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Endoplasmic reticulum (ER) stress-induced apoptosis may arise from multiple environmental and pharmacological causes, but the precise mechanism(s) involved are not completely known. Members of Bcl-2 protein family are important regulators of apoptosis. In this study, we report that in a process dependent on the proapoptotic Bcl-2 members Bax and Bak, exogenously expressed fluorescent protein localized to the ER lumen is released into the cytosol in cells undergoing ER stress. Upon ER stress induction, endogenous ER luminal proteins are also released into the cytosol in a similar manner accompanied by translocation and anchorage of Bax to the ER membrane. In addition, Bax and truncated-Bid (tBid) mediate a global increase in ER membrane permeability to ER luminal proteins in vitro. Importantly, antiapoptotic Bcl-XL antagonizes the effects of proapoptotic Bcl-2 proteins on ER membrane permeability. Consistent with Bax translocation to the ER membrane in whole apoptotic cells, there is also increased tight association of Bax with the ER membrane correlated with the increase in ER membrane permeability in vitro. Overall, these data suggest that the regulation of ER membrane permeability by Bcl-2 proteins could be an important molecular mechanism of ER stress-induced apoptosis.
Bcl-2 proteins are among the major regulators of apoptotic signaling.1, 2, 3, 4 The Bcl-2 protein family includes both antiapoptotic and proapoptotic members that share one or more homologous domains, named the Bcl-2 homology (BH) domains. There are two groups of proapoptotic Bcl-2 proteins: multiple BH domain proteins and BH3 domain-only proteins. Multiple BH domain members Bak and Bax are redundant promoters of cell death and either Bak or Bax is sufficient to trigger apoptosis under apoptotic conditions.5, 6 BH3-only Bcl-2 proteins are usually kept inactive by different mechanisms. In response to various death stimuli, BH3-only proteins are activated and function as effectors of apoptotic signaling pathways.1, 2 Bcl-2 proteins differentially interact with each other and with other regulators of apoptotic signaling, thus profoundly influencing the regulation of survival and death.
Bcl-2 proteins have been shown to localize to the mitochondria, the endoplasmic reticulum (ER), and the nuclear envelope.7 Most studies have focused on how Bcl-2 proteins function on mitochondria. A common theme is that permeabilization of the mitochondrial outer membrane (MOM) causes the release of apoptogenic factors from mitochondria into the cytosol.2, 4, 8 These apoptogenic factors subsequently activate a class of aspartate-directed cysteine proteases (caspases), which exist in healthy cells as inactive proenzymes and are activated through a cascade of caspase cleavages.9 It is generally believed that antiapoptotic Bcl-2 family members help to maintain MOM integrity during apoptosis, whereas proapoptotic Bcl-2 proteins facilitate its disintegration.2, 4
A large body of evidence indicates that Bcl-2 proteins can sense damage to the ER and either transmit death signals to mitochondria or directly function at the ER level to regulate apoptosis.10, 11 Recent studies have shown that ER membrane permeability to Ca2+ is altered by activation of the proapoptotic Bcl-2 proteins in cells undergoing apoptosis.12, 13, 14, 15 However, it is not known whether ER membrane permeability to luminal proteins is altered in apoptotic cells. In this study, we explored the role of Bcl-2 proteins in the regulation of ER membrane permeability to luminal proteins. We observed that ER luminal proteins were released into the cytosol during ER stress-induced apoptosis in a Bak/Bax-dependent manner. Furthermore, Bcl-2 proteins were also able to regulate ER membrane permeability in vitro in a manner similar to their activities on the MOM. Our findings provide evidence that Bcl-2 proteins regulate ER membrane permeability to luminal proteins during apoptosis.
To monitor ER membrane permeability in cells undergoing apoptosis, time-lapse confocal microscopy was used to image the localization of an ER luminal marker in single living cells. Wild-type mouse embryonic fibroblast (MEF) cells were transiently transfected with a plasmid encoding yellow fluorescence protein targeted to the ER lumen (ER-YFP). Cells were treated with ER stress inducers thapsigargin (an inhibitor of sarco/ER Ca2+ ATPase) or tunicamycin (an inhibitor of N-linked glycosylation) and an image field was acquired over the course of 10h (Figure 1a; Supplementary Movies 1 and 2). Before thapsigargin or tunicamycin application, the cellular fluorescence distribution was punctate and reticular, consistent with ER luminal localization.16 Addition of thapsigargin or tunicamycin induced a redistribution of YFP in about 40 and 50% of cells, respectively (Figure 1c), from an ER localization to an evenly dispersed cytoplasmic and nuclear distribution (Figure 1a). In addition, both reagents were able to release YFP with identical kinetics (Figure 1d). The release of ER-YFP is unlikely to be the result of simple ER membrane destruction, as wild-type MEFs treated with thapsigargin or tunicamycin for 10h had similar ER membrane topology to untreated cells when examined using electron microscopy (Supplementary Figure 1). When experiments were repeated using Bak−/−Bax−/− MEF cells, in contrast to wild-type cells, redistribution of ER-YFP occurred in less than 5% of treated cells (Figure 1b and c; Supplementary Movies 3 and 4). Taken together, these observations are consistent with the hypothesis that Bak/Bax-dependent changes in ER permeability during ER stress can facilitate the release of high molecular weight ER luminal proteins.
These findings raise the possibility that such a mechanism may not be limited to ER stress, but represent a common feature of multiple apoptotic paradigms. To explore this prospect further, the effect of the genotoxic reagent actinomycin D on ER membrane permeability was tested on wild-type MEF cells expressing ER-YFP (Figure 1e; Supplementary Movie 5). Actinomycin D induced shrinkage and detachment of all cells within the 10h recording period. No redistribution of ER-localized YFP was observed during this time, with the YFP being retained within highly localized puncta until cell detachment. These data suggest that early, Bak/Bax-dependent alterations in ER membrane permeability may be an exclusive feature of ER stress-induced apoptosis.
The role of Bak/Bax in MOM permeabilization and the release of cytochrome c are well established for many apoptotic paradigms.2, 4 The requirement for Bak/Bax to increase ER permeability during ER stress could either be because of the direct actions of Bak/Bax at the membrane or reflect signaling events secondary to Bak/Bax-dependent effects at mitochondria. If the former possibility was correct, then ER and mitochondrial membrane disruption should occur simultaneously, whereas MOM permeabilization would be expected to occur upstream of any ER-localized event in the latter case. To discriminate between these possibilities, the time course of mitochondrial membrane potential changes and ER-YFP release were monitored simultaneously during thapsigargin exposure. Mitochondrial membrane potential dissipates during apoptosis as a direct result of increased MOM permeability and has been widely used as an index of cytochrome c release.17, 18, 19 Wild-type MEF cells expressing ER-YFP were loaded with the mitochondrial membrane potential probe, tetramethylrhodamine, ethyl ester (TMRE), and with cells treated with thapsigargin (Figure 2a). The time-lapse data show that mitochondrial membrane potential dissipation is initiated in parallel with the redistribution of ER-localized YFP (Figure 2b), showing that disruption of both mitochondrial and ER membrane integrity occurs concomitantly. This is consistent with the hypothesis that Bak/Bax activation alone is sufficient to trigger ER membrane disruption, which is unlikely to be downstream of MOM permeabilization.
To determine whether ER membrane permeability to endogenous ER luminal proteins is also altered on ER stress, both wild-type and Bak−/−Bax−/− MEF cells were treated with thapsigargin, and the presence of the ER luminal proteins protein disulfide isomerase (PDI) and 78-kDa glucose-regulated protein (GRP78/BiP) in cytosolic fractions was determined by western blotting (Figure 3a). The levels of PDI and GRP78/BiP in cytosolic fractions of Bak−/−Bax−/− cells were unchanged upon thapsigargin treatment. However, the amounts of PDI and GRP78/BiP were notably higher in cytosolic fractions of thapsigargin-treated wild-type cells than those of untreated cells, consistent with previously observed redistribution of ER-localized YFP in cells under ER stress (Figure 1). The absence of ER membrane protein calnexin indicated that cytosolic fractions were free of ER/microsomal contamination. Similar results were obtained with interleukin 3 (IL-3)-dependent hematopoietic cells, suggesting that increased ER membrane permeability in response to ER stress is independent of cell lineage (Supplementary Figure 2). Importantly, when wild-type MEF cells were treated with thapsigargin, tunicamycin, actinomycin D, or staurosporine at the time points in which the levels of cell death were similar (Figure 3b), PDI and GRP78/Bip release was observed in cells treated with thapsigargin or tunicamycin, but not in those treated with actinomycin D or staurosporine (Figure 3c), providing further evidence that alterations in ER membrane permeability occur only in ER stress-induced apoptosis. To determine whether ER luminal proteins are released into the cytosol as monomeric proteins or in macromolecular complexes, we loaded cytosolic fractions of thapsigargin-treated wild-type MEF cells onto a FPLC Superose 6 gel filtration column (GE Healthcare, Piscataway, NJ, USA). PDI and GRP78/Bip were exclusively detected in late elution fractions containing molecules whose molecular weights are within the same range of those of PDI and GRP78/Bip, indicating that released ER luminal proteins were not in macromolecular complexes, such as lipid vesicles (Figure 3d). Overall, these data suggest that ER membrane permeability to endogenous luminal proteins is altered during ER stress-induced cell death, which depends on the presence of Bax and Bak.
As Bax translocation to mitochondria has been shown to be critical to MOM permeabilization,4, 20 we explored the role of Bax translocation to the ER membrane in ER membrane permeabilization. We carried out subcellular fractionation of both wild-type MEF cells (Figure 4a) and IL-3-dependent cells reexpressing Bak and Bax (Figure 4b) under ER stress. ER/microsomal fractions were not contaminated with mitochondria, as indicated by the absence of Tom20. In agreement with previous reports,13, 21 a modest amount of Bax was detected on the ER membrane in untreated cells. However, the levels of Bax in ER/microsomal fractions of cells under ER stress were higher than those of untreated cells. This trend was also observed when ER/microsomal fractions were incubated with 100mM Na2CO3 (pH 11.3) to remove peripheral ER membrane proteins,22 suggesting that translocated Bax was tightly anchored to the ER membrane. Furthermore, Bax oligomerization on the ER membrane in IL-3-dependent cells was also increased by thapsigargin, indicated by the increased intensity of slowly migrating bands recognized by Bax antibodies after treatment with the chemical cross-linking agent bismaleimidohexane (BMH) (Supplementary Figure 3). These results reveal a correlation between Bax translocation to the ER membrane and the increase in ER membrane permeability.
To investigate the direct effects of Bcl-2 proteins on ER membrane permeability to luminal proteins, we developed a reconstituted in vitro assay to recapitulate the alterations in ER membrane permeabilization observed in intact cells under ER stress. Studies were focused on Bax and an active form of the BH3-only protein Bid, truncated-Bid (tBid), which has been shown to translocate to the ER and mediate ER stress-induced apoptosis.23, 24 As the ER luminal protein PDI was released during ER stress-induced cell death (Figure 3a), we investigated the permeability of ER/microsomal vesicles to PDI as an index of ER membrane permeabilization in vitro. ER/microsomal vesicles were purified from Bak−/−Bax−/− IL-3-dependent cells or MEF cells and incubated with Bax alone, tBid alone or with both at 37°C. After ultracentrifugation, the amount of PDI in both the supernatant (released fraction) and the pellet (ER vesicle membrane fraction) was detected by western blotting (Figure 5). Bax or tBid alone did not enhance PDI release. However, combining Bax and tBid led to a considerable increase in PDI in the supernatant fraction and a decrease in PDI in the vesicle membrane fraction, indicating an increase in ER vesicle membrane permeability. Furthermore, the ER membrane protein, calnexin, was retained in the membrane fraction, suggesting that the increased ER membrane permeability did not lead to the overall destruction of ER membrane structure.
To further characterize the increase in ER membrane permeability mediated by proapoptotic Bcl-2 proteins in vitro, we examined whether ER luminal proteins other than PDI were also released. ER/microsomal vesicles isolated from Bak−/−Bax−/− IL-3-dependent cells were incubated with Bax and different dosages of tBid (Figure 6). As the tBid concentration increased, more PDI was released from microsomal vesicles. Two other ER luminal proteins, ERp29 and GRP78/BiP, were also detected in the released fractions. Importantly, tBid was equally effective in releasing all three ER luminal proteins in the presence of Bax, suggesting that the permeation channels formed by Bax and tBid on the ER membrane are nonselective (Figure 6b). This observation is consistent with the reported Bax/tBid-mediated supramolecular permeation channels formed in the MOM,25 suggesting that proapoptotic Bcl-2 proteins mediate ER membrane permeabilization in a manner similar to their activities on the MOM.
In addition to Bid, several other BH3-only Bcl-2 proteins have also been implicated in ER stress-induced apoptosis.26, 27, 28 As BH3 regions of BH3-only proteins have been shown to differentially regulate Bax-mediated MOM permeabilization,29, 30 we sought to study the ability of various BH3-only Bcl-2 proteins to modulate Bax-mediated ER membrane permeabilization. ER/microsomal vesicles isolated from Bak−/−Bax−/− IL-3-dependent cells were incubated with Bax and various BH3 peptides. ER membrane permeability assays were carried out (Supplementary Figure 4). Although some BH3 peptides (Bid, Bim, and Bmf) were able to function with Bax to release ER luminal proteins, others (Bnip3, Bik, Hrk, and Bad) failed to permeabilize the ER membrane in the presence of Bax (Table 1). The effects of BH3 peptides on Bax-dependent ER membrane permeabilization are largely consistent with their activities in permeabilizing the MOM,29, 30 suggesting that BH3 peptides derived from BH3-only members function through a similar mechanism to activate Bax on the ER membrane and the MOM.
Next, we investigated whether antiapoptotic Bcl-XL was able to maintain ER membrane permeability in vitro under apoptotic conditions. ER membrane permeability assays were carried out using ER/microsomal vesicles isolated from Bak−/−Bax−/− IL-3-dependent cells or Bak−/−Bax−/− MEFs (Figure 7). The addition of Bcl-XL alone had a minimal effect on the release of PDI. In the presence of both Bax and tBid, Bcl-XL was able to prevent PDI release, reminiscent of its ability to maintain MOM integrity under similar conditions.20, 30 Our data suggest that Bcl-XL maintains ER membrane permeability to ER luminal proteins in a manner similar to its activity on MOM permeability.
As Bax translocation to the ER membrane correlated with increased ER membrane permeability in cells under ER stress (Figure 4), we examined whether it could be recapitulated in vitro. ER/microsomal vesicles isolated from IL-3-dependent Bak−/−Bax−/− cells were first incubated with various combinations of Bcl-2 proteins (Figure 8a–c). Then microsomal vesicles were treated with 100mM Na2CO3 to remove proteins loosely attached to vesicles. The presence of Bax, PDI, GRP78/BiP, calnexin, and Tom20 was analyzed by western blotting. Tom20 was not detected in ER vesicle samples, indicating that ER vesicles had little mitochondria contamination. There was some Bax tightly attached to the ER membrane when ER vesicles were incubated with Bax alone, which could be caused by the precipitation of Bax protein after ultracentrifugation, as reported previously.30 Nevertheless, the basal amount of Bax on the ER membrane was not sufficient to mediate ER membrane permeabilization. The addition of tBid promoted the release of PDI and GRP78/BiP from vesicles and increased Bax association with the ER membrane, which is consistent with Bax translocation to the ER membrane in cells undergoing apoptosis (Figure 4). Although the addition of Bcl-XL reversed PDI and GRP78/BiP release caused by Bax and tBid, it did not affect the amount of Bax in the ER membrane. One possible scenario is that Bcl-XL interferes with a late step of ER membrane permeabilization, but not with the initial insertion of Bax into the ER membrane. Comparable results were also obtained using ER/microsomal vesicles isolated from Bak−/−Bax−/− MEFs (Figure 8d, e and f), providing more evidence to the hypothesis that the activities of Bcl-2 proteins on the ER membrane are cell lineage-independent.
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+ ionophore treatment.31 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−/−Bax−/− cells,21 but here, it only affected ER protein localization in wild-type cells (Figure 1). 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 (Figure 1). 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 40min 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 release.32 Our data provide evidence that the initiation of increase in ER membrane permeability and mitochondrial membrane potential dissipation occurs concomitantly (Figure 2), 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 40min, it took more than 120min 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.
Immortalized IL-3-dependent Bak−/−Bax−/− hematopoietic cells were cultured as described previously.37 Wild-type murine Bak and Bax cDNAs were reexpressed in IL-3-dependent Bak−/−Bax−/− cells by retroviral infection and stable clones expressing Bak and Bax were selected as described previously.37 Wild-type and Bak−/−Bax−/− MEF cells were cultured as described previously.13 Antibodies used were anti-actin mAb (Sigma, St. Louis, MO, USA), anti-Bax pAb (Santa Cruz, Santa Cruz, CA, USA), anti-Calnexin pAb (Assay Designs, Ann Arbor, MI, USA), anti-cytochrome c mAb (Becton Dickinson, Franklin Lakes, NJ, USA), anti-ERp29 pAb (Affinity BioReagents, Rockford, IL, USA), anti-GRP78/BiP mAb (Assay Designs), anti-PDI pAb (Becton Dickinson), anti-Tom20 pAb (western blotting; Santa Cruz), Alexafluor 488 Goat anti-mouse (Molecular Probes, Eugene, OR, USA), and Alexafluor 594 Goat anti-rabbit (Molecular Probes). Recombinant Bcl-2 proteins (hBcl-XL, htBid, and hBax) were acquired as described previously.38, 39 All BH3 peptides were purchased from AnaSpec (San Jose, CA, USA) with over 95% purity and dissolved in solvents recommended by the manufacturer at the concentration of 5mM.
Cells were transiently transfected with the plasmid pEYFP-ER (Clontech, Mountain View, CA, USA) using an Amaxa nucleofector device (Lonza, Allendale, NJ, USA) following the manufacturer's instructions and seeded into glass-coverslip-bottomed 35mm dishes (WPI Inc, Sarasota, FL, USA). pEYFP-ER is constructed with a calreticulin ER-targeting sequence at its N-terminus and the ER-retention sequence, KDEL, at its C-terminus. After 24h in culture, the dishes were placed into a miniature tissue culture incubator (DH-40i; Warner Instruments, Hamden, CT, USA) mounted onto an Olympus IX71 inverted microscope (Olympus, Center Valley, PA, USA). Individual cells were visualized with a × 60 oil immersion objective and confocal images acquired using a VT-Infinity 3 scanning unit (VisiTech, Alexandria, VA, USA). A motorized stage enabled the capture of multiple image fields from the same dish. Cells were treated with apoptotic stimuli and confocal images acquired for each field at 10min intervals for up to 12h. Images were analyzed using Simple PCI 6 (Hamamatsu, Billerica, MA, USA) and the time course of YFP release from the ER quantified by calculating the punctate/diffuse index, originally described by Douglas Green's group, and defined as the standard deviation (S.D.) of the mean pixel intensity of each cell.18, 40 Briefly, when YFP fluorescence is highly localized within the ER compartment, cells have a punctate staining pattern, thus the S.D. of the average pixel intensity will be high. In contrast, when YFP is distributed throughout the cell, the S.D. will be low. Thus, a change in the S.D. (punctate/diffuse index) from high to low is an index of YFP redistribution from the ER to the cytoplasm. To simultaneously image ER and mitochondria, ER-YFP-transfected cells were loaded with the mitochondrial membrane potential indicator TMRE (50nM for 20min at 37°C; Invitrogen, Carlsbad, CA, USA).
MEF cells were fixed with 3% glutaraldehyde in 0.1M sodium cacodylate at 4°C overnight. Before embedding, cells were treated with 2% osmium tetroxide followed by an increasing gradient dehydration step using ethanol and propylene oxide. Cells were then embedded in LX-112 epoxy plastic (Ladd Research, Williston, VT, USA). Ultrathin sections of 80nm were cut, placed on uncoated copper grids and stained with lead citrate and saturated aqueous uranyl acetate. Images were examined with a Philips CM12 transmission electron microscope at 80kV (Philips, Andover, MA, USA).
A FPLC Superose 6 10/30 gel filtration column (GE Healthcare) was equilibrated using the equilibrium buffer (20mM Tris-HCl (pH7.4) and 500mM KCl) on a Biologic DuoFlow chromatography system (Bio-Rad, Hercules, CA, USA). The Superose 6 column was calibrated using gel filtration standards (Bio-Rad). Cytosolic fractions of MEF cells treated with thapsigargin were dialyzed against the equilibrium buffer and very little protein precipitation was detected after the dialysis. Samples (250μl) were loaded onto the equilibrated Superose 6 column. Fractions were eluted at a rate of 0.4ml/min in 1ml fractions and 23 fractions were collected. A volume of 10.5μl of each fraction was loaded on a 4–12% NuPAGE gradient gel (Invitrogen). The amounts of ER luminal proteins were detected by western blotting.
MEF cells were collected and washed with 1 × PBS, and resuspended in MS buffer (5mM Tris-HCl (pH 7.5), 210mM mannitol, 70mM sucrose, and 1mM EDTA) supplemented with protease inhibitors (Complete, Roche Diagnostics, Indianapolis, IN, USA). The cells were broken open by passing them through a ball-bearing homogenizer. The whole cell lysate was centrifuged at 80 × g using a Sorvall Legend RT centrifuge (Thermo Scientific) for 10min at 4°C to eliminate large cellular debris and nuclei. An aliquot of each supernatant was reserved as whole cell lysate. The remaining supernatants were centrifuged in a TLA 100.2 rotor using a TL-100 tabletop ultracentrifuge (Beckman, Fullerton, CA, USA) at 10000 × g for 25min at 4°C and subsequently at 350000 × g for 20min at 4°C to obtain the cytosolic supernatant fractions. Equivalent total protein amounts of whole cell lysates and corresponding volume equivalents of cytosolic fractions were electrophoresed in a 4–12% NuPAGE gel (Invitrogen). The presence of ER proteins was detected by western blotting.
MEFs or IL3-dependent cells were collected and broken open as described in the ‘Detection of ER luminal protein release' section. The whole cell lysate was centrifuged at 80 × g for 10min at 4°C and the supernatants were centrifuged at 10000 × g for 20min at 4°C. The supernatants were then centrifuged at 350000 × g for 20min at 4°C and ER/microsomal vesicle pellets were resuspended in either MS buffer or 100mM Na2CO3 (pH 11.3). After incubation on ice for 30min, samples were centrifuged at 350000 × g for 20min and resuspended in MS buffer. The protein concentrations of purified ER/microsomal vesicles were measured by Bradford assay (Bio-Rad). Equal amounts of ER vesicles (10μg) were loaded in a 4–12% NuPAGE gradient gel (Invitrogen) and Bax was detected by western blotting. For cross-linking experiments, 0.1mM BMH was added to whole cell lysate and incubated at 25°C for 30min. Then, the ER fractions were obtained from the cell lysate as described earlier. A measure of 10μg of total protein was separated on a 4–12% gradient NuPAGE gel. A polyclonal anti-Bax antibody was used to detect Bax.
Cells were collected and broken open as described in the ‘Detection of ER luminal protein release' section, except that the supernatants were centrifuged twice at 900 × g for 10min at 4°C after large cellular debris and nuclei were removed. The supernatants were again centrifuged at 350000 × g for 20min at 4°C and ER/microsomal vesicles were obtained by resuspending pellets in MS buffer. The protein concentrations of purified ER vesicles were measured by Bradford assay. Purified ER vesicles (0.5μg/μl) were incubated with different combinations of recombinant Bcl-2 proteins in buffer containing 12mM HEPES (pH 7.5), 1.7mM Tris-HCl, (pH 7.5), 100mM KCl, 140mM mannitol, 23mM sucrose, 2mM KH2PO4, 1mM MgCl2, 0.67mM EGTA and 0.6mM EDTA. After 37°C incubation for 60min (vesicles isolated from IL-3-dependent cells) or 30min (vesicles isolated from MEFs), the samples were centrifuged at 350000 × g for 20min. The supernatants (released fractions) and the pellets (ER/microsomal vesicle fractions) were separated and resuspended in equal volumes of 1 × LDS sample buffer (Invitrogen). The presence of ER luminal proteins was detected by western blotting. For alkali treatment, after incubation with various Bcl-2 proteins, ER/microsomal vesicles were resuspended in 100μl of 100mM Na2CO3 (pH 11.3) and put on ice for 30min. Treated ER/microsomal vesicles were centrifuged at 350000 × g for 20min and vesicles were dissolved in 1 × LDS sample buffer (Invitrogen). Proteins in the vesicle and supernatant fractions were detected by western blotting. The protein intensity was determined using ImageJ software (NIH, Bethesda, MA, USA). To normalize sample-loading variation, the relative levels of PDI were first calculated by dividing the PDI value into the corresponding value for calnexin. The normalized values of PDI levels were then calculated as a percentage of relative PDI in the control sample (buffer incubation only). Similarly, the relative levels of Bax were first measured as described for PDI, and then normalized as a percentage of the relative Bax level in the sample incubated with Bax only.
We are grateful to Drs John Eaton and Brian Wattenberg for advice. We also thank the Calcium Imaging Research Support Laboratory, Department of Physiology & Biophysics, Rosalind Franklin University. This work was supported by Schweppe Foundation and Rosalind Franklin University (CW) and NIH grants CA106599 and RR018733 and funding from JG Brown Cancer Center (CL).
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd)
Edited by C Borner