ER stress-induced apoptosis is a well-described phenomenon, but the precise mechanisms through which ER stress is coupled with ultimate cell death are not well understood. In this study, we investigated the role of Bcl-2 proteins in modulating ER membrane permeability to luminal proteins during apoptosis. To monitor the distribution of exogenously expressed YFP targeted to the ER lumen in asynchronized cell populations, we used a time-lapse confocal microscope approach. ER stress inducers, but not the genotoxic reagent actinomycin D, induced the release of ER-YFP in a Bak/Bax-dependent manner. Similarly, endogenous ER luminal proteins seemed to be released into the cytosol on ER stress treatment. Furthermore, the flux of ER luminal proteins likely occurred independently of MOM permeabilization. In vitro ER membrane permeability assays showed that proapoptotic Bcl-2 proteins induced ER membrane permeabilization to luminal proteins, whereas antiapoptotic Bcl-XL inhibited the release. The increased ER membrane permeability mediated by proapoptotic Bcl-2 proteins seemed to be nonselective to luminal proteins, suggesting that Bcl-2 proteins regulate ER membrane permeability in vitro in a manner similar to their activities on the MOM. Together, our studies provide molecular evidence that Bcl-2 proteins regulate ER membrane permeability during ER stress-induced apoptosis.
Although MOM permeabilization has been well studied, little is known about the modulation of ER membrane permeability to luminal proteins during apoptosis. The redistribution of ER luminal proteins from the ER to the Golgi apparatus has been observed previously during ER stress induced by Ca2+
However, this process is likely to be different from Bcl-2 protein-mediated ER luminal protein release described here. First, the changes in luminal protein distribution originally reported were associated with ER Ca2+
store depletion, as tunicamycin failed to induce the translocation. In the present experiments, luminal protein release occurred independently of changes in ER Ca2+
concentration, as thapsigargin is known to be equally effective in depleting ER Ca2+
store in both wild-type and Bak−/−
but here, it only affected ER protein localization in wild-type cells (). Second, ER-YFP proteins released from the ER lumen are evenly distributed in the cytosol and nuclei without obvious Golgi localization (Supplementary Movies 1 and 2). Finally, Ca2+
ionophore-induced ER luminal protein depletion was not detected in SV40-transformed fibroblasts, whereas ER luminal protein release mediated by proapoptotic Bcl-2 proteins occurred in SV40-transformed MEFs. Thus, the luminal protein release described here is likely different from Ca2+
ionophore-induced luminal protein secretion reported previously.
Live cell imaging of cells undergoing apoptosis revealed that the kinetics of exogenously expressed ER-YFP release was almost identical in cells treated with two different ER stress inducers (). As expected, the increase in ER membrane permeability to luminal proteins was dependent on Bak and Bax. The majority of ER-YFP release occurred within 40
min in MEF cells, suggesting that a secondary event, as proposed to be involved in the release of AIF from mitochondria, is not required.18
The rate of ER-YFP release was slower than the previously reported release of several apoptogenic proteins from mitochondria in HeLa cells undergoing apoptosis.17, 18
It has been proposed that the speed of proapoptotic Bcl-2 protein-mediated formation of permeation channels on the MOM determines the release of mitochondrial protein.18
Because previous studies indicate that only about 10% of Bax and 15% of Bak are localized on the ER membrane,13, 21
it is conceivable that the relatively slower release of ER luminal proteins could be attributed to lower concentrations of Bak or Bax proteins on the ER membrane. In addition, lipid components of intracellular organelle membrane could also influence the speed of permeation channel formation on the particular organelle membrane.25
MOM permeabilization has been proposed to be a committed no-return point for apoptosis.4
However, irreversible damage to the membrane of other intracellular organelles independently of MOM permeabilization could also represent a no-return point for cell death. One example is oxidative stress-induced lysosome rupture and subsequent release of lysosomal enzymes, which in turn directly impinge on mitochondria, resulting in cytochrome c
Our data provide evidence that the initiation of increase in ER membrane permeability and mitochondrial membrane potential dissipation occurs concomitantly (), implying that ER membrane permeability alterations could also serve as a committed no-return point for ER stress-induced apoptosis. Interestingly, the kinetics of ER-YFP redistribution and mitochondrial membrane potential dissipation in thapsigargin-treated MEFs was significantly different. Although the redistribution of most ER-YFP proteins occurred within 40
min, it took more than 120
min for the complete dissipation of mitochondrial membrane potential, suggesting that mitochondria lose their function more slowly in thapsigargin-treated MEF cells.
Currently, the exact role of ER stress-induced increase in ER membrane permeability to luminal proteins in apoptotic signaling remains unknown. Although unlikely, it is possible that ER stress renders the lipid environment of the ER membrane more susceptible to Bax insertion and/or Bak activation, resulting in ER membrane permeabilization irrelevant to apoptotic signaling. Many ER luminal proteins function as chaperones to facilitate appropriate protein folding and are generally considered to be prosurvival by assisting cells to adapt to unfolded protein response induced by ER stress.33
However, recent studies have shown that ER luminal chaperones localized outside the ER lumen exhibit unique proapoptotic activities in various apoptotic signaling pathways. For instance, the ER chaperone calreticulin expressed on the cell surface is a general recognition ligand to initiate clearance of dying cells through phagocytosis.34
Moreover, rapid translocation of calreticulin to the plasma membrane determines the cancer cell immunogenicity during apoptosis.35
Another ER luminal chaperone possessing proapoptotic activities is GRP78/BiP. In cells under ER stress induced by extracellular apoptotic stimuli, GRP78/BiP is translocated to the plasma membrane, where it serves as a cell surface receptor for the proapoptotic protein Par-4 to activate FADD/caspase8/caspase3 apoptotic signaling pathway.36
It is conceivable that certain luminal chaperones released from the ER lumen during ER stress could translocate to the cell surface and function in a variety of ways to facilitate apoptotic signaling cascade. Taken together, we suggest that the regulation of ER membrane permeability to luminal proteins by Bcl-2 proteins may be involved in ER stress-induced apoptosis.