Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M313420200 on May 11, 2004

J. Biol. Chem., Vol. 279, Issue 29, 30081-30091, July 16, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/29/30081    most recent
M313420200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Terrones, O.
Right arrow Articles by Basañez, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Terrones, O.
Right arrow Articles by Basañez, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Lipidic Pore Formation by the Concerted Action of Proapoptotic BAX and tBID*

Oihana Terrones{ddagger}§, Bruno Antonsson¶, Hirohito Yamaguchi||, Hong-Gang Wang||, Jihua Liu**, Ray M. Lee**, Andreas Herrmann{ddagger}{ddagger}, and Gorka Basañez{ddagger}§§

From the {ddagger}Unidad de Biofísica (Centro Mixto Consejo Superior de Investigaciones Cientificas-Universidad del Pais Vasco/Euskal Herriko Unibertsitatea), Universidad del Pais Vasco/Euskal Herriko Unibertsitatea, P. O. Box 644, 48080 Bilbao, Spain, Serono Pharmaceutical Research Institute, Serono International S.A., 14, chemin des Aulx, CH-1228 Plan-les Ouates, Geneva, Switzerland, the ||Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, the **Huntsman Cancer Institute and Departments of Internal Medicine and Oncological Sciences, University of Utah, Salt Lake City, Utah 84112, and the {ddagger}{ddagger}Institut für Biologie, Molekulare Biophysik, Humboldt-Universität Berlin, Invalidenstr. 42, D-10115, Berlin, Germany

Received for publication, December 9, 2003 , and in revised form, April 15, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
BCL-2 homology 3 (BH3)-only proteins of the BCL-2 family such as tBID and BIMEL assist BAX-type proteins to breach the permeability barrier of the outer mitochondrial membrane, thereby allowing cytoplasmic release of cytochrome c and other active inducers of cell death normally confined to the mitochondrial inter-membrane space. However, the exact mechanism by which tBID and BIMEL aid BAX and its close homologues in this mitochondrial protein release remains enigmatic. Here, using pure lipid vesicles, we provide evidence that tBID acts in concert with BAX to 1) form large membrane openings through both BH3-dependent and BH3-independent mechanisms, 2) cause lipid transbilayer movement concomitant with membrane permeabilization, and 3) disrupt the lipid bilayer structure of the membrane by promoting positive monolayer curvature stress. None of these effects were observed with BAX when BIMEL was substituted for tBID. Based on these data, we propose a novel model in which tBID assists BAX not only via protein-protein but also via protein-lipid interactions to form lipidic pore-type non-bilayer structures in the outer mitochondrial membrane through which intermembrane prodeath molecules exit mitochondria during apoptosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Mitochondria usually play a crucial role in the cellular commitment to apoptosis through the release of a variety of prodeath molecules from the intermembrane space into the cytosol (1). This process is tightly controlled by BCL-2 family proteins, which exert their function primarily, although not exclusively, at the level of the OMM1 (2–4). Members of the BCL-2 family possess up to four conserved regions called BCL-2 homology (BH) domains and can be either proapoptotic or antiapoptotic. Based on these criteria, BCL-2 family members can be divided into three subgroups. Members of the first subgroup, exemplified by BCL-2, contain four BH domains and act predominantly as death inhibitors. Members of the second subgroup, exemplified by BAX, contain BH1–BH3 domains and promote apoptosis in most cellular contexts. Finally members of the third subgroup share only the BH3 domain (BH3-only proteins) and appear to function invariably as death agonists. Two of the most highly studied and important BH3-only proteins are BID and BIM.

BID and BIM must cooperate with multidomain proapoptotic members to kill cells (5–7). However, it is unclear exactly how BID and BIM function in concert with BAX-type proteins to induce the release of mitochondrial intermembrane apoptogenic factors. One popular model holds that BID and BIM share a common mode of action via BH3-mediated binding to BAX-type proteins at the OMM (8). This physical interaction is believed to trigger a conformational change of multidomain proapoptotic members, resulting in their intramembraneous oligomerization and OMM permeabilization. Other not necessarily mutually exclusive mechanisms of action proposed for BID and BIM include (i) binding to and neutralization or reversal of prosurvival BCL-2-type family member function (6, 7, 9, 10), (ii) modulation of resident mitochondrial channels such as voltage-dependent anion channel and adenine nucleotide translocator (11–13), and (iii) autonomous pore-forming activity (14–20).

Accumulating evidence indicates that lipids can play important roles at different stages of the molecular pathway driven by proapoptotic BCL-2 family members culminating with OMM permeabilization and release of intermembrane apoptogenic factors. First, an early, reversible conformational change of BAX associated with apoptosis induction that occurs prior to BAX oligomerization can be reproduced in vitro upon interaction of monomeric BAX with purely lipidic vesicles (21). Second, specific lipids have been shown to favor membrane association of activated forms of BAX and BID. For example, apoptogenic fatty acids augment binding to liposomes of an oligomeric form of BAX capable of inducing mitochondrial cytochrome c release (22). Upon engagement of cell surface death receptors, BID is proteolytically cleaved by activated caspase-8, generating caspase-8-cleaved BID (cBID), which subsequently dissociates into a truncated carboxyl-terminal fragment (tBID) harboring the full apoptogenicity of the molecule and an amino-terminal inert fragment (23–25). Cardiolipin (CL) increases binding of both cBID and tBID to pure lipid vesicles as well as to the OMM (26, 27), and myristoylation of tBID further enhances its membrane avidity (28). Third, BID and cBID possess lipid transfer activity, raising the possibility that BID/cBID may transport specific lipids into or out of the OMM during apoptosis to facilitate breaching of this permeability barrier (17, 29–31). Finally lipids have been implicated in creating a permeability pathway at the OMM through which intermembrane apoptogenic factors are released into the cytosol. BAX-type proteins destabilize planar lipid bilayers through reduction of membrane line tension and permeabilize large unilamellar vesicles (LUVs) to cytochrome c through a mechanism sensitive to changes in membrane monolayer curvature (32–34). Based on these findings, we proposed that BAX-type proteins increase OMM monolayer curvature stress, promoting the formation of a large pore with its surface covered, at least partially, by polar head groups of bent mitochondrial lipids (lipidic pore model) (32–34). Along the same lines, using vesicular systems of decreasing complexity, Kuwana et al. (35) showed that activated BAX forms very large pores in a CL-dependent manner and hypothesized that CL cooperates with BAX to form highly curved non-bilayer lipid structures, such as lipidic pores or inverted micelles at the OMM. Interestingly, in the course of apoptosis, BAX-type proteins co-localize with components of the mitochondrial fusion and fission machineries at sites of OMM scission in which lipids are likely to adopt non-bilayer dispositions (36).

In the present study, we used LUVs to investigate the mechanism(s) through which biologically active forms of BID and BIM assist BAX to create a membrane passageway large enough to permit transit of mitochondrial intermembrane prodeath proteins. Our results support the notion that tBID acts in concert with BAX to form lipidic pores in the membrane of LUVs allowing for the release of macromolecules of the size of cytochrome c and other larger mitochondrial apoptogenic factors. tBID appears to act not only by triggering BAX intramembranous oligomerization via its BH3 domain but also by directly assisting BAX to form these large lipidic pores, utilizing a non-BH3 portion of the molecule. None of these effects were observed with BIMEL, but BIMEL reversed the inhibitory effect of BCL-2 on LUV permeabilization.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Materials—Recombinant full-length monomeric BAX with an amino-terminal His6 tag (BAX) (37), His6-tagged monomeric BAX with the 20-amino acid carboxyl-terminal domain truncated (BAX{Delta}C) (38), cBID with an amino-terminal His6 tag (15), cBID mutant mIII-2 (93IGDE96 -> AAAA) (15), cBID mutant mIII-3 (Gly94 -> Ala) (15), tBID (amino acids 60–195) (28), the cardiolipin-binding domain of BID (amino acids 103–162) (CBD) (27), BIMEL with an amino-terminal His6 tag and devoid of the carboxyl-terminal hydrophobic domain (20), and BCL-2 devoid of the carboxyl-terminal hydrophobic domain (38) were obtained as described previously. All proteins were >90% pure electrophoretically. Octylglucoside (OG)-activated BAX (OG-BAX) was obtained by incubating BAX in 100 mM KCl, 10 mM HEPES, 0.1 mM EDTA, pH 7.0 buffer (KHE buffer) containing OG (2%, w/v) for 1 h at 4 °C. Dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylglycerol (DOG), oleoylphosphatidylcholine (O-LPC), oleoylphosphatidylethanolamine (O-LPE), tetraoleoylphosphatidylglycerol (cardiolipin) (TOPG), heart CL, egg phosphatidylcholine (PC), egg phosphatidylethanolamine (PE), egg phosphatidylglycerol (PG), brain phosphatidylserine (PS), and liver phosphatidylinositol (PI) were purchased from Avanti Polar Lipids (Alabaster, AL). KCl, HEPES, EDTA, dodecyl octaethylene glycol mono ether (C12E8), D2O, melittin, Staphylococcus aureus {alpha}-toxin, and fluorescein isothiocyanate-labeled dextrans (FDs) of 10 kDa (FD-10) and 70 kDa (FD-70) were obtained from Sigma. 8-Aminonaphtalene-1,3,6-trisulfonate (ANTS) and p-xylene-bispyridinium bromide (DPX) were from Molecular Probes (Eugene, OR).

Release of Cytochrome c from Isolated Mitochondria—Mitochondria were isolated from livers of male Harlan Sprague-Dawley rats as described in Basañez et al. (33). Isolated mitochondria (200 µg of protein/ml) were incubated for 20 min with recombinant proteins in 50 µlof125 mM KCl, 5 mM KH2PO4, 25 µM EGTA, 5 mM succinate, 5 µM rotenone, and 10 mM HEPES-KOH, pH 7.2. Reaction mixtures were centrifuged at 10,000 x g for 10 min. Supernatant fractions (20 µl) were subjected to 4–20% SDS-PAGE followed by Western blotting using anti-cytochrome c 7H8.2C-12 antibody (Pharmingen) and visualization by the ECL method (Amersham Biosciences). Mitochondria were kept on ice and used within 3 h of preparation.

Preparation of LUVs—Lipid mixtures at the indicated ratios were co-dissolved in chloroform/methanol (2:1). Organic solvents were removed by evaporation under an argon stream followed by incubation under vacuum for 2 h. Dry lipid films were resuspended in the following buffers: for assays of release of vesicular contents, KHE buffer supplemented with either 100 mg/ml FDs or 12.5 mM ANTS, 45 mM DPX, 20 mM KCl, 10 mM HEPES, 0.1 mM EDTA, pH 7.0; for assays of vesicular size, BAX oligomerization, and transbilayer lipid redistribution, KHE buffer; for assays of membrane binding and integral membrane insertion of BAX, KHE buffer using D2O instead of H2O. LUVs were formed by the method of Mayer et al. (39) using 20 freeze/thaw cycles and two polycarbonate membranes of 0.2-µm pore size for extrusion (Nucleopore, San Diego, CA). Untrapped ANTS/DPX and FDs were removed by gel filtration in Sephadex G-25 and Sephacryl S-500 HR columns, respectively, with KHE running as elution buffer. Lipid concentration was determined by the method of Bartlett et al. (40). Unless otherwise stated, liposome composition was DOPC/TOPG (cardiolipin) (80:20, mol/mol).

Release of LUV-entrapped Markers—Release of LUV-encapsulated fluorescent markers was monitored in an SLM-2 Aminco-Bowman luminescence spectrometer (Spectronic Instruments, Rochester, NY) in a thermostatted 1-cm path length cuvette with constant stirring at 37 °C. For FDs, {lambda}ex was 490, and {lambda}em was 520 nm (slits, 4 nm); for ANTS/DPX, {lambda}ex was 350 nm, and {lambda}em was 520 nm (slits, 8 nm). A 515 nm cut-off filter was placed between the sample and the emission monochromator to avoid scattering interferences. The extent of marker release was quantified on a percentage basis according to the equation: (Ft – F0/F100 – F0) x 100 where Ft is the measured fluorescence of protein-treated LUVs at time t, F0 is the initial fluorescence of the LUV suspension before protein addition, and F100 is the fluorescence value after complete disruption of LUVs by addition of C12E8 (final concentration, 0.5 mM). Unless otherwise stated, lipid concentration was 20 µM.

Assays of BAX Binding to and Insertion into the LUV Membrane—To measure the amount of BAX bound to LUVs, a method was used based on the fact that lipid-associated protein, but not free protein, floats in D2O-based KHE buffer. Briefly proteins and LUVs were incubated together at 37 °C in D2O-based KHE buffer (final volume of reaction mixture, 100 µl) followed by centrifugation of the mixture for 1 h at 100,000 x g at room temperature. Under these conditions, LUVs remained in the upper fraction of the buffer, whereas free BAX protein sedimented. The top 20-µl fraction of the gradient, corresponding to the lipid-associated fraction, was subjected to SDS-PAGE in 15% Tris-glycine gels followed by BAX visualization by Western blotting using N20 anti-BAX polyclonal antibody (Santa Cruz Biotechnology, Inc.).

To discriminate between protein inserted into and only peripherally adsorbed to the membrane surface, the same protocol as above was followed except that a second incubation was performed for 30 min at pH 11.5 with the alkaline pH being maintained during sample centrifugation. Upon alkaline pH incubation, the fraction of protein integrated into the membrane hydrophobic matrix remains associated to LUVs, whereas the fraction of protein only peripherally associated with the membrane is detached from vesicles.

Size Exclusion Chromatography Analysis of BAX Oligomerization in LUVs—Experiments were performed in a Superdex-200 (15 x 45 cm) column equilibrated with 100 mM KCl, 10 mM HEPES, 0.2 mM EDTA (pH 7.0) with or without 2% (w/v) CHAPS (J. T. Baker Inc.) at a 1 ml/min flow rate. The column was calibrated using protein gel filtration standards (Bio-Rad). Samples of 300 µl were loaded onto the column followed by collection of 2-ml elution fractions. Aliquots of individual fractions were subjected to SDS-PAGE in 15% Tris-glycine gels followed by visualization of BAX using N20 anti-BAX antibody.

Measurements of LUV Size by Quasielastic Light Scattering—Vesicle size was determined by quasielastic light scattering at a fixed angle of 90° and room temperature using a Malvern Zetasizer 4 instrument (Malvern, UK). A 64-channel correlator was used capable of estimating particle sizes in the range from 5 to 5000 nm. Data were analyzed by the cumulant method using Malvern application software. The hydrodynamic radius of the particle was obtained from the first cumulant, whereas the polydispersity of the sample was obtained from the second cumulant.

Assays of Lipid Transbilayer Movement in LUVs—Assays of lipid transbilayer motion were done using 1-lauroyl-2-(1'-pyrenebutyroyl)-sn-glycero-3-phosphocholine (pyPC) as described before (41, 42). Briefly pyPC dissolved in an ethanolic solution was added externally to the liposome suspension at a final concentration of 5 mol % (pyPC/total lipid), leading to its incorporation only in the external monolayer of the membrane. When pyPC translocates from the external membrane monolayer to the internal membrane monolayer the probe is diluted, leading to a decrease in the ratio of fluorescence intensities of pyPC excimers to pyPC monomers (IE/IM). The degree of pyPC transbilayer redistribution (q) was estimated from measured IE/IM ratios using a calibration curve obtained with LUVs containing different molar amounts of pyPC as described before (41). Fluorescence was monitored in an SLM-2 Aminco-Bowman luminescence spectrometer (Spectronic Instruments) in a thermostatted 1-cm path length cuvette with constant stirring at 37 °C. {lambda}ex was set at 345 nm, {lambda}emM was set at 395 nm, and {lambda}emE was set at 465. Unless otherwise stated, lipid concentration was 20 µM.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
tBID, but Not BIMEL, Works in Conjunction with BAX to Form Large Membrane Openings in LUVs—To elucidate the mechanism(s) by which BID and BIM assist BAX in the release of mitochondrial intermembrane apoptogenic factors, we first used an in vitro reconstitution system consisting of mitochondria (which contain minimal amounts of BAX and BAK) freshly isolated from rat liver (43) and various combinations of recombinant purified proapoptotic proteins. We used physiologically relevant concentrations of full-length monomeric BAX (44) together with increasing concentrations of tBID or a form of BIM previously shown to induce BAX-dependent mitochondrial cytochrome c release in cultured cells (BIMEL) (20). Soluble fractions were separated from mitochondria by centrifugation and assayed for the presence of freed cytochrome c by immunoblotting. As shown in Fig. 1A, the combination of BAX with either tBID or BIMEL led to release of mitochondrial cytochrome c, whereas none of these proteins alone released substantial cytochrome c under the conditions tested (Fig. 1A, and data not shown). However, tBID released mitochondrial cytochrome in the presence of BAX with ~5-fold higher potency compared with BIMEL (Fig. 1A).



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 1.
Influence of recombinant BCL-2 family proteins on the release of both cytochrome c from isolated mitochondria and fluorescent markers from LUVs. A, freshly isolated rat liver mitochondria were incubated with the indicated amounts of recombinant apoptotic proteins for 20 min at 30 °C. The 10,000 x g supernatants (sup.) were collected and analyzed by Western blotting for cytochrome c (Cyt. c) release. Total cytochrome c release was determined by 0.5% Triton X-100 solubilization of mitochondria. B, representative time courses of FD-70 release from LUVs elicited by BAX (20 nM) combined with either tBID (20 nM), cBID (20 nM), or BIMEL (200 nM). The arrow denotes the time of addition of apoptotic proteins to the liposome suspension. Similar results were obtained when LUVs were incubated first with BAX alone and then with tBID/cBID/BIMEL or vice versa. C, freshly isolated mitochondria from rat liver were incubated for 5 min with antiapoptotic BCL-2 prior to treatment with BAX plus tBID followed by analysis of freed cytochrome c as explained in A. D, dose-dependent inhibition by BCL-2 of the vesicular FD-70 release induced by BAX and tBID. Vesicles were incubated for 5 min with BCL-2 prior to treatment with BAX (20 nM) plus tBID (20 nM). Maximum extents of marker release were obtained when the fluorescence signal reached a plateau. Mean values ± S.E. are shown for three independent experiments. Appropriate controls demonstrated that none of the protein buffers induced significant release of mitochondrial cytochrome c or LUV-entrapped markers at conditions used in these assays.

 
Next we wished to test whether, in the absence of additional proteins, tBID and/or BIMEL can assist BAX to release macromolecules of the size of cytochrome c (~12 kDa) as well as other larger prodeath proteins released from mitochondria during apoptosis, i.e. Smac/DIABLO (~23 kDa), endonuclease G (~30 kDa), Omi/HtrA2 (~37 kDa), and apoptosis-inducing factor (~57 kDa) (4). To this aim, we used LUVs loaded with self-quenching concentrations of FD-70. Release of LUV-entrapped FD-70 was monitored as an increase in the fluorescence signal due to marker dilution in the external medium. None of the proapoptotic proteins alone caused significant FD-70 release from LUVs (data not shown). However, when LUVs were treated with a BAX + tBID mixture efficient efflux of vesicular FD-70 ensued (Fig. 1B). Kuwana et al. (35) have shown previously that the combination of BAX and cBID can elicit the release of 2000-kDa dextrans from pure lipid vesicles. We found that the combination of BAX and tBID is more potent in the release of vesicular FD-70 than the BAX + cBID combination (Fig. 1B). This finding is consistent with evidence showing that BID possesses an autoinhibitory amino-terminal domain that remains attached to the apoptogenic carboxyl-terminal part of the molecule after caspase-8 cleavage (15). In sharp contrast to the situation found with tBID/cBID, addition of BIMEL together with BAX resulted in virtually no vesicular FD-70 release even at concentrations of BIMEL 10-fold higher than those used in the tBID/cBID assays (Fig. 1B).

Antiapoptotic proteins such as BCL-2 antagonize the release of mitochondrial prodeath factors during apoptosis (1–4, 7). To analyze this issue in our in vitro reconstitution systems, isolated rat liver mitochondria and LUVs were incubated with BCL-2 prior to the BAX + tBID treatment. BCL-2 effectively blocked the release of mitochondrial cytochrome c as well as that of LUV-entrapped FD-70 induced by the BAX + tBID mixture (Fig. 1, C and D). Moreover, the finding that BCL-2 inhibition occurred with a similar dose dependence in both experimental setups adds credit to the physiological significance of the LUV system.

BH3-only proteins are thought to heterodimerize with other BCL-2 family members through insertion of their BH3 domain into a hydrophobic groove localized on the surface of binding partners (9). The three-dimensional structure of BAX revealed that a carboxyl-terminal helix occupies the groove to which BH3 ligands presumably bind (45). To test whether this could explain the lack of effect of the BAX + BIMEL mixture in LUVs, we used a deletion mutant of BAX lacking its carboxyl-terminal hydrophobic region (BAX{Delta}C). Additionally we decided to examine the size of the membrane lesion caused by various combinations of apoptotic proteins in this system. To achieve this, LUVs were prepared that encapsulated markers of different molecular masses, i.e. ANTS/DPX (~0.4 kDa), FD-10, and FD-70. When BIMEL was added together with BAX{Delta}C to the liposome suspension, significant amounts of vesicular ANTS but not FD-10 or FD-70 was released (Fig. 2, left panel). However, considering that the OMM is normally permeable to molecules <5kDa, the physiological significance of this vesicular ANTS release induced by BAX + BIMEL, if any, remains to be determined. On the other hand, when either BAX or BAX{Delta}C was added together with tBID or cBID to LUVs not only ANTS but also FD-10 and FD-70 were released (Fig. 2, central and right panels). Surprisingly, despite the availability of the BH3-binding pocket in the BAX{Delta}C molecule, tBID/cBID induced vesicular marker release with lower efficiency in the presence of BAX{Delta}C relative to its full-length counterpart.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 2.
Influence of the carboxyl-terminal hydrophobic domain of BAX on the release of vesicular markers. The effects of different combinations of proapoptotic proteins on the release of vesicular ANTS/DPX (0.4 kDa) (black bars), FD-10 (light gray bars), and FD-70 (dark gray bars) are shown. Mean values ± S.E. are shown for three to five independent experiments. BAX, BAX{Delta}C, tBID, and cBID concentrations were 20 nM, and BIMEL concentration was 200 nM.

 
In summary, these results indicate that cBID and more efficiently tBID cooperates with BAX in pure lipid vesicles to form a membrane lesion large enough to allow transit of mitochondrial intermembrane apoptogenic proteins. In sharp contrast, BIMEL lacks this capacity.

tBID Can Collaborate with BAX in a BH3-independent Manner to Induce Membrane Damage—In healthy cells BAX exists in a monomeric inactive state localized in the cytosol or loosely attached to the OMM (3). BAX activation during apoptosis has been related to a change in conformation of the protein leading to its outer membrane insertion and oligomerization, and cBID/tBID has been suggested to trigger both events through binding to BAX via its BH3 domain (46–48). To analyze the importance of the BH3 domain of BID in assisting BAX to form large membrane openings in LUVs, we used two BH3 mutants of BID with reduced affinity for BAX: cBid mIII-2 (93IGDE96 -> AAAA) and cBid mIII-3 (Gly94 -> Ala) (46, 49). We compared the effects of increasing concentrations of cBID, cBID mIII-2, and cBID mIII-3 on the release of FD-70 from LUVs treated with fixed amounts of BAX. cBID mIII-2 showed ~3–4-fold reduced efficiency in vesicular FD-70 release relative to cBID, whereas cBID mIII-3 showed similar efficiency in FD-70 release relative to cBID (Fig. 3). Previous studies have shown that BID mIII-3 and BID mIII-2 possess at least 10-fold lower affinity for BAX (46). This observation, together with the paradoxical finding that exposure of the BH3-binding cleft of BAX did not increase but actually decreased tBID/cBID-assisted release of vesicular contents, prompted us to investigate the possibility that tBID utilizes BH3-independent mechanisms to aid BAX in membrane damage induction.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 3.
Role of the BH3 domain of cBID on vesicular FD-70 release. LUVs were treated with BAX (20 nM) together with increasing concentrations of wild-type cBID or cBID mutants mIII-2 and mIII-3, and extents of vesicular FD-70 release were determined when the fluorescent signal reached a plateau. Data correspond to means (±S.E.) of at least three independent experiments.

 
Specific detergents such as OG can induce BAX membrane integration and oligomerization (21, 34, 35, 38). Several groups have shown that such OG-activated BAX increases the permeability of both outer mitochondrial (35, 38, 50, 51) as well as pure lipid membranes (13, 22, 34, 35, 38). Moreover OG-activated BAX and tBID-activated BAX display similar degrees of membrane integration and oligomerization in LUVs (Ref. 21 and data not shown). Thus, we decided to use such OG-activated BAX as a tool to test whether tBID can assist BAX to form large membrane openings in LUVs independently of BH3-mediated BAX intramembranous oligomerization. OG-activated BAX alone released LUV-entrapped FD-70 efficiently (data not shown), consistent with previous findings (35). Remarkably, when LUVs were treated with suboptimal doses of OG-activated BAX, subsequent addition of tBID, but not BIMEL or tBID buffer alone, led to additional vesicular FD-70 release (Fig. 4A). Since, with or without tBID, OG-activated BAX inserted to the same degree in the LUV membrane in an alkali-resistant manner (Fig. 4D) and formed multimers of similar sizes in CHAPS-solubilized LUVs (Fig. 4E), we concluded that tBID can assist BAX to cause vesicular FD-70 release without altering the membrane insertion capacity or oligomeric status of BAX.



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 4.
tBID can cooperate with BAX in a BH3-independent manner to induce LUV permeabilization. A, B, and C, tBID and CBD release FDs from LUVs pretreated with OG-BAX. A, FD-70-loaded LUVs were treated as follows: OG-BAX + tBID: first OG-BAX (large arrow) and then tBID (small arrow); OG-BAX: first OG-BAX (large arrow) and then tBID buffer (small arrow); OG-BAX + BIMEL: first OG-BAX (large arrow) and then BIMEL (short arrow); tBID: first OG-BAX buffer (large arrow) and then tBID (short arrow). Concentrations of OG-BAX, tBID, and BIMEL were 20, 150, and 200 nM, respectively. B, FD-10-loaded LUVs were treated as follows: OG-BAX + CBD: first OG-BAX (large arrow) and then CBD (short arrow); OG-BAX: first OG-BAX (large arrow) and then CBD buffer (short arrow); CBD: first OG-BAX buffer (large arrow) and then CBD (short arrow); BAX + CBD: first monomeric BAX (large arrow) and then CBD (short arrow). OG-BAX, monomeric BAX, and CBD concentrations were 20, 20, and 150 nM, respectively. C, dose dependence of the tBID- and CBD-mediated vesicular FD release in LUVs pretreated with OG-BAX (20 nM) as explained in A and B. tBID/CBD concentrations were 20 nM (black bars), 50 nM (light gray bars), and 150 nM (dark gray bars). Data represent means (±S.E.) of at least two independent experiments. D, effect of tBID and CBD on the membrane binding and membrane insertion capacities of OG-BAX. LUVs (40 µM) were treated first with OG-BAX (40 nM) and subsequently with tBID/CBD (300 nM), as described in A and B. "Total" corresponds to a sample of 40 nM OG-BAX. E, migration patterns of apoptotic proteins after incubation with LUVs as described in A and B followed by solubilization of membranes with 2% (w/v) CHAPS. Samples containing the indicated proteins were subjected to gel filtration in the presence of 2% (w/v) CHAPS, elution fractions of 2 ml were collected, and BAX migration profiles were determined by immunoblotting. Arrows denote elution peaks of standard proteins: thyroglobulin (670 kDa), gamma globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa). OG-BAX, tBID, and CBD protein concentrations were 80, 600, and 600 nM, respectively. Lipid concentration was 80 µM.

 
One possibility to explain the above observations is that, in addition to the BH3 domain, tBID also utilizes an intramembranous region to cooperate with BAX in membrane damage induction. A region was identified in tBID possessing potential for membrane insertion that targets the molecule to CL-containing membranes, the so-called CBD (26, 27). Similar to tBID, addition of CBD to LUVs pretreated with suboptimal concentrations of OG-activated BAX caused further release of vesicular markers (Fig. 4B), although CBD did not alter significantly the degree of membrane insertion or multimerization of BAX (Fig. 4, D and E). However, CBD was less efficient than tBID in eliciting the release of FD-10 and FD-70 from LUVs pretreated with suboptimal concentrations of OG-activated BAX (Fig. 4C). CBD did not induce substantial marker release in LUVs pretreated with OG-activated BAX in which CL had been replaced by equimolar amounts of PS (data not shown), consistent with the selective preference of CBD for CL (26, 27). CBD also did not elicit substantial marker release in LUVs pretreated with monomeric BAX (Fig. 4B) in agreement with the notion that the BH3 domain of BID (absent in CBD) is required for triggering BAX membrane insertion and oligomerization.

Collectively the above described results suggest that tBID utilizes two distinct mechanisms to assist BAX to form large openings in the membrane: 1) activation of BAX integral membrane insertion and oligomerization via physical interaction of the BH3 domain of tBID with BAX and 2) a heterodimerization-independent mechanism via non-BH3 regions of tBID apparently including, but not limited to, the CBD.

Lipidic Pore Formation by the Concerted Action of BAX and tBID—The finding that the membrane lesion caused by BAX in conjunction with tBID/cBID did not discriminate permeants according to size (Fig. 2) raised the possibility that proapoptotic proteins may act by solubilizing LUVs in a "detergent-like" manner. For example, BAX together with tBID/cBID may induce micellization of the membrane of LUVs, breaking it up into small fragments or disk-like structures as seen with a number of membrane-disrupting antimicrobial peptides, including melittin (52). Evidence for such a detergent-like action can be obtained by analyzing changes in the size distribution of LUVs using quasielastic light scattering. This approach was utilized here to evaluate whether proapoptotic proteins caused FD-10 release through vesicle fragmentation. Results were compared with those obtained with melittin, which was used as a positive reference. As shown in Table I, addition of melittin caused, as expected, a decrease in the size of vesicles together with near complete release of vesicular FD-10. However, at concentrations of either BAX + tBID or BAX + cBID causing comparable FD-10 release, the size distribution of LUVs did not change significantly with respect to untreated samples.


View this table:
[in this window]
[in a new window]
  		TABLE I
Effects of proapoptotic proteins and melittin on LUV size and on vesicular FD-10 release

 
An alternative mechanism to explain the large size and non-selectivity of the membrane lesion caused by BAX together with tBID/cBID is that proapoptotic proteins disrupt the bilayer structure of the membrane by forming non-lamellar lipid structures. In this scenario, proapoptotic proteins would act either globally or in a localized manner on the membrane to change its material elastic properties, forcing membrane lipids to adopt a curved, non-bilayer disposition. Two main types of non-bilayer structures have been related to BAX-induced membrane permeabilization: (i) inverted-type lipid structures, such as inverted intrabilayer micelles (35, 53), and (ii) lipidic pores (1, 2, 32–35, 44, 53). One distinguishing feature between these two types of non-bilayer structures is their net curvature. By definition, inverted-type lipid structures possess net negative curvature (54). In opposition, lipidic pores possess net positive curvature (55). Hence increasing membrane monolayer curvature toward more negative values should lower the energy required to form inverted-type lipid structures, whereas increasing membrane monolayer curvature toward more positive values is predicted to favor lipidic pore formation.

To assess whether membrane monolayer curvature is an important contributor to the vesicular FD-70 release elicited by BAX and tBID/cBID and to try to distinguish between the inverted micelle model and the lipidic pore model, non-bilayer lipids of known intrinsic curvature were used. Positive curvature-inducing lipids such as O-LPC and O-LPE potentiated vesicular FD-70 release, whereas negative curvature-inducing lipids such as DOG and DOPE inhibited vesicular FD-70 release (Fig. 5A, Table II, and data not shown). O-LPC possesses an intrinsic curvature more positive than O-LPE (56), and O-LPC potentiated vesicular FD-70 release to a higher extent than O-LPE (Table II). DOG possesses an intrinsic curvature more negative than DOPE (57), and DOG inhibited vesicular FD-70 release to a higher extent than DOPE (Table II). Since the intrinsic curvature of O-LPC is opposite to that of DOG, next we examined whether O-LPC and DOG cancel each other's effect on LUV permeabilization. The levels of vesicular FD-70 release obtained in the presence of equimolar amounts of O-LPC and DOG were intermediate between those obtained with either DOG or O-LPC alone (Table II). Changes in either the degree of BAX membrane insertion or its oligomeric status could not provide a complete explanation for the effects of non-bilayer lipids on vesicular FD-70 release (data not shown). Based on analogous results, we previously proposed that OG-activated BAX induces lipidic pore formation by imposing positive membrane monolayer curvature stress (34). We now extend this proposal to the more physiologically relevant form of BAX activated by tBID/cBID. Importantly O-LPC and DOG also potentiated and decreased, respectively, tBID-induced FD-70 release from LUVs treated with OG-activated BAX without producing major changes in the assembly of intramembranous BAX oligomers (Fig. 5B, Table II, and data not shown). These results suggest that the mechanism unrelated to triggering of BAX intramembranous oligomerization by which tBID can assist BAX in LUV permeabilization corresponds to generation of positive membrane monolayer curvature stress. In apparent contradiction with our proposal, however, is the finding that cBID can decrease the bilayer-to-non-bilayer lipid phase transition temperature implying that cBID promotes negative, not positive, membrane monolayer curvature stress (19). However, such experiments were done in the absence of BAX using DOPC/DOPE/TOPG (1:1:1) lipid mixtures and with millimolar calcium concentrations in the medium. Biophysical studies are in progress in our laboratory to determine the concerted effect of BAX and tBID/cBID on bilayer-to-non-bilayer lipid phase transitions under more physiologically relevant conditions. Finally the effects of non-bilayer lipids on release of vesicular contents were tested for S. aureus {alpha}-toxin, a bacterial toxin known to form purely proteinaceous channels (58). Unlike the situation found with proapoptotic proteins, O-LPC did not affect and DOG promoted the release of vesicular contents induced by S. aureus {alpha}-toxin (Table II).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 5.
tBID cooperates with BAX to release vesicular FD-70 through a mechanism sensitive to intrinsic membrane monolayer curvature. A, typical kinetics of vesicular FD-70 release induced by the concerted action of BAX and tBID in DOPC/TOPG (8:2) (Control), DOPC/TOPG/O-LPC (7:2:1) (O-LPC), and DOPC/TOPG/DOG (7:2:1) (DOG) LUVs. BAX and tBID concentrations were 20 and 10 nM, respectively. B, representative kinetics of vesicular FD-70 release in LUVs treated first with OG-BAX (20 nM) (large arrow) and then with tBID (50 nM) (small arrow).

 


View this table:
[in this window]
[in a new window]
  		TABLE II
Effect of non-bilayer lipids on the release of vesicular contents and on the transbilayer lipid redistribution induced by apoptotic proteins and by S. aureus {alpha}-toxin

ND, not determined. Data correspond to means ±S.E. of two to five independent measurements.

 
If, as suggested by the results described above, the concerted action of BAX and tBID creates lipidic pores in the membrane, its constituent monolayers would become continuous via the pore-lining lipids. Thus, one implication of the lipidic pore model is that such structure should allow the movement of lipid molecules from one monolayer of the bilayer to the other. To test for this possibility, we applied an assay developed by Muller et al. (41) recently used in our laboratory (42). In this assay, LUVs are labeled with the fluorescent phosphatidylcholine analogue pyPC exclusively in the external monolayer of the membrane. Membrane-incorporated pyPC displays two distinct peaks in the fluorescence spectrum, one arising from excited monomeric pyPC molecules and the other arising from excited dimeric (excimer) pyPC molecules. Any redistribution of pyPC to the internal monolayer is accompanied by changes of analogue concentration and, by that, of the excimer to monomer ratio in each monolayer. Thus, changes in the ratio of the excimer to monomer fluorescence intensity signals of pyPC (IE/IM) can be used to monitor the outward-inward transbilayer movement of the PC analogue.

Addition of BAX together with tBID to LUVs containing pyPC localized only in the external monolayer led to a rapid decrease of IE/IM, indicating transfer of the analogue to the internal monolayer (Fig. 6A). Addition of BAX together with cBID also triggered pyPC transfer to the internal monolayer albeit to a lower extent. In sharp contrast, the BAX + BIMEL mixture caused negligible transbilayer movement of pyPC. Of note, the time courses of pyPC transbilayer redistribution induced by BAX together with either tBID or cBID were comparable to those obtained in analogous experiments of vesicular FD-70 release (compare Fig. 1B and Fig. 6A). Next the degree of pyPC transbilayer redistribution between outer and inner monolayer (q) was determined. The results obtained with different protein combinations correlated with those obtained in analogous experiments of vesicular FD-70 release (Fig. 6B). Last the influence of membrane monolayer curvature on pyPC transbilayer movement was analyzed. As in the case of release of vesicular contents, positive and negative membrane monolayer curvature increased and decreased, respectively, the degree of pyPC transbilayer motion induced by tBID together with monomeric BAX or with OG-activated BAX (Table II). These observations strongly suggest that the release of vesicular contents induced by the concerted action of BAX and tBID is mechanistically related to lipid transbilayer movement. Importantly S. aureus {alpha}-toxin induced little pyPC transbilayer redistribution under conditions in which it caused extensive release of vesicular contents, indicating that the opening of a purely proteinaceous channel in the membrane of LUVs does not cause substantial lipid transbilayer redistribution (Table II). Taken together, these results support the hypothesis that tBID cooperates with BAX at different stages of a molecular pathway culminating with formation of a large, lipid-containing pore having net positive curvature.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 6.
Effects of BCL-2 family proteins on transbilayer lipid redistribution. A, representative time courses of pyPC transbilayer redistribution in LUVs after addition of BAX combined either with cBID, tBID, or BIMEL. LUVs were labeled exclusively in the outer leaflet by external addition of pyPC (5 mol % of total lipid) to the liposome suspension. After a 5-min preincubation of the lipid mixture, proteins were added (corresponding to time = 0 s), and the ratio of excimer to monomer fluorescence intensity signals (IE/IM) was recorded at any given time. Values of IE/IM are given normalized to those obtained in pure lipid vesicles. BAX, cBID, and tBID concentrations were 20 nM. The concentration of BIMEL was 200 nM. B, comparison of the degree of pyPC transbilayer redistribution elicited by various combinations of apoptotic proteins. The degree of pyPC transbilayer redistribution was estimated from IE/IM values as explained under "Experimental Procedures." Proapoptotic protein concentrations were as follows: BAX + BIMEL, 20 + 200 nM; BAX + cBID, 20 + 20 nM; OG-BAX, 20 nM; OG-BAX + tBID, 20 + 50 nM; and BAX + tBID, 20 + 20 nM. Data represent means (±S.E.) of two to four experiments. Appropriate controls demonstrated that (a) none of the buffers in which the proteins were suspended caused substantial pyPC transbilayer movement at conditions used in these assays and (b) addition of proteins to liposomes symmetrically labeled with pyPC caused much lower changes in the IE/IM ratio as compared with asymmetrically labeled liposomes.

 
Studies with Biomembrane Mimetic LUVs of OMM Contact Site Composition—BAX (59, 60), tBID (26, 61), and BCL-2 (62, 63) have been shown to localize at zones of close proximity between the outer and the inner mitochondrial membranes, the so-called mitochondrial membrane contact sites. Thus, we decided to examine the effect of these proteins in LUVs bearing the phospholipid composition determined for the OMM at such contact sites (64). Neither BAX nor tBID alone had any effect on such LUVs (data not shown). However, addition of the two proteins together caused vesicular FD-70 release and pyPC transbilayer redistribution with similar time courses and comparable susceptibilities to inhibition by BCL-2 (Fig. 7A and Table III). cBID functioned together with BAX in a similar manner causing both FD-70 release and lipid transbilayer movement although with reduced potency compared with tBID (Fig. 7B and Table III). Once again, however, BIMEL was unable to cooperate with BAX to induce any of these effects in LUVs of OMM contact site composition (Table III).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 7.
Effects of BCL-2 family proteins on LUVs reflecting the physiological phospholipid composition of OMM contact sites. A, PG can replace CL in the vesicular FD-70 release and the transbilayer pyPC redistribution induced by the concerted action of BAX and tBID. LUVs were prepared that mimicked the phospholipid composition of outer mitochondrial membrane contact sites including CL (26:21:8:20 PC/PE/PI/CL by weight) (64) with CL being replaced by equal molar amounts of PG (26:21:8:10 PC/PE/PI/PG by weight), PI (26:21:20 PC/PE/PI by weight), or PS (26:21:8:11 PC/PE/PI/PS by weight) or excluding all acidic lipids (26:21 PC/PE by weight) (–). Then BAX (20 nM) and tBID (20 nM) were added to the vesicle suspensions (arrow), and the time dependence of vesicular FD-70 release (continuous lines) in each type of LUVs as well as the transbilayer redistribution of pyPC in CL-containing LUVs (filled circles) and PG-containing LUVs (empty circles) was monitored. Experiments were performed three times, each yielding similar results. B, effects of various BCL-2 protein combinations on the release of FD-70 from LUVs of OMM contact site composition (26:21:8:20 PC/PE/PI/CL by weight). Data represent means (±S.E.) of two independent experiments.

 


View this table:
[in this window]
[in a new window]
  		TABLE III
A comparison of the vesicular FD-70 release and the lipid transbilayer redistribution induced by different combinations of BCL-2 family proteins in CL- and PG-containing LUVs emulating the composition of OMM contact sites

BAX, tBID, and cBID concentrations were 20 nM, BIMEL concentration was 200 nM, and BCL-2 concentration was 300 nM. Data correspond to mean values ± S.E. of two to three independent experiments. ND, not determined.

 
Next we sought to determine whether CL is required for the membrane actions of BAX and tBID. To this aim, LUVs of OMM contact site composition but excluding all acidic lipids and with CL being substituted by PS, PI, or PG (at the same mole ratios of acidic lipids) were prepared. The BAX + tBID mixture did not permeabilize LUVs of OMM contact site composition in which all acidic lipids had been excluded (Fig. 7A). Substitution of CL by PI or PS caused much reduction in the release of vesicular FD-70 elicited by BAX together with tBID. Interestingly, when CL was substituted by PG, the BAX + tBID mixture caused vesicular FD-70 release with only slightly lower efficiency as compared with CL-containing LUVs (Fig. 7A). Moreover pyPC transbilayer redistribution accompanied the release of vesicular FD-70 in PG-containing LUVs, and both processes were inhibited by BCL-2 (Fig. 7A and Table III). Thus, we concluded that PG can replace CL in formation of large lipidic pores by BAX and tBID. This finding may be of physiological relevance considering that (i) PG and CL are structurally and biosynthetically related lipids primarily localized to mitochondrial membranes (65), (ii) mitochondrial PG levels increase early during Fas- and radiation-induced apoptosis (66), and (iii) BAX can induce cytochrome c release in mitochondria from mutant yeast lacking CL but containing increased PG levels (67).

What remains to be addressed is the origin of the CL/PG selectivity in the vesicular permeabilization elicited by the concerted action of BAX and tBID. Because CL, PG, PS, and PI all possess near zero intrinsic curvature under the conditions tested here (68), changes in membrane monolayer curvature implicated in lipidic pore formation are unlikely to be the source of the CL/PG preference over PS/PI. Interestingly recent mass spectrometry studies in cells of different origin showed that during Fas-induced apoptosis the amount of mitochondrial CL decreases, whereas the amounts of monolysocardiolipin, lysophosphatidylglycerol, and lysophosphatidylcholine increase coincidentally with cytochrome c release (29–31). Considering that lysolipids, in general, possess positive intrinsic curvature (54), it is tempting to speculate that during apoptosis such lysoderivatives rather than their parental species may be sequentially and/or cooperatively implicated in lipidic pore formation by BAX and tBID/cBID.

Finally we performed additional studies to gain more insight on the mechanism of action of BIMEL. Since no evidence was found that BIMEL can assist BAX in the release of large molecular weight dextrans from LUVs yet BIMEL was able to assist BAX in cytochrome c release from isolated rat liver mitochondria, we reasoned that additional endogenous mitochondrial factors may be required to reconstitute the BIMEL-BAX cooperation in LUVs. It has been proposed that BH3-only proteins can be subdivided into two classes: those that can bind to and activate BAX-type proteins directly and those that can render cells more susceptible to apoptogenic stimuli through binding to and inactivation of BCL-2-type proteins (8, 10). Although it has been difficult to capture BIMEL-BAX complexes (8, 20, 69–73), evidence indicates that BIMEL can bind to BCL-2 in different cellular contexts (69–73). Thus, we assessed whether, by virtue of its reported capacity to bind to BCL-2, BIMEL could reverse the inhibitory effect of BCL-2 on LUV permeabilization. BCL-2 exerted a dose-dependent inhibition on the release of vesicular FD-70 elicited by the BAX + cBID mixture in LUVs of OMM contact site composition (Fig. 7B). Addition of equimolar concentrations of BIMEL compared with cBID did not affect vesicular FD-70 release. However, augmentation of BIMEL concentration led to a progressive increase of vesicular FD-70 release, and at a 6-fold higher concentration of BIMEL relative to cBID a complete reversal of the BCL-2 inhibition was achieved (Fig. 6B). BIMEL did not enhance the release of vesicular FD-70 mediated by the BAX + cBID combination in the absence of BCL-2, arguing that BIMEL acts through BCL-2 rather than through BAX and/or cBID.

Concluding Remarks—In summary, we showed that tBID and BIMEL use different mechanisms to aid BAX in making lipid vesicles permeable to macromolecules as large as prodeath proteins released from mitochondria during apoptosis. Several findings support the idea that tBID directly cooperates with BAX to induce LUV permeabilization through formation of large, lipid-containing pores: 1) the observation that BAX together with tBID induces the release of all molecules entrapped within LUVs irrespective of their size, 2) the strong coupling between this release of vesicular contents and the redistribution of lipids from one monolayer of the membrane to the other, 3) the dependence of both release of vesicular contents and lipid transbilayer redistribution on membrane monolayer curvature, and (4) the lack of effect of proapoptotic proteins on LUV size, which argues against a detergent-like mechanism of action. An additional important highlight of our study is that tBID utilizes BH3-dependent as well as BH3-independent mechanisms to assist BAX in LUV permeabilization. The BH3-independent effect of tBID appears to be due to promotion of positive membrane monolayer curvature stress. Based on these observations, we propose a new model in which tBID aids BAX to permeabilize the OMM in a dual manner: 1) triggering BAX intramembranous oligomerization via the BH3 domain and 2) reducing the energetic cost to form a large lipidic pore, utilizing regions other than the BH3 domain.

In contrast to tBID, our results in LUVs suggest that BIMEL aids BAX to permeabilize the OMM in an indirect way through binding to and neutralization of BCL-2-type proteins. However, we cannot rule out the possibility that other isoforms of BIM or a post-translationally modified form of BIMEL may assist BAX in a more direct manner. Additionally it is possible that mitochondrial components unrelated to BCL-2 family proteins (e.g. voltage-dependent anion channel) play an important role in BIMEL bioactivity (12). Further studies are required to test these possibilities as well as to examine the applicability of the lipidic pore model and its postulates for the increasing number of molecules proposed to collaborate with BAX-type proteins in OMM permeabilization during apoptosis (36, 74–77).


   FOOTNOTES
 
Addendum—While this manuscript was in preparation Epand et al. (78) reported that cBID-activated BAX promotes transbilayer lipid diffusion in LUVs.

* This work was supported in part by funds from the Ministerio de Ciencia y Tecnología, Spain, Grant BMC 2002-00784. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ A predoctoral student supported by the Basque Government. Back

§§ To whom correspondence should be addressed. Tel.: 34-946013355; Fax: 34-946013360; E-mail: gbzbaasg{at}lg.ehu.es.

1 The abbreviations used are: OMM, outer mitochondrial membrane; LUV, large unilamellar vesicle; cBID, caspase-8-cleaved BID; tBID, the carboxyl-terminal fragment of caspase-8-cleaved BID; OG, octylglucoside; DOPC, dioleoylphosphatidylcholine; DOPE, dioleoylphosphatidylethanolamine; DOG, dioleoylglycerol; O-LPC, oleoylphosphatidylcholine; O-LPE, oleoylphosphatidylethanolamine; TOPG, tetraoleoylphosphatidylglycerol (cardiolipin); CL, cardiolipin; CBD, cardiolipin-binding domain of BID; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; C12E8, dodecyl octaethylene glycol monoether; FD, fluorescein isothiocyanate-labeled dextran; FD-10, fluorescein isothiocyanate-labeled dextrans of 10-kDa average molecular mass; FD-70, fluorescein isothiocyanate-labeled dextrans of 70-kDa average molecular mass; ANTS, 8-aminonaphtalene-1,3,6-trisulfonate; DPX, p-xylene-bispyridinium bromide; pyPC, 1-lauroyl-2-(1'-pyrenebutyroyl)-sn-glycero-3-phosphocholine; BH, BCL-2 homology; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Dr. B. Polster for critically reading the manuscript and Dr. M. D. Esposti for helpful comments.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Newmeyer, D. D., and Ferguson-Miller, S. (2003) Cell 112, 481–490[CrossRef][Medline] [Order article via Infotrieve]
  2. Kuwana, T., and Newmeyer, D. D. (2003) Curr. Opin. Cell Biol. 15, 691–699[CrossRef][Medline] [Order article via Infotrieve]
  3. Sharpe, J. C., Arnoult, D., and Youle, R. J. (2004) Biochim. Biophys. Acta 1644, 107–113[Medline] [Order article via Infotrieve]
  4. Festjens, N., van Gurp, M., van Loo, G., Saelens, X., and Vandenabeele, P. (2004) Acta Haematol. (Basel) 111, 7–27
  5. Wei, M. C., Zong, W. X., Cheng, E. H., Lindsten, T., Panautsakopoulou, V., Ross, A. J., Roth, K. A., MacGregor, G. R., Thompson, C. B., and Korsmayer, S. J. (2001) Science 292, 727–730[Abstract/Free Full Text]
  6. Zong, W. X., Lindsten, T., Ross, A. J., MacGregor, G. R., and Thompson, C. B. (2001) Genes Dev. 15, 1481–1486[Abstract/Free Full Text]
  7. Cheng, E. H., Wei, M. C., Weiler, S., Flavell, R. A., Mak, T. W., Lindsten, T., and Korsmeyer, S. J. (2001) Mol. Cell 8, 705–711[CrossRef][Medline] [Order article via Infotrieve]
  8. Letai, A., Bassik, M. C., Walensky, L. D., Sorcinelli, M. D., Weiler, S., and Korsmeyer, S. J. (2002) Cancer Cell 2, 183–192[CrossRef][Medline] [Order article via Infotrieve]
  9. Puthalakath, H., and Strasser, A. (2002) Cell Death Differ. 9, 505–512[CrossRef][Medline] [Order article via Infotrieve]
  10. Terradillos, O., Montessuit, S., Huang, D. C., and Martinou, J. C. (2002) FEBS Lett. 522, 29–34[CrossRef][Medline] [Order article via Infotrieve]
  11. Zamzami, N., El Hamel, C., Maisse, C., Brenner, C., Muñoz-Pinedo, C., Belzacq, A. S., Costantini, P., Vieira, H., Loeffler, M., Molle, G., and Kroemer, G. (2000) Oncogene 19, 6342–6350[CrossRef][Medline] [Order article via Infotrieve]
  12. Sugiyama, T., Shimizu, S., Matsuoka, Y., Moneda, Y., and Tsujimoto, Y. (2002) Oncogene 21, 4944–9456[CrossRef][Medline] [Order article via Infotrieve]
  13. Rostovtseva, T. K., Antonsson, B., Suzuki, M., Youle, R. J., Colombini, M., and Bezrukov, S. (2004) J. Biol. Chem. 279, 13575–13583[Abstract/Free Full Text]
  14. Schendel, S. L., Azimov, R., Pawlowski, K., Godzik, A., Kagan, B. L., and Reed, J. C. (1999) J. Biol. Chem. 274, 21932–21936[Abstract/Free Full Text]
  15. Kudla, G., Montessuit, S., Eskes, R., Berrier, C., Martinou, J. C., Ghazi, A., and Antonsson, B. (2000) J. Biol. Chem. 275, 22713–22718[Abstract/Free Full Text]
  16. Zhai, D., Miao, Q., Xin, X., and Yang, F. (2001) Eur. J. Biochem. 268, 48–55[Medline] [Order article via Infotrieve]
  17. Esposti, M. D., Erler, J. T., Hickman, J. A., and Dive, C. (2001) Mol. Cell. Biol. 21, 7268–7276[Abstract/Free Full Text]
  18. Grinberg, M., Sharig, R. Zaltsman, Y., Frumkin, D., Grammatikakis, N., Reuveny, E., and Gross, A. (2002) J. Biol. Chem. 277, 12237–12245[Abstract/Free Full Text]
  19. Epand, R. F., Martinou, J. C., Fornallaz-Mulhauser, M., Hughes, D. W., and Epand, R. M. (2002) J. Biol. Chem. 277, 32632–32639[Abstract/Free Full Text]
  20. Yamaguchi, H., and Wang, H. G. (2002) J. Biol. Chem. 277, 41604–41612[Abstract/Free Full Text]
  21. Yethon, J. A., Epand, R. F., Leber, B., Epand, R. M., and Andrews, D. W. (2003) J. Biol. Chem. 278, 48935–48941[Abstract/Free Full Text]
  22. Epand, R. F., Martinou, J.-C., Montessuit, S., and Epand, R. M. (2004) Biochem. J. 377, 509–516[CrossRef][Medline] [Order article via Infotrieve]
  23. Luo, X., Budlhardjo, I., Zou, H., Slaughter, C., and Wang, X. (1998) Cell 94, 481–490[CrossRef][Medline] [Order article via Infotrieve]
  24. Li, H., Zhu, H., Xu, C.-J., and Yuan, J. (1998) Cell 94, 491–501[CrossRef][Medline] [Order article via Infotrieve]
  25. Gross, A., Yin, X.-M., Wang, K., Wei, M. C., Jockel, J., Milliman, C., Erdjument-Bromage, H., Tempst, P., and Korsmeyer, S. J. (1999) J. Biol. Chem. 274, 1156–1163[Abstract/Free Full Text]
  26. Lutter, M., Fang, M., Luo, X., Nishijima, M., Xie, X., and Wang, X. (2000) Nat. Cell Biol. 2, 754–761[CrossRef][Medline] [Order article via Infotrieve]
  27. Liu, J., Weiss, A., Durrant, D., and Lee, R. M. (2004) Apoptosis, in press
  28. Zha, J., Weiler, S., Oh, K. J., Wei, M. C., and Korsmeyer, S. J. (2000) Science 290, 1761–1765[Abstract/Free Full Text]
  29. Esposti, M. D., Cristea, I. M., Gaskell, S. J., Nakao, Y., and Dive, C. (2003) Cell Death Differ. 10, 1300–1309[CrossRef][Medline] [Order article via Infotrieve]
  30. Cristea, I. M., and Esposti, M. D. (2004) Chem. Phys. Lipids 129, 133–160[CrossRef][Medline] [Order article via Infotrieve]
  31. Sorice, M., Circella, A., Cristea, I. M., Garofalo, T., Renzo, L. D., Alessandri, C., Valesini, G., and Esposti, M. D. (2004) Cell Death Differ., in press
  32. Basañez, G., Nechushtan, A., Drozhinin, O., Chanturiya, A., Choe, E., Tutt, S., Wood, K. A., Hsu, Y., Zimmerberg, J., and Youle, R. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 5492–5497[Abstract/Free Full Text]
  33. Basañez, G., Zhang, J., Chau, B. N., Maksaev, G. I., Frolov, V. A., Brandt, T. A., Burch, J., Hardwick, J. M., and Zimmerberg, J. (2001) J. Biol. Chem. 276, 31083–31091[Abstract/Free Full Text]
  34. Basañez, G., Sharpe, J. C., Galanis, J., Brandt, T. B., Hardwick, J. M., and Zimmerberg, J. (2002) J. Biol. Chem. 277, 49360–49365[Abstract/Free Full Text]
  35. Kuwana, T., Mackey, M. R., Perkins, G., Ellisman, M. H., Latterich, M., Schneiter, R., Green, D. R., and Newmeyer, D. D. (2002) Cell 111, 331–342[CrossRef][Medline] [Order article via Infotrieve]
  36. Karbowski, M., Lee, Y.-J., Gaume, B., Jeong, S.-Y., Frank, S., Nechushtan, A., Santel, A., Fuller, M., Smith, C. L., and Youle, R. J. (2002) J. Cell Biol. 159, 931–938[Abstract/Free Full Text]
  37. Montessuit, S., Mazzei, G., Magnenat, E., and Antonsson, B. (1999) Protein Expr. Purif. 15, 202–206[CrossRef][Medline] [Order article via Infotrieve]
  38. Antonsson, B., Montessuit, S., Lauper, S., Eskes, R., and Martinou, J.-C. (2000) Biochem. J. 345, 271–278[CrossRef][Medline] [Order article via Infotrieve]
  39. Mayer, L. D., Hope, M. J., and Cullis, P. R. (1986) Biochim. Biophys. Acta 858, 161–168[Medline] [Order article via Infotrieve]
  40. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466–468[Free Full Text]
  41. Muller, P., Schiller, S., Wieprecht, T., Dathe, M., and Herrmann, A. (2000) Chem. Phys. Lipids 106, 89–99[CrossRef][Medline] [Order article via Infotrieve]
  42. Basañez, G., Shinnar, A. E., and Zimmerberg, J. (2002) FEBS Lett. 532, 115–120[CrossRef][Medline] [Order article via Infotrieve]
  43. Polster, B. M., Kinnally, K. W., and Fiskum, G. (2001) J. Biol. Chem. 276, 37887–37894[Abstract/Free Full Text]
  44. Polster, B. M., Basañez, G., Young, M., Suzuki, M., and Fiskum, G. (2003) J. Neurosci. 23, 2735–2743[Abstract/Free Full Text]
  45. Suzuki, M., Youle, R. J., and Tjandra, N. (2000) Cell 103, 645–654[CrossRef][Medline] [Order article via Infotrieve]
  46. Desagher, S., Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S., Maundrell, K., Antonsson, B., and Martinou, J. C. (1999) J. Cell Biol. 144, 891–901[Abstract/Free Full Text]
  47. Eskes, R., Desagher, S., Antonsson, B., and Martinou, J.-C. (2000) Mol. Cell. Biol. 20, 929–935[Abstract/Free Full Text]
  48. Roucou, X., Montessuit, S., Antonsson, B., and Martinou, J.-C. (2002) Biochem. J. 368, 915–921[CrossRef][Medline] [Order article via Infotrieve]
  49. Wang, K., Yin, X. M., Chao, D. T., Milliman, C. L., and Korsmeyer, S. J. (1999) Genes Dev. 15, 2859–2869
  50. Ott, M., Robertson, J. D., Gogvadze, V., Zhivotovsky B., and Orrenius, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 1259–1263[Abstract/Free Full Text]
  51. Arnoult, D., Gaume, B., Karbowski, M., Sharpe, J. C., Cecconi, F., and Youle, R. J. (2003) EMBO J. 22, 4385–4399[CrossRef][Medline] [Order article via Infotrieve]
  52. Dufourcq, J., Faucon, J.-F., Fourche, G., Dasseaux, J.-L., Le Maire, M., and Gulik-Krzywicki, T. (1986) Biochim. Biophys. Acta 859, 33–48[Medline] [Order article via Infotrieve]
  53. Hardwick, J. M., and Polster, B. M. (2002) Mol. Cell 10, 963–965[CrossRef][Medline] [Order article via Infotrieve]
  54. Basañez, G. (2002) Cell. Mol. Life Sci. 59, 1478–1490[CrossRef][Medline] [Order article via Infotrieve]
  55. Chernomordik, L. V., Melikyan, G. B., and Chizmadzhev, Y. A. (1987) Biochim. Biophys. Acta 906, 309–352[Medline] [Order article via Infotrieve]
  56. Fuller, N., and Rand, R. P. (2001) Biophys. J. 81, 243–254[Medline] [Order article via Infotrieve]
  57. Szule, J. A., Fuller, N. L., and Rand, R. P. (2002) Biophys. J. 83, 977–984[Medline] [Order article via Infotrieve]
  58. Menestrina, G., Serra, M. D., and Prevost, G. (2001) Toxicon 39, 1661–1672[Medline] [Order article via Infotrieve]
  59. Zamzami, N., Brenner, C., Marzo, I., Susin, S. A., and Kroemer, G. (1998) Oncogene 30, 2265–2282
  60. Capano, M., and Crompton, M. (2002) Biochem. J. 367, 169–178[CrossRef][Medline] [Order article via Infotrieve]
  61. Lutter, M., Perkins, G. A., and Wang, X. (2001) BMC Cell Biol. 2, 22[CrossRef][Medline] [Order article via Infotrieve]
  62. Krajeski, S., Tanaka, S., Takayama, S., Schiber, M. J., Fenton, W., and Reed, J. C. (1993) Cancer Res. 53, 4701–4714[Abstract/Free Full Text]
  63. de Jong, D., Prins, F. A., Mason, D. Y., Reed, J. C., van Ommen, G. B., and Kluin, P. M. (1994) Cancer Res. 54, 256–260[Abstract/Free Full Text]
  64. Ardail, D., Privat, J. P., Egret-Charlier, M., Levrat, C., Lerme, F., and Louisot, P. (1990) J. Biol. Chem. 265, 18797–18802[Abstract/Free Full Text]
  65. Malisan, F., and Testi, R. (2003) Curr. Med. Chem. 10, 1573–1580[CrossRef][Medline] [Order article via Infotrieve]
  66. Matsko, C. M., Hunter, O. C., Rabinowich, H., Lotze, M. T., and Amoscato, A. A. (2001) Biochem. Biophys. Res. Commun. 287, 1112–1120[CrossRef][Medline] [Order article via Infotrieve]
  67. Iverson, S. L., Enoksson, M., Gogvadze, V., Ott, M., and Orrenius, S. (2004) J. Biol. Chem. 279, 1100–1107[Abstract/Free Full Text]
  68. Cullis, P. R., Tilcock, C. P., and Hope, M. J. (1999) in Membrane Fusion (Wilschut, J., and Hoekstra, D., eds) pp. 35–64, Marcel Dekker Inc., New York
  69. O'Connor, L. A., Strasser, A., O'Reilly, L. A., Hausmann, G., Adams, J. M., Cory, S., and Huang, D. (1998) EMBO J. 17, 384–395[CrossRef][Medline] [Order article via Infotrieve]
  70. Sheau, Y. H., Lin, P., and Hsueh, J. W. (1998) Mol. Endocrinol. 12, 1432–1440[Abstract/Free Full Text]
  71. Marani, M., Tenev, T., Hancock, D., Downward, J., and Lemoine, N. R. (2002) Mol. Cell. Biol. 11, 3577–3589
  72. Mouhamad, S., Besnault, L., Auffredou, M. T., Leprince, C., Bourgeade, M. F., Leca, G., and Vazquez, A. (2004) J. Immunol. 172, 2084–2091[Abstract/Free Full Text]
  73. Zhu, Y., Swanson, B. J., Wang, M., Hildeman, D. A., Schaefer, B. C., Liu, X., Suzuki, H., Mihara, K., Kappler, J., and Marrack, P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 7681–7686[Abstract/Free Full Text]
  74. Konishi, A., Shimizu, S., Hirota, J., Takao, T., Fan, Y., Matsuoka, Y., Zhang, L., Yoneda, Y., Fujii, Y., Skoultchi, A. I., and Tsujimoto, Y. (2003) Cell 114, 673–688[CrossRef][Medline] [Order article via Infotrieve]
  75. Chipuk, J. E., Kuwana, T., Bouchier-Hayes, L., Droin, N. M., Newmeyer, D. D., Schuler, M., and Green, D. R. (2004) Science 13, 1010–1014
  76. Leu, J. I., Dumont, P., Hafey, M., Murphy, M. E., and George, D. L. (2004) Nat. Cell Biol. 6, 443–450[CrossRef][Medline] [Order article via Infotrieve]
  77. Lin, Z., Kolluri, S. K., Lin, F., Liu, W., Hang, Y.-H., Cao, X., Dawson, M. I., Reed, J. C., and Zhang, X. (2004) Cell 116, 527–541[CrossRef][Medline] [Order article via Infotrieve]
  78. Epand, R. F., Martinou, J.-C., Montessuit, S., and Epand, R. M. (2003) Biochemistry 42, 14576–14582[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Biol. CellHome page
B. Schafer, J. Quispe, V. Choudhary, J. E. Chipuk, T. G. Ajero, H. Du, R. Schneiter, and T. Kuwana
Mitochondrial Outer Membrane Proteins Assist Bid in Bax-mediated Lipidic Pore Formation
Mol. Biol. Cell, April 15, 2009; 20(8): 2276 - 2285.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Etxebarria, O. Terrones, H. Yamaguchi, A. Landajuela, O. Landeta, B. Antonsson, H.-G. Wang, and G. Basanez
Endophilin B1/Bif-1 Stimulates BAX Activation Independently from Its Capacity to Produce Large Scale Membrane Morphological Rearrangements
J. Biol. Chem., February 13, 2009; 284(7): 4200 - 4212.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Qian, W. Wang, L. Yang, and H. W. Huang
Structure of transmembrane pore induced by Bax-derived peptide: Evidence for lipidic pores
PNAS, November 11, 2008; 105(45): 17379 - 17383.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Yamaguchi, N. T. Woods, J. F. Dorsey, Y. Takahashi, N. R. Gjertsen, T. Yeatman, J. Wu, and H.-G. Wang
Src Directly Phosphorylates Bif-1 and Prevents Its Interaction with Bax and the Initiation of Anoikis
J. Biol. Chem., July 4, 2008; 283(27): 19112 - 19118.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Montero, A. Morales, L. Llacuna, J. M. Lluis, O. Terrones, G. Basanez, B. Antonsson, J. Prieto, C. Garcia-Ruiz, A. Colell, et al.
Mitochondrial Cholesterol Contributes to Chemotherapy Resistance in Hepatocellular Carcinoma
Cancer Res., July 1, 2008; 68(13): 5246 - 5256.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
L. Zhou and D. C. Chang
Dynamics and structure of the Bax-Bak complex responsible for releasing mitochondrial proteins during apoptosis
J. Cell Sci., July 1, 2008; 121(13): 2186 - 2196.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
O. Terrones, A. Etxebarria, A. Landajuela, O. Landeta, B. Antonsson, and G. Basanez
BIM and tBID Are Not Mechanistically Equivalent When Assisting BAX to Permeabilize Bilayer Membranes
J. Biol. Chem., March 21, 2008; 283(12): 7790 - 7803.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. J. Siskind, L. Feinstein, T. Yu, J. S. Davis, D. Jones, J. Choi, J. E. Zuckerman, W. Tan, R. B. Hill, J. M. Hardwick, et al.
Anti-apoptotic Bcl-2 Family Proteins Disassemble Ceramide Channels
J. Biol. Chem., March 14, 2008; 283(11): 6622 - 6630.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. Wang, Y. Wang, H. P. Kim, K. Nakahira, S. W. Ryter, and A. M. K. Choi
Carbon Monoxide Protects against Hyperoxia-induced Endothelial Cell Apoptosis by Inhibiting Reactive Oxygen Species Formation
J. Biol. Chem., January 19, 2007; 282(3): 1718 - 1726.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. J. Oh, S. Barbuto, K. Pitter, J. Morash, L. D. Walensky, and S. J. Korsmeyer
A Membrane-targeted BID BCL-2 Homology 3 Peptide Is Sufficient for High Potency Activation of BAX in Vitro
J. Biol. Chem., December 1, 2006; 281(48): 36999 - 37008.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. A. Sobko, E. A. Kotova, Y. N. Antonenko, S. D. Zakharov, and W. A. Cramer
Lipid Dependence of the Channel Properties of a Colicin E1-Lipid Toroidal Pore
J. Biol. Chem., May 19, 2006; 281(20): 14408 - 14416.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
Y. Takahashi, M. Karbowski, H. Yamaguchi, A. Kazi, J. Wu, S. M. Sebti, R. J. Youle, and H.-G. Wang
Loss of Bif-1 Suppresses Bax/Bak Conformational Change and Mitochondrial Apoptosis
Mol. Cell. Biol., November 1, 2005; 25(21): 9369 - 9382.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
L. M. Dejean, S. Martinez-Caballero, L. Guo, C. Hughes, O. Teijido, T. Ducret, F. Ichas, S. J. Korsmeyer, B. Antonsson, E. A. Jonas, et al.
Oligomeric Bax Is a Component of the Putative Cytochrome c Release Channel MAC, Mitochondrial Apoptosis-induced Channel
Mol. Biol. Cell, May 1, 2005; 16(5): 2424 - 2432.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
R. Rotem, A. Heyfets, O. Fingrut, D. Blickstein, M. Shaklai, and E. Flescher
Jasmonates: Novel Anticancer Agents Acting Directly and Selectively on Human Cancer Cell Mitochondria
Cancer Res., March 1, 2005; 65(5): 1984 - 1993.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. M. Polster, G. Basanez, A. Etxebarria, J. M. Hardwick, and D. G. Nicholls
Calpain I Induces Cleavage and Release of Apoptosis-inducing Factor from Isolated Mitochondria
J. Biol. Chem., February 25, 2005; 280(8): 6447 - 6454.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
S. Lucken-Ardjomande and J.-C. Martinou
Newcomers in the process of mitochondrial permeabilization
J. Cell Sci., February 1, 2005; 118(3): 473 - 483.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. J. Oh, S. Barbuto, N. Meyer, R.-S. Kim, R. J. Collier, and S. J. Korsmeyer
Conformational Changes in BID, a Pro-apoptotic BCL-2 Family Member, upon Membrane Binding: A SITE-DIRECTED SPIN LABELING STUDY
J. Biol. Chem., January 7, 2005; 280(1): 753 - 767.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Enoksson, J. D. Robertson, V. Gogvadze, P. Bu, A. Kropotov, B. Zhivotovsky, and S. Orrenius
Caspase-2 Permeabilizes the Outer Mitochondrial Membrane and Disrupts the Binding of Cytochrome c to Anionic Phospholipids
J. Biol. Chem., November 26, 2004; 279(48): 49575 - 49578.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/29/30081    most recent
M313420200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Terrones, O.
Right arrow Articles by Basañez, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Terrones, O.
Right arrow Articles by Basañez, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement