of apoptosis in mammals.
Human calnexin and S. pombe
calnexin are very similar in their structure (Jannatipour and Rokeach, 1995
). Here we report that like S. pombe
calnexin, the overexpression of its human orthologue in the fission yeast induces cell death with typical apoptotic features. These results indicate that human calnexin interacts with S. pombe
apoptotic machinery and overrides the mechanisms that regulate apoptosis involving endogenous calnexin. Collectively, these observations argue for the conservation between the mammalian and the S. pombe
mechanisms of apoptosis involving calnexin and validate S. pombe
as a model organism.
Most of our current knowledge on the apoptotic response due to ER stress derives from studies with mammalian cells (Breckenridge et al., 2003
; Xu et al., 2005
; Kim et al., 2006
; Szegezdi et al., 2006
). Evidence accumulated in the last 10 years has proven that yeasts are interesting models to investigate the mechanism of apoptosis (Ink et al., 1997
; Madeo et al., 1997
; Ligr et al., 1998
; Madeo et al., 1999
; Madeo et al., 2002
; Priault et al., 2003
; Hardwick and Cheng, 2004
; Madeo et al., 2004
; Rodriguez-Menocal and D'Urso, 2004
; Burhans and Weinberger, 2007
; Frohlich et al., 2007
; Almeida et al., 2008
). Although human and S. pombe
calnexin share significant sequence similarity (Jannatipour and Rokeach, 1995
), the S. cerevisiae
molecule (Cne1p; Parlati et al., 1995b
) is the most distant known homologue. Thus, the study of the role of calnexin in the mechanisms of ER-stress apoptosis in S. pombe
should bring crucial information regarding this cellular process in mammals.