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Vol. 20, Issue 8, 2276-2285, April 15, 2009
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*Department of Pathology, University of Iowa Carver College of Medicine, Iowa City, IA 52242 National Resource for Automated Molecular Microscopy, Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037 Division of Biochemistry, Department of Medicine, University of Fribourg, Fribourg CH1700, Switzerland Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105
Submitted October 22, 2008;
Revised February 11, 2009;
Accepted February 13, 2009
Monitoring Editor: Thomas D. Fox
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Kuwana and Newmeyer, 2003; Youle and Strasser, 2008). They share multiple BH (Bcl-2 homology) domains (BH1–3) and regulate apoptosis predominantly by controlling the permeability of the MOM. Proapoptotic Bcl-2 family members promote permeabilization, which leads to the release of apoptogenic proteins, including cytochrome c (Liu et al., 1996), SMAC/DIABLO (Du et al., 2000; Verhagen et al., 2000), and Omi/HtrA (Suzuki et al., 2001), from the intermembrane space. Antiapoptotic family members inhibit this process. Proapoptotic multidomain Bcl-2 family members Bax and Bak are essential effectors of mitochondrial outer membrane permeabilization (MOMP) (Lindsten et al., 2000; Wei et al., 2001), and when they are activated, they change the conformation and oligomerize in the membrane to promote MOMP (Nechushtan et al., 1999, 2001; Antonsson et al., 2001; Kuwana et al., 2002).
The molecular mechanisms that underlie MOMP have been a subject of intensive study, because this step is deemed to have high potential for therapeutic intervention in cancer (Lowe et al., 2004; Brown and Attardi, 2005; Fesik, 2005; Green and Kroemer, 2005). Once MOMP occurs, cells permanently cease to proliferate (Xiang et al., 1996; McCarthy et al., 1997; Chipuk and Green, 2005; Brown et al., 2007; Knudson and Brown, 2008), except under unusual circumstances (Colell et al., 2007). However, the molecular mechanism whereby the MOM is permeabilized upon Bax and Bak activation remains obscure. Bax-mediated membrane permeabilization has been studied in in vitro liposome systems that faithfully recapitulate some aspects of MOMP. Such studies have revealed a size-indiscriminatory release of contents upon the addition of proapoptotic Bax and caspase-8–cleaved Bid and inhibition of this release by antiapoptotic family members (Basanez et al., 2002; Kuwana et al., 2002, 2005; Roucou et al., 2002b; Yethon et al., 2003; Terrones et al., 2004). It is also established that liposomes require at least 7 mol % of cardiolipin in their membranes to undergo optimal permeabilization induced by cleaved Bid and Bax (Kuwana et al., 2002; Yethon et al., 2003; Terrones et al., 2004). The nature of this permeabilization remains unclear. Some studies suggest that it may involve the formation of proteinaceous channels by Bax molecules, either by themselves (Saito et al., 2000) or together with other MOM-resident membrane proteins, such as voltage-dependent anion channel (VDAC) (Shimizu et al., 1999, 2000). However, that double knockout mice lacking VDAC isoforms 1 and 3 do not show the apoptotic defect argues against the involvement of these proteins (Baines et al., 2007), and the other studies suggest that the pores may be lipidic in nature (Basanez et al., 2002; Hardwick and Polster, 2002; Terrones et al., 2004).
The physiological relevance of cardiolipin in MOMP, however, remains controversial. Cardioipin is suggested as a receptor for Bid in mitochondria (Lutter et al., 2000) and is shown to facilitate Bax insertion and oligomerization in liposomes (Lucken-Ardjomande et al., 2008a). In contrast, some studies conclude that cardiolipin is not required for Bax-mediated killing of yeast (Iverson et al., 2004; Polcic et al., 2005), although molecular mechanisms of yeast "death" induced by Bax may not be mediated through MOMP, or the mechanism of yeast MOMP may not be the same as that of mammalian mechanism (Roucou et al., 2002b).
Here, we report on a series of studies investigating the nature of membrane permeabilization induced by cleaved Bid and Bax by using vesicle systems. We have developed proteo-liposome systems that reproduce permeabilization induced by Bax and cleaved Bid with more physiological material. These proteo-liposomes are comprised solely of native MOM components, with cardiolipin present only in trace amounts or absent. It therefore seems that the MOM contains at least one factor other than cardiolipin that helps mediate Bid/Bax-induced membrane permeabilization. Given that this factor was not extracted by organic solvents, it is likely proteinaceous. We also found that cardiolipin dependence was only seen when Bax was activated by cleaved Bid but not by Bid or Bim BH3 peptide or oligomerized Bax. Thus, the proteinaceous factor may be a "receptor" for cleaved Bid. Finally, cryoelectron microscopy (cryo-EM) has revealed pore-like openings in cardiolipin-liposomes after their permeabilization by cleaved Bid and Bax. These data provide long-sought visual evidence consistent with a lipidic pore model, suggested previously based on biochemical observations (Basanez et al., 1999; Kuwana et al., 2002; Terrones et al., 2004). We conclude that MOM proteins assist cleaved Bid in inducing Bax-dependent lipidic pore formation in the MOM.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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130 µg of protein) were solubilized in 100 µl of KKE containing 4% OG for 1 h at 4°C with mixing, and ReOMVs were formed following the procedures as described above (Figure 2). For OmpA loading, OmpA in 2% OG-KKE (50 µg) was added to lipids (500 µg) resuspended in 2% OG-KKE, detergent-solubilized OMVs (130 µg of protein), or LMVs (130 µg of protein). The mixture was stirred for 1 h at 4°C, followed by the same procedures described above. OMVs and LMVs were isolated from mitochondria and from endoplasmic reticulum (ER) fractions of Xenopus egg extracts as reported previously (Kuwana et al., 2002).
Formation of ExOMVs
OMVs (780 µg of protein) were mixed with 1 ml of chloroform/methanol (1:1) after which 1 ml of water was added, and the samples were vortexed. The suspension was spun at 500 x g for 10 min in a tabletop centrifuge, and the organic solvent layer was discarded. One milliliter of chloroform was added to the aqueous layer, and the lipids were extracted again. The aqueous solution containing precipitated membrane proteins was mixed with 1.5 ml of pentane and dried under argon. Dried proteins were resuspended in 200 µl of 8 M urea and centrifuged at 150,000 x g for 30 min at 4°C, and the supernatant was collected. The urea solution was diluted threefold with 4% OG-KKE, and the mixture was dialyzed against 2% OG-KKE to completely remove the urea. The dialysate was centrifuged at 150,000 x g for 30 min to remove nonrenatured precipitates in the pellet. A mixture of 100 µg of phospholipids (PC:PE:PI:PS, 59:23:16:2 mol %) was dried and resuspended with the supernatant of the dialysate containing refolded membrane proteins. The lipid–protein–detergent mixture was stir-mixed for 1 h at 4°C, and detergent was removed to form ExOMVs as described above. The final pellet was resuspended in 40–80 µl of KKE and used in the dextran release assay.
OmpA Production
Escherichia coli BL21(DE3) cells were transformed with pET3b-OmpA171 and induced according to the methods described in Pautsch et al. (1999). The bacterial pellet was sonicated in 20 mM Tris, pH 8.5, and the supernatant was discarded. The pellet was resuspended in the same buffer plus 2% NP-40 and spun down to collect the inclusion bodies, which were solubilized in 6 M guanidine-HCl, 20 mM Tris, pH 8.5, in a shaker at 37°C for 2 h. Solubilized inclusion bodies were diluted fourfold with 4% OG-KKE and dialyzed in 2% OG-KKE at 4°C. The purity of the OmpA obtained was >95%, according to a Coomassie-stained gel.
Generation of Recombinant Bcl-2-Family Proteins, BH3 Peptides, Dextran Release Assays, Protein Assay, and Lipid Composition Analysis by Thin Layer Chromatography (TLC)
These experimental procedures were performed as described previously (Kuwana et al., 2002, 2005), except that, in the dextran release assays, at the end of the incubation, vesicles were pelleted at 150,000 x g for 10 min to collect the released dextrans in the supernatant. Data shown for each release assay are representative of >10 independent experiments unless otherwise stated, and all the bar graphs are presented as mean ± SEM.
Protein Identification by Mass Spectrometry
The protein bands were excised from Coomassie Blue-stained SDS-polyacrylamide gel electrophoresis (PAGE) gels, reduced, and alkylated with iodoacetamide and tryptic digest was prepared. Mass spectrometric analysis was performed using a model 4700 Proteomics Analyzer from Applied Biosystems (Foster City, CA). This instrument uses matrix-assisted laser desorption/ionization in conjunction with tandem time-of-flight mass analyzers. The digest was introduced into the instrument in a crystalline matrix of a-cyano-4-hydroxycinnamic acid containing 2 mM ammonium citrate to suppress ionization of matrix clusters. Database searches were performed with Applied Biosystem's GPS explorer software that uses the Mascot search engine. Protein assignments were made on the basis of both mass spectrometry and tandem mass spectrometry spectra. NCBInr was used for protein identification.
Mass Spectrometry for Cardiolipin Quantification
For the internal standards, 5 µg of C14 1,1',2,2'-tetramyristoyl cardiolipin (Avanti Polar Lipids) was added as an internal standard to 100 µg of OMV preparations before lipid extraction with CHCl3:MeOH:0.15 M NaCl; 4:2:1, per vol (Sorice et al., 2004). Lipids were resuspended in chlorofom:methanol:H2O (10:9:1, per vol) and analyzed in the negative ion mode on a Bruker Esquire HCT ion trap mass spectrometer electrospray ionization at a flow rate of 120 µl/h and a capillary tension of –250 V. Ion fragmentation was induced by argon as collision gas at a pressure of 8 mbar. [M-2H]2-ions of cardiolipin were quantified relative to the internal standard.
Cryo-EM
OG-LUVs were formed as described above but in the absence of fluorescein-dextrans. Typically, 2 mg of phospholipids were dried, and the final pellet was resuspended in 15 µl of KKE to obtain the high concentration that is required for visualization by cryo-EM. Nontreated OG-LUVs were mixed with 4 µM glutathione transferase to control for the presence of proteins. For Bax, tBid, and Bcl-xL treatment, OG-LUVs (6 µl) were incubated with Bax (3.8 µM), N/C-Bid (2.3 µM), or Bcl-xL (26 µM) in 12 µl. The recombinant protein concentrations were increased by 23- to 30-fold, because of the high concentration of vesicles present in the sample (
100 times more than in the standard dextran release assay). The salt concentration was adjusted by adding KCl to 40–50 mM. For each time point, 3-µl aliquots of each sample were applied to a C-flat holey carbon film (Protochips, Raleigh, NC) supported on a 400 mesh copper grid. These grids were blotted for 2–4 s and plunge-frozen in ethane in a humidity (95%)- and temperature (25°C)-controlled chamber using a Vitrobot automated blotting device (FEI, Eindhoven, The Netherlands). Grids were cleaned before vitrification, using a Solarus Plasma Cleaner (25% O2, 75% argon). The vitreous ice grids were transferred into the electron microscope using a Gatan cryostage, which maintains the grids at a temperature below –170°C. Transmission electron microscopy was performed using low dose methods on a Tecnai F20 microscope (FEI, Hillsboro, OR) operating at 120 KeV, and at a nominal magnification of between 29,000x and 80,000x. Images were acquired using a Tietz slow scan charge-coupled device camera and the Leginon software (Suloway et al., 2005).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Yethon et al., 2003; Terrones et al., 2004). We therefore examined the possibility that cardiolipin is required for the response of native MOMs to N/C-Bid. Cardiolipin amounts in Xenopus egg OMVs, however, were below the limits of detection for TLC (Figure 1A), and quantitative lipid mass spectrometry for cardiolipin revealed the presence of this lipid at only 0.26 ± 0.14 mol % (n = 5; Figure 1B). This low level could represent the real amount of cardiolipin, or it could theoretically result from minor contamination of our OMV preparations with inner membranes that are enriched with cardiolipin.
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7 mol % cardiolipin (Kuwana et al., 2002) (Figure 1C). That the OMVs responded readily to N/C-Bid, despite their low content of cardiolipin, suggests that other OMV components may compensate for the reduced amounts of cardiolipin or that the cardiolipin present in the MOM is concentrated into small functional domains, as has been suggested for contact sites between the outer and inner membranes (Ardail et al., 1990; Lutter et al., 2001). All the reconstituted vesicles reported in this study are listed in Figure 1D.
ReOMVs with Greatly Reduced Amounts of Cardiolipin Respond Well to Bid/Bax
To determine biochemically whether MOM components facilitate Bid/Bax-mediated pore formation, it was necessary to disassemble the OMV components by using detergent. To this end, we first solubilized OMVs by using OG, removed OG by dialysis, and then allowed vesicles to form (Figure 2A). We observed that both endogenous membrane proteins and lipids assembled into proteo-liposomes, as the detergent was gradually removed. The resulting proteo-liposomes, termed ReOMVs, were permeabilized after the addition of a combination of N/C-Bid and Bax, and this permeabilization was inhibited by the presence of antiapoptotic Bcl-xL or Mcl-1, as in cardiolipin-liposomes (Kuwana et al., 2002). Notably, although the OMVs became permeabilized in the presence of N/C-Bid alone, the ReOMVs did not, but rather required both N/C-Bid and Bax. Presumably, this was because the ReOMVs lacked a Bax-like molecule that is present in the OMVs but is lost during the solubilization and reconstitution process.
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; Pautsch et al., 1999), into liposomes lacking cardiolipin, and also into detergent-solubilized LMVs, which consist mainly of ER membranes. As shown in Figure 2, C and E, OmpA loading did not confer Bid/Bax responsiveness to noncardiolipin-bearing liposomes generated by the use of detergent (OG-LUVs; Figure 2A) or ReLMVs. Instead, OmpA loading was inhibitory in those vesicles at higher doses. In ReOMVs, however, OmpA loading did not inhibit dextran release, even though more OmpA was present with respect to the estimated amount of lipid in the ReOMVs (Figure 2, D and E). Thus, OmpA does not interfere with the function of N/C-Bid and Bax. We also found that exogenous addition of nonpermeabilizing levels of OG (0.001–0.1%) did not affect Bid/Bax-dependent permeabilization of cardiolipin-liposomes generated by extrusion method that does not involve detergent (data not shown). This experiment was to mimic the situation in which a small amount of OG is present in liposome membranes after dialysis. We also analyzed the cardiolipin content in ReOMVs by two-dimensional TLC to rule out the possibility that cardiolipin is enriched in these vesicles and responsible for their permeabilization. Cardiolipin was under the detection limit (data not shown), confirming that the content of this lipid in ReOMVs is minimal, like that of OMVs. Taken together, our data suggest that native MOMs have components that substitute for cardiolipin, or possibly rearrange small amounts of this lipid into local domains, to promote Bid/Bax-induced membrane permeabilization.
MOM Proteins Are Sufficient to Confer Bid/Bax-Responsiveness to Proteo-Liposomes
We next asked whether proteins or lipids are responsible for Bid/Bax-induced permeabilization. Experiments using protease treatment were inconclusive, because for unknown reasons, this treatment led to the loss of both MOM proteins and endogenous lipids. Thus, we instead used organic solvents to separate MOM proteins from lipids. Endogenous lipids were extracted from the OMVs as in the experiment shown in Figure 1C, except that the aqueous phase containing the precipitated membrane proteins was collected. These proteins were lyophilized under a stream of argon and then resuspended in 8 M urea. The urea-denatured membrane proteins were refolded in 2% OG and then loaded onto liposomes with a lipid composition that mimicked that of the OMVs (PC:PE:PI:PS, 59:23:16:2 mol %; Figure 3A); note that cardiolipin was absent. We found that this strategy led to the loading of some proteins into the final liposomes (Figure 3B) and that these proteo-liposomes, termed extraction OMVs (ExOMVs), responded to N/C-Bid and Bax to the same extent as do ReOMVs and cardiolipin-bearing liposomes (Figure 3C). This indicates that the factors relevant to permeabilization are not organic-solvent extractable, and likely proteinaceous. Thus, we conclude that cardiolipin is not required for Bid/Bax-mediated permeabilization of the native MOM and that under physiological conditions, unidentified MOM proteins alone can facilitate this process without cardiolipin.
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; Gross, 2005), we identified MOM proteins around the size of Mtch2 (33 kDa) present in ReOMVs and ExOMVs. As shown in Figure 4C, mass spectroscopy detected Mtch2 in ReOMVs, but not in ExOMVs, suggesting that the proteinaceous factor in ExOMVs is not Mtch2.
Cryo-EM Reveals Features of Bid/Bax-mediated Permeabilization of the Lipid Membrane
To investigate the nature of the "openings" in the membrane, we chose to visualize the membrane by cryo-EM. In contrast to conventional transmission electron microscopy (TEM), cryo-EM allows the sample to be captured instantaneously in a hydrated state; thus, it is widely applied to visualize liposomes (Almgren, 2000; Johnsson and Edwards, 2003; Frederik and Hubert, 2005). The drawback of this technique, however, is that it is not amenable to the study of larger and more physiologically relevant objects, such as OMVs or mitochondria (500 nm), as the specimens must be contained in a thin vitrified ice slab that is penetrable by the electron beam. We found that OG-LUVs (Figure 2A) are well suited for cryo-EM because of their small diameter (50–100 nm). OG-LUVs containing 7 mol % cardiolipin responded to Bcl-2 family proteins in a manner similar to LUVs generated by the extrusion method (Kuwana et al., 2002) (Figure 5A). An earlier study showed that in individual apoptotic cells, the release of green fluorescent protein-cytochrome c from mitochondria is complete within 6 min (Goldstein et al., 2000), but we found the release from liposomes slower, reaching its peak after 1 h (Figure 5B). We speculate that this is because the liposome system lacks factors that facilitate Bax translocation and that the association of Bax with the liposome membrane is thus limited by diffusion.
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). Untreated vesicles seemed perfectly round and the membrane was smooth and continuous (Figure 6A). In contrast, vesicles treated with N/C-Bid and Bax exhibited discontinuity in the membrane, and pore-like structures with diameters of 25–100 nm became visible over time (Figures 6, B and C, and 7, A and B). It seems from the images that the pores start to form from one or more foci in a vesicle and then dilate (Figure 7A). The presence of Bcl-xL prevented pore formation (Figure 6D), and the percentage of pored vesicles correlated well with the dextran release curves (Figures 5B and 7C), arguing strongly that the pores are a result of the permeabilizing effect of activated Bax on the membrane. The edges of the pores seemed smooth or rough, suggestive of a mainly lipidic composition. Based on the variation in size and shape, the pores are unlikely to be proteinaceous channels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; McCarthy et al., 1997; Chipuk and Green, 2005). How Bcl-2 family proteins interact with one another to promote or inhibit apoptosis during MOMP has been studied extensively, but the final membrane permeabilization step remains a mystery. Bax and Bak are effector proteins in MOMP and are activated by certain BH3-only proteins. Thus, a synergy between Bax and BH3-only proteins is observed in in vitro systems (Kuwana et al., 2002; Roucou et al., 2002a; Roucou et al., 2002b; Yethon et al., 2003; Terrones et al., 2004). As a result of MOMP, several intermembrane space proteins are released into the cytoplasm.
In our previous studies, we found that the presence of 7 mol % cardiolipin optimizes the permeabilization of pure lipid vesicles by N/C-Bid and Bax, but we did not address the physiological relevance of cardiolipin (Kuwana et al., 2002). Cardiolipin facilitates Bax insertion, oligomerization, and permeabilization of the membrane in liposomes (Kuwana et al., 2002; Terrones et al., 2004; Lucken-Ardjomande et al., 2008b) and truncated Bid (a carboxy-terminal portion after the cleavage) targeting to mitochondria (Lutter et al., 2000, 2001). In contrast, the physiological relevance of cardiolipin in Bax-mediated cell death has been questioned by the studies that use yeast systems (Iverson et al., 2004; Polcic et al., 2005; Ott et al., 2007). Here, we addressed this question biochemically. Because OMVs contain little cardiolipin, and because extracted OMV lipids did not by themselves support permeabilization, we hypothesized that MOM proteins may act either by substituting for cardiolipin or by arranging the membrane in such a way that the local concentration of this lipid is sufficiently high to facilitate permeabilization. Our discovery that proteo-liposomes formed solely from OMV lipids and proteins (ReOMVs) did respond efficiently to N/C-Bid and Bax supports this idea. However, we could not rule out the possibility that trace amounts of cardiolipin were present in ReOMVs and contributed to permeabilization, or that some other endogenous lipid(s) mediated this effect.
Subsequently, we separated OMV proteins and lipids through organic solvent-based extraction. After the lipids were extracted, we followed a standard method for purifying and incorporating membrane proteins into a lipid bilayer. This approach has been particularly successful in the study of membrane proteins with a β-barrel structure (Surrey and Jahnig, 1992; Pautsch and Schulz, 1998; Pautsch et al., 1999; Tamm et al., 2004; Arnold et al., 2007; Shanmugavadivu et al., 2007). Our finding that the resulting ExOMVs respond to N/C-Bid- and Bax-mediated permeabilization strongly supports the notion that the responsible factor is proteinaceous. Moreover, as these vesicles possess no cardiolipin, we can conclude that one or more MOM proteins substitute functionally for cardiolipin rather than rearranging it to increase the local concentration of the lipid. As controls, we tested ReLMVs and OmpA-loaded liposomes, and the inability of these proteo-liposomes to be permeabilized by Bid/Bax argues that the permeabilization we observed in ExOMVs is dependent on specific proteins present rather than proteo-liposome formation per se.
Bid or Bim BH3 peptide and Bax together permeabilized noncardiolipin containing liposomes equally well as cardiolipin-containing liposomes. Also, detergent-oligomerized Bax displayed only slight cardiolipin dependence. These findings suggest that N/C-Bid is the component that is dependent on cardiolipin in our liposome system. Therefore, the proteinaceous factor that confers responsiveness to N/C-Bid and Bax might be a "Bid receptor." A report on an in vitro liposome system has shown that cleaved Bid induces binding of Bax and Bcl-xL to the membrane (Billen et al., 2008). Our putative Bid receptor hypothesis fits with this scenario. Billen et al. (2008) used tBid, the C-fragment portion of cleaved Bid, which might have mitigated the strict requirement for cardiolipin like that for N/C-Bid. Still, the idea of Bid association with the membrane preceding Bax binding to the membrane agrees with our finding. Recently, Gross and colleagues have reported that Mtch2, a MOM protein, binds to cleaved Bid and regulates MOMP (Grinberg et al., 2005; Gross, 2005). This protein might be a good candidate for our proteinaceous factor; however, we did not detect Mtch2 in our ExOMVs. Although it might play some role in ReOMVs, we suspect that Mtch2 is not essential in our ExOMV permeabilization. The identity of the factor together with its physiological relevance and the detailed molecular mechanisms through which this factor helps Bid activate Bax will be addressed in the future studies. Our results also suggest that activated Bax may not require other specific components in the lipid bilayer to permeabilize it, so the critical apoptotic regulations may be at the point of Bax activation (Leber et al., 2007).
Earlier studies addressing the nature of the Bax pore have generated conflicting evidence, some suggesting that it is proteinaceous (Shimizu et al., 1999; Saito et al., 2000) and others that it is lipidic (Basanez et al., 1999, 2002; Terrones et al., 2004). Although our previous study failed to detect gross morphological changes in the OMV membranes after they were treated with N/C-Bid (Kuwana et al., 2002), it was inconclusive because it relied on conventional TEM, which requires chemical fixation and dehydration that could destroy subtle membrane changes. We avoided such potential artifacts in the current study by applying cryo-EM, which does not require harsh treatment of the vesicles and is able to capture transient changes. When permeabilized by N/C-Bid and Bax, the liposomes exhibited pore-like openings at one or more foci. The finding that the diameter of these pores was 25–100 nm accounted for our previous observation that different-sized dextrans are released from permeabilized vesicles (Kuwana et al., 2002). Such pores might also explain the large channel activity recorded in mitochondria during MOMP (Pavlov et al., 2001; Guihard et al., 2004; Dejean et al., 2005). Given that we observed a good correlation between the number of vesicles with pores and the amount of dextran release, we conclude that dextrans escape through these pores. Based on our images, which reveal variability in size and relatively smooth but irregular edges, we propose that the openings induced by activated Bax are mostly lipidic, consistent with the earlier proposal by Hardwick and Polster (2002). The lipidic-pore (or toroidal) model (Shai, 1999; Sobko et al., 2004) for Bax-dependent membrane permeabilization has been suggested by the studies using a Bax 
5 peptide (a 34 mer taken from helix 5 of Bax) (Garcia-Saez et al., 2007; Qian et al., 2008). Indeed, our Bax-pores seem different from proteinaceous pores formed by a bacterial toxin pneumolysin, which is visualized by cryo-EM (Tilley et al., 2005). Pneumolysin pore formation is supposed to follow the barrel stave model because of its β-barrel structure (Tilley and Saibil, 2006). These pores are uniform in size and the toxin molecules are visible all around the edges of the pore. We did not see any protein structures in the vicinity of the Bax pore in our liposomes. Unfortunately, OMVs cannot be visualized at all by cryo-EM, because of their large size. ReOMVs are smaller than OMVs and our preliminary results suggest that the ReOMVs undergo a membrane destabilization similar to that observed with OG-LUVs (data not shown). Visualization of pores in such objects using a more suited EM technique should answer whether the pores are also present in vivo.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Tomomi Kuwana (t-kuwana{at}uiowa.edu)
Abbreviations used: ExOMV, proteo-liposomes composed of lipid-extracted outer membrane vesicle; LMV, light membrane vesicle; LUV, large unilamellar vesicle; MOM, mitochondrial outer membrane; MOMP, mitochondrial outer membrane permeabilization; OG, octylglucoside; OMV, outer membrane vesicle; ReOMV, reformed outer membrane vesicle.
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