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Mitochondrial intermembrane junctional complexes and their involvement in cell death
Biochimie 84 (2002) 143–152
Mitochondrial intermembrane junctional complexes and their involvement in cell death Martin Crompton *, Emma Barksby, Nicholas Johnson, Michela Capano Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK Received 31 August 2001; accepted 6 November 2001
Abstract Mitochondria establish contact sites between the inner and outer membranes. The contact sites are held together by junctional complexes of the adenine nucleotide translocase (ANT; inner membrane) and the voltage-dependent anion channel (VDAC; outer membrane). The junctional complexes act as multifunctional recruitment centres, binding a range of proteins according to the function to be executed. Some of these, involving kinases and enzymes of lipid transfer, are readily understood as ongoing functions in energy and lipid metabolism. But the roles of other proteins recruited to the junctional complexes are less well defined. Here, we focus on the complexes formed with Bax and with cyclophilin-D, and their possible roles in apoptotic and necrotic cell death. We have isolated both types of complexes using glutathione-S-transferase fusion proteins of Bax and of cyclophilin-D. The VDAC/ANT/cyclophilin-D complex reconstitutes Ca2+- and cyclosporin A-sensitive permeability transition pore activity when incorporated into proteoliposomes. The complex forms readily in the absence of factors required for pore opening in isolated mitochondria, suggesting that these factors act on the preexisting complex, rather than drive its assembly, and that the complex is a physiological entity in healthy cells. © 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Permeability transition; Cyclophilin-D; Bax; Apoptosis; Necrosis
1. Introduction The voltage-dependent anion channel (VDAC) and adenine nucleotide translocase (ANT) are mitochondrial proteins that perform well-established roles in the daily life of the cell. VDAC, or mitochondrial porin, is a large H2Ofilled pore that allows low Mr solutes to permeate freely across the outer membrane and to gain access to the solute-specific transport systems of the inner membrane. ANT mediates ADP–ATP exchange across the inner membrane, essential for the basic bioenergetic function of the organelle in supplying ATP to the cytosol. These two proteins also form a complex that establishes contact sites between the outer and inner membranes. Since VDAC and ANT comprise the major proteins of their respective mem-
Abbreviations: ANT, adenine nucleotide translocase; CSA, cyclosporin A; CyP-D, cyclophilin-D; GST, glutathione-S-transferase; PT, permeability transition; VDAC, voltage dependent anion channel * Corresponding author. Tel.: +44-20-7679-2207; fax: +44-20-7679-7193. E-mail address: [email protected] (M. Crompton).
branes many such junctional complexes can be formed. The junctional complexes in turn recruit other proteins. Some of these proteins perform readily understood functions that facilitate the export of mitochondrial high energy phosphates to the cytosol. But other proteins attracted to the junctional complexes are implicated in cell death, and here the role of the VDAC/ANT complex is more obscure. These proteins include the proapoptotic Bax, which migrates to mitochondria under apoptotic stimuli, and cyclophilin-D (CyP-D), an essential constituent of the permeability transition (PT) pore. This article outlines the evidence for the formation of VDAC/ANT complexes with Bax and with CyP-D, in vitro and in vivo, and considers their possible roles in apoptotic and necrotic cell death.
2. VDAC/ANT complexes in energy and lipid metabolism Contact sites between the mitochondrial inner and outer membranes were first observed in electron micrographs of
© 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 1 3 6 8 - 8
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isolated mitochondria over 30 years ago [1]. Subsequently, the outer membrane protein VDAC was found to be preferentially localised at these sites [2]. The capacity of VDAC to bind strongly to the inner membrane was first revealed in the work of Ono and Tuboi [3], who synthesised radiolabelled VDAC in vitro and imported the protein into isolated mitochondria. Following digitonin fractionation and separation of the inner and outer membrane fractions, a large part of the VDAC was recovered fused with the inner membrane. Subsequently, McEnery et al. [4] purified a complex of VDAC and the inner membrane protein ANT from detergent extracts of mitochondria thereby establishing the molecular nature of the contact sites as junctional complexes of VDAC and ANT. It has been estimated that VDAC accounts for about 0.3% of mitochondrial protein [5], which is equivalent to about 100 pmol VDAC mg–1 of mitochondrial protein. This amounts to about one-tenth of the amount of ANT, so that only a small fraction of ANT can be complexed to VDAC in this way. VDAC/ANT complexes can recruit a range of other proteins indicating that they participate in a number of cellular functions. Some of this relates to the efficiency of cellular energy metabolism. Mitochondria provide the bulk of cellular ATP in most types of cell. Yet ADP is poorly diffusible in the cytoplasm, since most is bound to protein. Mitochondria need to work in tandem, therefore, with systems for the efficient distribution of ‘ATP energy’
through the cell. One way is to generate a more diffusible energy currency. The VDAC/ANT complex binds creatine kinase in the mitochondrial intermembrane space [6]. This creates a microdomain in which ATP translocated via ANT generates creatine phosphate, which is then exported to the cytosol via VDAC. In the cytosol, creatine and creatine phosphate are highly diffusible. A second ploy is to bring sites of ATP usage in the cytosol very close to the sites of mitochondrial ATP export. Thus, VDAC recruits hexokinases and glycerol kinase from the cytosol (Fig. 1). Hexokinase is bound according to its catalytic activity. Evidently, the binding occurs to the junctional complexes, since hexokinase copurifies with the VDAC/ANT complex, and immunogold studies show enrichment of hexokinase at contact sites between the outer and inner membranes [7]. In addition, cells have evolved a sophisticated system for the distribution of the primal mitochondrial energy currency, the inner membrane proton electrochemical gradient. Thus, mitochondria in situ can form tight intermitochondrial junctions, providing ionic continuity between the matrix spaces of the conjugated mitochondria, and allowing the proton electrochemical gradient to be conducted from one mitochondrion to the next. In many cells, conjugated mitochondria form large interconnected networks for the efficient distribution of the proton motive force throughout the cell [8]. Immunogold studies with antiVDAC antibodies show localisation of gold particles at the junctions between
Fig. 1. VDAC/ANT complexes as multifunctional recruitment centres. VDAC and ANT bind to each other and to a number of other proteins with diverse functions. Under pathological conditions, one of these complexes (VDAC/ANT/CyP-D), can deform (asterisk) into the PT pore, which permeabilises the inner membrane to low Mr solutes.
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closely apposed mitochondria, indicating enrichment of VDAC, or exposure of the N-terminal epitope, at these sites. Either way the data point to a role for VDAC in establishing the intermitochondrial junctions [9]. The nature of the ion conduit between conjugated mitochondria has not been resolved; but VDAC/ANT complexes can form open pores in the inner membrane when associated with CyP-D (the PT pore, below), and it would seem relevant to investigate the possible, physiological function of the VDAC/ANT/CyP-D complex in establishing these conduits [10]. The junctional complexes have also been implicated in lipid metabolism. Thus, the complex copurifies with the peripheral benzodiazepine receptor [4]; this is believed to facilitate the transfer of cholesterol to the inner membrane in steroidogenic cells [11]. An enrichment of carnitine-acyl transferases at the contact sites has also been reported [12]. Different functions of the VDAC/ANT complexes may utilise different isoforms. Three ANT isoforms have been identified as products of different genes [13]. Three VDAC isoforms have also been recognised [14]. There is evidence that hexokinases bind specifically to VDAC1 [7].
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3. VDAC/ANT interaction with cyclophilin-D Quite a pure form of the VDAC/ANT complex can be obtained using CyP-D (Fig. 2). Since CyP-D is resident in the mitochondrial matrix [15], it presumably binds to the ANT component of the VDAC/ANT complex. All indications are that CyP-D binds to the complex very tightly indeed since, in the ‘pull down’ experiments (Fig. 2), the sepharose beads and attached proteins can be washed repeatedly without detectable loss of VDAC/ANT [16]. VDAC and ANT have almost the same Mr values, but on long SDS–PAGE gels the two proteins can be separated, and from the relative intensities of Coomassie blue staining, it is apparent that VDAC and ANT are retained in the approximate ratio 1:1. The reason why CyP-D binds to ANT is not clear. But the amount of CyP-D in heart mitochondria, for example, is < 5% of the amount of ANT, and only a small fraction of ANT in the inner membrane can be complexed with CyP-D. Evidently, CyP-D is not involved in the basic function of ANT in mediating ADP/ATP exchange across the inner membrane.
Fig. 2. VDAC/ANT complexes bind CyP-D and Bax. Detergent extracts of heart mitochondrial membranes were mixed with GST–CyP-D (b–d) or GST–Bax (e, g) fusion proteins, or GST (f, h), immobilised on glutathione-S-sepharose. The sepharose beads were washed five times, and then the attached proteins were displaced by glutathione and analysed by SDS–PAGE and immunoblotting. Lane (a), unfractionated membrane extract (Coomassie blue stained). Lane (b), proteins displaced from the GST–CyP-D matrix, i.e. GST–CyP-D (upper band, 48 kDa) and superimposed VDAC and ANT (lower band, 32 kDa), Coomassie blue stained. Lanes (c) and (d), ANT and VDAC immunoblots, respectively, of (b). Lanes (e) and (f), VDAC immunoblots of proteins displaced from the GST–Bax matrix (e) and a GST matrix (f). Lanes (g) and (h), ANT immunoblots of proteins displaced from the GST–Bax matrix (g) and a GST matrix (h). Lanes (b–d) and lanes (e–h) were obtained with different membrane extracts. The data show that both GST–CyP-D and GST–Bax selectively ‘pull down’ VDAC/ANT complexes from mitochondrial membrane extracts, whereas GST does not, confirming that the VDAC/ANT complexes bind to CyP-D and to Bax. Further details of protocols are given in [16].
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Cyclophilins are found throughout nature and present a number of isoforms with defined subcellular locations, e.g. CyP-A and CyP-40 (cytosol), CyP-B and CyP-C (endoplasmic reticulum), CyP-D (mitochondria). They catalyse the rotation of Xaa–Pro peptide bonds in proteins. In general most peptide groups assume the trans configuration, which is more stable (about 8 kJ mol–1) because of steric hindrance. The energy difference between the cis and trans configurations of peptidylprolyl bonds is much less, how ever, so that a significant fraction of Pro residues in proteins follow a cis peptide bond. The peptidylprolyl cis–transisomerase (PPIase) activity of CyP facilitates the folding of nascent proteins. For example, the folding of newly imported mitochondrial proteins is impeded in deletion mutants (yeast) lacking mitochondrial cyclophilin [17]. But CyPs also form complexes with mature, fully folded proteins. Examples include CyP-40 and the oestrogen receptor [18], CyP-A and the antioxidant protein, Aop1 [19] and, as described above, CyP-D and VDAC/ANT. In these cases, it seems reasonable to consider that the CyP facilitates conformational change of the complexed protein by catalysing the rotation of surface-exposed peptidyl prolyl bonds. In addition, cyclophilins (at least, CyP-A in E. coli ) preferentially bind cis-Pro isomers. Thus, CyPs may catalyse the formation of, and stabilise, a particular conformation of the target protein [20]. If CyP-D establishes a particular conformation of ANT then, clearly, it would need to bind to ANT via its active (PPIase) site. In this case, CyP-D binding to ANT would be expected to be blocked by CSA, since CSA occludes the active site in CyP. Moreover, CSA occlusion of the active site is accompanied by very little conformational change in CyP so that CSA inhibition of ANT binding to CyP could reasonably be interpreted that the active site is involved. It has been reported that CyP-D binding to ANT alone is reduced by CSA [21]. In our hands, however, CSA produced negligible inhibition of CyP-D binding to the VDAC/ANT complex [16]. In subsequent experiments (unpublished), we have detected some reduction in VDAC/ANT binding to CyP-D when the [CSA] is raised to around 20 µM, which is > 104 times the KD value of free CyP-D and CSA. If this does reflect competition between CSA and VDAC/ANT for the PPIase active site, then it is clear that the VDAC/ANT complex, at least when isolated, must have a very high affinity for CyP-D. Alternatively, the VDAC/ANT complex may bind to a site on CyP-D distal from the active site, which would explain the poor ability of CSA to prevent VDAC/ANT binding. This particular issue has not been settled. The use of a photoactive CSA derivative has allowed the interactions between CyP-D, CSA and competing proteins to be analysed in intact mitochondria [22,23]. Mitochondria were preincubated with a radiolabelled, photoactive CSA in the dark, allowing it to equilibrate with CyP-D, before photactivation and covalent radiolabelling of the CyP-D associated with the CSA (as shown in Fig. 3). We used a
CSA derivative with a photoactive group and spacer at position 8 of the ring; substituents here do not interfere with CSA binding to CyP (involving residues 1–3 and 9–11), and the derivative photolabels CyP quite efficiently (e.g. [22,23]). If CyP were substantially free in the matrix, then its photolabelling would not be expected to be influenced by reagents that interact with ANT. However, we found that photolabelling of intramitochondrial CyP-D by photoactive CSA was markedly enhanced by extramitochondrial ADP [22,23] (Fig. 3). Since the interaction of free CyP-D with CSA is unaffected by ADP, the result means that some CyP-D in mitochondria must be associated with an ADPbinding protein. Moreover, this protein would need to bind ADP on the outside of the inner membrane. The obvious candidate is ANT. Thus, we arrive at the rather important conclusion that a fraction of ANT (presumably VDAC/ANT) is associated with CyP-D in isolated mitochondria. In other words, it appears that the VDAC/ANT/CyP-D complex may form under normal physiological conditions.
Fig. 3. CyP-D associates with ANT in intact mitochondria. Upper panel: a radiolabelled, photoactive CSA derivative was reacted with isolated heart mitochondria in the presence (closed circles) and absence (open circles) of 1 mM ADP. The medium also contained 1 mM EGTA to remove Ca2+. The mitochondrial membranes were extracted with 6% Chaps as detergent, and the extracts were analysed by gel filtration. Lower panel: CyP-D (PPIase) was assayed in the fractions obtained by gel filtration. The data show that ADP promotes CSA interaction (photolabelling) with CyP-D (recovered in fractions 9 and 10), indicating that CyP-D associates with an ADP-binding protein (ANT) in intact mitochondria. Further details of protocols are given in [22,23].
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Other observations made with the isolated VDAC/ANT/CyP-D complex are consistent with the notion that this complex is a physiological entity. In particular, assembly of the complex does not appear to be influenced by factors associated with PT pore opening. Thus the retention of VDAC/ANT complexes by CyP-D (GST fusion; as in Fig. 2) is unaffected by the presence of Ca2+, ADP, ATP, and dithiothreitol to maintain reduced thiols ([16]; unpublished data). Although low ATP, high Ca2+ and oxidative stress (thiol oxidation) trigger PT pore opening, these studies with the isolated complex suggest that these factors act on the preexisting VDAC/ANT/CyP-D complex, rather than drive its assembly from the separate components.
4. VDAC/ANT/CyP-D complexes and the PT pore PT pore activity has been reconstituted from purified VDAC, ANT, and recombinant CyP-D [16] (Fig. 4). VDAC/ANT/CyP-D complexes in Chaps were mixed with phospholipids and incorporated into proteoliposomes by detergent dialysis. Fluorescein sulphonate was introduced into the vesicles (brief sonication) and the vesicles were separated out by gel filtration. PT pore opening was assayed from the loss of fluorescein sulphonate to the medium (increased fluorescence due to decreased quenching on dilution). CSA-sensitive PT pore opening was observed on addition of Ca2+ in the presence of Pi [16] (Fig. 4). These conditions correspond to those that activate the PT pore in isolated mitochondria when both Ca2+ and Pi are required [24]. These results provide good evidence that VDAC, ANT and CyP-D are all that is required to form the PT pore. Of these, CyP-D is clearly a component, since the reconstituted activity is blocked by CSA. But the reconstitutions do not tell us whether the PT pore comprises ANT/CyP-D or VDAC/ANT/CyP-D, or both. PT pore activity has also been reconstituted from protein fractions that contain VDAC, ANT and CyP-D along with a number of other proteins including hexokinase [25]. Hexokinase associates reversibly with VDAC according to its activity. Thus, hexokinase binding to VDAC is increased in the presence of the substrates ATP and glucose and is decreased in the presence of the product inhibitor glucose6-phosphate. ATP and glucose were found to inhibit the reconstituted PT pore activity, and the inhibition was reversed by glucose-6-phosphate [25]. The obvious interpretation is that the reconstituted PT pore contained VDAC as the site of hexokinase attachment, and that attached hexokinase blocked the pore. These findings can be related to earlier observations on the capacity of extramitochondrial adenine nucleotides to prevent pore opening in isolated mitochondria. In the presence of Mg2+, > 1.5 mM ATP (buffered enzymatically) was required to block PT pore opening (inner membrane depolarisation) [26]. ADP (1 mM; buffered enzymatically) produced no inhibition of PT pore opening. These concentrations bear no relation to the binding affinities of
Fig. 4. Reconstitution of PT pore activity from purified VDAC, ANT, and recombinant CyP-D. VDAC, ANT and CyP-D (GST fusion) were purified as in Fig. 2, lane (b), and incorporated into proteoliposomes containing fluorescein sulphonate. The vesicles were incubated in medium containing 1 mM Pi and in the presence (lower trace) and absence of 1 µM CSA. Ca2+ of 100 µM was added at the arrows. Increase in fluorescence indicates release of fluorescein from the vesicles (decreased fluorescence quenching on dilution). The data show that CSA-sensitive PT pore opening can be reconstituted from purified VDAC, ANT and CyP-D. Further details of protocols are given in [16].
ANT for ADP and ATP and suggest that the inhibitory action of ATP may have been mediated by kinase attachment to VDAC. From this it seems that the VDAC/ANT/CyP-D complex can form the PT pore. But it does not rule out the possibility that ANT/CyP-D can do likewise.
5. VDAC/ANT complexes and the action of Bax Mitochondria amplify the apoptotic signalling pathway by releasing apoptogenic proteins from the intermembrane space. These proteins include cytochrome c, AIF (apoptosis inducing factor) and some procaspases. Current indications are that large holes or breaks appear in the mitochondrial
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outer membrane, since the apoptogenic proteins are released along with other intermembrane space proteins, some of which are quite large, e.g. sulphite oxidase [27]. Outer membrane permeabilisation is elicited by proapoptotic Bcl-2 family members, notably Bid, Bak and Bax. In non-apoptosing cells, these proteins reside in the cytosol, but under apoptotic stimuli they migrate to mitochondria leading to outer membrane permeabilisation. Bid is resident in the cytosol as an inactive precurser; this is cleaved by caspase-8 to produce a 15 kDa C-terminal fragment that binds tightly to mitochondria. Bax is retained in the cytosol of healthy cells as an inactive monomer. The mechanisms that trigger Bax translocation to mitochondria are unclear, but it seems that on association with mitochondria it undergoes conformational change [28] and oligomerisation [29]. These changes may be facilitated by Bid on the mitochondrial surface. The question arises whether these proapoptotic Bcl-2 family proteins target specific receptor proteins on the mitochondrial outer membrane. As yet, no such receptor proteins have been unambiguously identified, and the question remains open. But there are indications that the junctional complexes may be involved at some stage in the action of Bax on mitochondria. Shimizu et al. [30] reported various lines of evidence that Bax can interact with VDAC. Thus recombinant Bax coimmunoprecipitates with VDAC. VDAC normally allows free permeation of sucrose, but VDAC proteoliposomes are poorly permeable to sucrose at low pH (pH 5.2) and, under these conditions, sucrose permeability is markedly enhanced by Bax, again indicating Bax–VDAC interactions. The VDAC liposomes also became permeable to cytochrome c on addition of Bax, although larger proteins were retained. Other investigators have used partially purified VDAC/ANT preparations containing Bax. As in isolated mitochondria, the ANT ligand atractylate induces PT pore opening in proteoliposomes containing the VDAC/ANT complex, evidently by establishing an ANT conformation prone to distortion into an open pore state. Immunodepletion of Bax from these complexes led to a loss of atractylate induced pore activity [31], pointing to an interaction , directly or indirectly, of Bax with ANT. Work in our laboratory broadly corroborates the above data. Thus, VDAC/ANT complexes are selectively retained by GST–Bax fusion protein immobilised on glutathione-Ssepharose (Fig. 2). In these experiments, we have detected no retention of CyP-D, suggesting that Bax/VDAC/ANT and VDAC/ANT/CyP-D are alternative complexes of VDAC/ANT. Other studies implicate the VDAC/ANT complex in apoptosis, without indicating the capacity in which it may be involved. Bauer et al. [32] identified ANT-1 as a proapoptotic protein from screening an expression library for dominant apoptosis-inducing genes. Overexpression of ANT-1 led to spontaneous apoptosis in a number of cell lines, whereas transfection of ANT-2 was innocuous. The
apoptogenic action of ANT-1 was unrelated to its basic function in ADP–ATP exchange, since transport-deficient mutants were also proapoptotic. Other indications have been derived from studies of viral and bacterial infected cells. The protein, vMIA, produced by human cytomegalovirus, blocks fas-mediated apoptosis. It inhibits cytochrome c release to the cytosol, but does not inhibit Bid cleavage, indicating that it blocks the action of truncated Bid on mitochondria. In line with this, vMIA locates to mitochondria (Immunogold) of transfected cells, and it coimmunoprecipitates ANT from cell extracts [33]. The proapoptotic HIV 1 protein R also binds ANT (surface plasmon resonance studies) [34]; the binding is inhibited by a peptide corresponding to a segment of ANT (amino acids 104–116) that loops into the intermembrane space. Other apoptoticregulatory, viral and bacterial proteins have been recognised that target VDAC. HBX is produced by hepatitis B virus infected liver cells, leading to apoptotic cell death. When transfected into cells, HBX colocalises with mitochondria (immunofluorescence) and coimmunoprecipitates VDAC from cell extracts [35]. Neisseria mengintidis infection inhibits apoptosis; the major outer membrane protein, Por B, does likewise when transfected into cells. Again, this protein translocates primarily with mitochondria and coimmunoprecipitates with VDAC from cell extracts [36]. In spite of these positive indications for interactions between Bax and VDAC/ANT, it is unclear at what step any such interaction takes place. On binding to mitochondria Bax changes conformation and oligomerises. The conformational changes lead to exposure of the N-terminal region, which becomes accessible to trypsin and to antibodies [28,37]. At the same time, Bax becomes cross-linkable, indicating oligomerisation [38]. This appears to be an essential step in the apoptogenic function of Bax. Cells transfected with a chimeric FK506 binding protein-Bax become apoptotic when dimerisation is promoted with a bivalent ligand [38]. Bax oligomers produced in vitro by the appropriate detergent are better able to release cytochrome c from isolated mitochondria. Antonsson and coworkers [29] have isolated the mitochondrial Bax oligomers from detergent extracts of staurosporine-treated cells. The oligomers/complexes displayed apparent Mr values of 96 and 260 kDa on gel filtration, but contained no ANT or VDAC. Thus, although Bax may well interact with the junctional complexes at some stage in its action on mitochondria, that stage has yet to be identified. The fact that Bax can interact (experimentally, at least) with the VDAC/ANT complex may account for numerous observations that added Bax promotes PT pore activity in isolated mitochondria (e.g. [39]). This behaviour has sometimes been interpreted to indicate that the physiological action of Bax in releasing cytochrome c is mediated via PT pore opening. A more conservative interpretation would be that Bax can bind to components of the PT pore (in line with the data discussed above) and, in so doing, modifies their properties, including ‘deformation’ of the complex into the
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PT pore under pathological conditions. It does not necessarily mean that PT pore opening is part of the normal action of Bax. In line with this, Bax can release cytochrome c from isolated mitochondria in the absence of Ca2+ [40], the basic trigger for PT pore opening, and the release occurs without the increase in matrix volume that accompanies PT pore opening in vitro [27].
6. The VDAC/ANT/CyP-D complex in ischaemic injury In isolated mitochondria, deformation of the VDAC/ANT/Cyp-D complex into the PT pore requires a particularly stringent set of conditions, namely, high intramitochondrial [Ca2+], high [Pi], oxidative stress, and low extramitochondrial [ATP] (reviewed in Ref. [10]). These factors operate synergistically, but in isolated mitochondria, at least, two conditions are paramount, namely high [Ca2+], and low [ATP]. In our experience, PT pore opening (as measured unambiguously by radiolabelled sucrose entry into the matrix space) has an absolute requirement for Ca2+,
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and PT pore opening under any condition is completely blocked by physiological concentrations of ATP [10]. If similar restrictions apply in vivo, and there is no reason to suggest that they do not, then we may anticipate PT pore opening only in extreme pathological states. Such conditions are found in ischaemia/reperfusion-induced injury. Ischaemia leads to ATP dissipation with consequent rises in cell Ca2+ and Pi, and oxidative stress occurs on reperfusion (reviewed in Ref. [10]). This striking correlation between the conditions required for PT pore opening and those that are known to provoke reperfusion injury first led to the hypothesis that PT pore opening may be a key lesion in this form of injury [41,42] (Fig. 5). The hypothesis stands up to fairly close scrutiny regarding the actual changes in ATP and Ca2+ that are required for injury and for PT pore opening. In heart cells irreversible injury follows when about two-thirds of cell ATP have been dissipated [43], which agrees reasonably well with the [ATP] below which PT pore opening can be detected in isolated mitochondria (1.5 mM ATP) [26]. Mitochondrial Ca2+ overload occurs when mitochondria are exposed to
Fig. 5. A model for the involvement of the PT pore in necrotic and apoptotic cell death induced by ischaemia and reperfusion. Ischaemia leads to ATP dissipation and consequent rises in cell Ca2+ and Pi. On reperfusion, the increased cytosolic Ca2+ leads to mitochondrial Ca2+ overload. These factors together with oxidative stress trigger PT pore opening. If widespread, PT pore opening overwhelms the capacity of the cell to maintain ATP and the cells become necrotic. If PT pore opening is limited (localised), allowing maintenance of cell ATP, outer membrane rupture and release of apoptogenic proteins from the intermembrane space may be sufficient to initiate the caspase cascade, leading to apoptotic cell death. The degree of PT pore opening may be a factor determining the proportion of necrotic and apoptotic cell death.
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maintained Ca2+ levels (not transients) exceeding 1–2 µM (discussed in Ref. [10]). This limiting value of resting cytosolic free Ca2+ consistent with mitochondrial viability correlates well with the [Ca2+] required to trigger irreversible cell injury (cardiomyocytes). Thus, when aequorinloaded (Ca2+ indicator) myocytes are subjected to anoxia, there is an eventual progressive rise in resting cytosolic Ca2+ [44]. The rise is reversed on reoxygenation provided that a limit of 1–3 µM resting Ca2+ is not exceeded. If this limiting value is exceeded, however, the myocytes undergo lethal injury. The hypothesis is also supported by various studies of PT pore opening in ischaemia, anoxia, and related conditions. In particular, the PT pore inhibitor. CSA, protects against ischaemia- or anoxia-induced injury in a number of systems (reviewed in Ref. [10]). The groups of Halestrap [45] and Lemasters [46] have demonstrated the movement of otherwise impermeant small molecules (e.g. 2-deoxyglucose, calcein) across the mitochondrial inner membrane in ischaemia/anoxia-stressed cells, consistent with PT pore opening. Ischaemia/reperfusion produces both necrotic and apoptotic cell death. Characteristically, the regions that suffer the most severely ischaemic conditions become necrotic, whereas the less stressed regions that escape necrosis succumb subsequently by apoptosis. Evidently, a period of ischaemia can engage the apoptotic signalling pathway in some way. In seeking to resolve the underlying mechanism it is important to distinguish between the physiological process of programmed cell death, which specifically selects apoptosis (‘authorised’ apoptosis), and the ‘accidental’ apoptosis that accompanies necrosis in ischaemic disease. As discussed above, ‘authorised’ apoptosis might utilise the VDAC/ANT complex in some capacity. But, it seems unlikely that this would involve the complex acting as a PT pore, since this would introduce the possibility of a rapid ATP dissipation, leading to cell necrosis, the undesired outcome. But when apoptosis (‘accidental’) and necrosis coexist, as they do in ischaemic disease, it is conceivable that they are a product of the same mechanism [47]. Extensive PT pore opening, producing rapid ATP dissipation, overwhelming the ATP generating capacity of the cell, would produce a rapid loss of cell viability. But a limited degree of PT pore opening could be tolerated. Cellular energy transduction would be less efficient, but there would be no immediate threat to the life of the cell provided that cellular ATP was maintained. However, any rupture of the mitochondrial outer membrane breakage under such conditions might be sufficient to trigger the caspase cascade (Fig. 5). PT pore opening in isolated mitochondria produces matrix swelling, which can result in physical rupture of the outer membrane [39]. Might limited PT pore opening in vivo do the same? Firstly, limited PT pore opening has been detected in vivo. When heart cells are loaded with positively charged rhodamines, the dyes accumulate electrophoretically according to the magnitude of the mitochondrial inner mem-
brane potential. This allows the potential to be monitored by fluorescence imaging techniques. The dyes also photodecompose, producing reactive oxygen species in the mitochondrial matrix. Under these conditions, a small number of mitochondria (e.g. in cardiomyocytes, astrocytes) are seen to undergo occasional, transient depolarisations. The depolarisations require mitochondrial Ca2+ uptake [48] and are blocked by CSA [49], indicating that they are due to transient PT pore opening. Most probably, the mitochondria which exhibit this behaviour are those located close to the Ca2+ release channels of the sarco(endo)plasmic reticulum, since the depolarisations are also blocked by agents that deplete the reticulum of Ca2+ (thapsigargin) and that inhibit Ca2+ release from the reticulum (ryanodine) [48]. Secondly, there is evidence that transient PT pore opening is sufficient to release cytochrome c. Gogvadze et al. [50] reported that relatively low doses of Ca2+, barely sufficient to cause detectable loss of the inner membrane potential in populations of isolated mitochondria, can induce cytochrome c release via PT pore opening. It was concluded that the behaviour reflected variations in the threshold for PT pore opening from one mitochondrion to the next, such that with limiting Ca2+, PT pore opening and cytochrome c release occurred from those most susceptible, whilst the potential (tetraphenylphosphonium ion uptake) was maintained by the resistant fraction. However, an alternative interpretation is possible based on the previous work in this laboratory and the phenomenon of transient PT pore opening [24]. In essence, by entrapping Ca2+ buffers in the matrix space, and with Ca2+ transport inhibitors present, it can be shown that PT pores open and close continuously at a frequency determined by the matrix [Ca2+], and that there is no critical [Ca2+] threshold. The relation between PT pore opening and matrix free [Ca2+] is linear up to 20 µM Ca2+ [24]. At any matrix [Ca2+] there is a dynamic steady state distribution between those mitochondria that have open pores and those that do not. At low Ca2+ loads, when only a small fraction of mitochondria have open pores at any point in time, the potential is maintained by the remainder. But added radiolabelled sucrose eventually enters all mitochondria (transient PT pore opening) and does so according to a single rate constant, i.e. the mitochondria behave homogeneously. When considered together, therefore, the two pieces of work [24,50], both carried out with liver mitochondria, indicate that transient PT pore opening can release cytochrome c from the intermembrane space of isolated mitochondria. The critical question, as yet unan swered, is whether transient PT pore opening in vivo can do the same.
Acknowledgements The authors’ work described was supported by the British Heart Foundation (awards PG /2000009, PG / 96162, FS /97072).
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Mitochondrial intermembrane junctional complexes and their involvement in cell death Martin Crompton *, Emma Barksby, Nicholas Johnson, Michela Capano Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK Received 31 August 2001; accepted 6 November 2001
Abstract Mitochondria establish contact sites between the inner and outer membranes. The contact sites are held together by junctional complexes of the adenine nucleotide translocase (ANT; inner membrane) and the voltage-dependent anion channel (VDAC; outer membrane). The junctional complexes act as multifunctional recruitment centres, binding a range of proteins according to the function to be executed. Some of these, involving kinases and enzymes of lipid transfer, are readily understood as ongoing functions in energy and lipid metabolism. But the roles of other proteins recruited to the junctional complexes are less well defined. Here, we focus on the complexes formed with Bax and with cyclophilin-D, and their possible roles in apoptotic and necrotic cell death. We have isolated both types of complexes using glutathione-S-transferase fusion proteins of Bax and of cyclophilin-D. The VDAC/ANT/cyclophilin-D complex reconstitutes Ca2+- and cyclosporin A-sensitive permeability transition pore activity when incorporated into proteoliposomes. The complex forms readily in the absence of factors required for pore opening in isolated mitochondria, suggesting that these factors act on the preexisting complex, rather than drive its assembly, and that the complex is a physiological entity in healthy cells. © 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Permeability transition; Cyclophilin-D; Bax; Apoptosis; Necrosis
1. Introduction The voltage-dependent anion channel (VDAC) and adenine nucleotide translocase (ANT) are mitochondrial proteins that perform well-established roles in the daily life of the cell. VDAC, or mitochondrial porin, is a large H2Ofilled pore that allows low Mr solutes to permeate freely across the outer membrane and to gain access to the solute-specific transport systems of the inner membrane. ANT mediates ADP–ATP exchange across the inner membrane, essential for the basic bioenergetic function of the organelle in supplying ATP to the cytosol. These two proteins also form a complex that establishes contact sites between the outer and inner membranes. Since VDAC and ANT comprise the major proteins of their respective mem-
Abbreviations: ANT, adenine nucleotide translocase; CSA, cyclosporin A; CyP-D, cyclophilin-D; GST, glutathione-S-transferase; PT, permeability transition; VDAC, voltage dependent anion channel * Corresponding author. Tel.: +44-20-7679-2207; fax: +44-20-7679-7193. E-mail address: [email protected] (M. Crompton).
branes many such junctional complexes can be formed. The junctional complexes in turn recruit other proteins. Some of these proteins perform readily understood functions that facilitate the export of mitochondrial high energy phosphates to the cytosol. But other proteins attracted to the junctional complexes are implicated in cell death, and here the role of the VDAC/ANT complex is more obscure. These proteins include the proapoptotic Bax, which migrates to mitochondria under apoptotic stimuli, and cyclophilin-D (CyP-D), an essential constituent of the permeability transition (PT) pore. This article outlines the evidence for the formation of VDAC/ANT complexes with Bax and with CyP-D, in vitro and in vivo, and considers their possible roles in apoptotic and necrotic cell death.
2. VDAC/ANT complexes in energy and lipid metabolism Contact sites between the mitochondrial inner and outer membranes were first observed in electron micrographs of
© 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 1 3 6 8 - 8
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isolated mitochondria over 30 years ago [1]. Subsequently, the outer membrane protein VDAC was found to be preferentially localised at these sites [2]. The capacity of VDAC to bind strongly to the inner membrane was first revealed in the work of Ono and Tuboi [3], who synthesised radiolabelled VDAC in vitro and imported the protein into isolated mitochondria. Following digitonin fractionation and separation of the inner and outer membrane fractions, a large part of the VDAC was recovered fused with the inner membrane. Subsequently, McEnery et al. [4] purified a complex of VDAC and the inner membrane protein ANT from detergent extracts of mitochondria thereby establishing the molecular nature of the contact sites as junctional complexes of VDAC and ANT. It has been estimated that VDAC accounts for about 0.3% of mitochondrial protein [5], which is equivalent to about 100 pmol VDAC mg–1 of mitochondrial protein. This amounts to about one-tenth of the amount of ANT, so that only a small fraction of ANT can be complexed to VDAC in this way. VDAC/ANT complexes can recruit a range of other proteins indicating that they participate in a number of cellular functions. Some of this relates to the efficiency of cellular energy metabolism. Mitochondria provide the bulk of cellular ATP in most types of cell. Yet ADP is poorly diffusible in the cytoplasm, since most is bound to protein. Mitochondria need to work in tandem, therefore, with systems for the efficient distribution of ‘ATP energy’
through the cell. One way is to generate a more diffusible energy currency. The VDAC/ANT complex binds creatine kinase in the mitochondrial intermembrane space [6]. This creates a microdomain in which ATP translocated via ANT generates creatine phosphate, which is then exported to the cytosol via VDAC. In the cytosol, creatine and creatine phosphate are highly diffusible. A second ploy is to bring sites of ATP usage in the cytosol very close to the sites of mitochondrial ATP export. Thus, VDAC recruits hexokinases and glycerol kinase from the cytosol (Fig. 1). Hexokinase is bound according to its catalytic activity. Evidently, the binding occurs to the junctional complexes, since hexokinase copurifies with the VDAC/ANT complex, and immunogold studies show enrichment of hexokinase at contact sites between the outer and inner membranes [7]. In addition, cells have evolved a sophisticated system for the distribution of the primal mitochondrial energy currency, the inner membrane proton electrochemical gradient. Thus, mitochondria in situ can form tight intermitochondrial junctions, providing ionic continuity between the matrix spaces of the conjugated mitochondria, and allowing the proton electrochemical gradient to be conducted from one mitochondrion to the next. In many cells, conjugated mitochondria form large interconnected networks for the efficient distribution of the proton motive force throughout the cell [8]. Immunogold studies with antiVDAC antibodies show localisation of gold particles at the junctions between
Fig. 1. VDAC/ANT complexes as multifunctional recruitment centres. VDAC and ANT bind to each other and to a number of other proteins with diverse functions. Under pathological conditions, one of these complexes (VDAC/ANT/CyP-D), can deform (asterisk) into the PT pore, which permeabilises the inner membrane to low Mr solutes.
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closely apposed mitochondria, indicating enrichment of VDAC, or exposure of the N-terminal epitope, at these sites. Either way the data point to a role for VDAC in establishing the intermitochondrial junctions [9]. The nature of the ion conduit between conjugated mitochondria has not been resolved; but VDAC/ANT complexes can form open pores in the inner membrane when associated with CyP-D (the PT pore, below), and it would seem relevant to investigate the possible, physiological function of the VDAC/ANT/CyP-D complex in establishing these conduits [10]. The junctional complexes have also been implicated in lipid metabolism. Thus, the complex copurifies with the peripheral benzodiazepine receptor [4]; this is believed to facilitate the transfer of cholesterol to the inner membrane in steroidogenic cells [11]. An enrichment of carnitine-acyl transferases at the contact sites has also been reported [12]. Different functions of the VDAC/ANT complexes may utilise different isoforms. Three ANT isoforms have been identified as products of different genes [13]. Three VDAC isoforms have also been recognised [14]. There is evidence that hexokinases bind specifically to VDAC1 [7].
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3. VDAC/ANT interaction with cyclophilin-D Quite a pure form of the VDAC/ANT complex can be obtained using CyP-D (Fig. 2). Since CyP-D is resident in the mitochondrial matrix [15], it presumably binds to the ANT component of the VDAC/ANT complex. All indications are that CyP-D binds to the complex very tightly indeed since, in the ‘pull down’ experiments (Fig. 2), the sepharose beads and attached proteins can be washed repeatedly without detectable loss of VDAC/ANT [16]. VDAC and ANT have almost the same Mr values, but on long SDS–PAGE gels the two proteins can be separated, and from the relative intensities of Coomassie blue staining, it is apparent that VDAC and ANT are retained in the approximate ratio 1:1. The reason why CyP-D binds to ANT is not clear. But the amount of CyP-D in heart mitochondria, for example, is < 5% of the amount of ANT, and only a small fraction of ANT in the inner membrane can be complexed with CyP-D. Evidently, CyP-D is not involved in the basic function of ANT in mediating ADP/ATP exchange across the inner membrane.
Fig. 2. VDAC/ANT complexes bind CyP-D and Bax. Detergent extracts of heart mitochondrial membranes were mixed with GST–CyP-D (b–d) or GST–Bax (e, g) fusion proteins, or GST (f, h), immobilised on glutathione-S-sepharose. The sepharose beads were washed five times, and then the attached proteins were displaced by glutathione and analysed by SDS–PAGE and immunoblotting. Lane (a), unfractionated membrane extract (Coomassie blue stained). Lane (b), proteins displaced from the GST–CyP-D matrix, i.e. GST–CyP-D (upper band, 48 kDa) and superimposed VDAC and ANT (lower band, 32 kDa), Coomassie blue stained. Lanes (c) and (d), ANT and VDAC immunoblots, respectively, of (b). Lanes (e) and (f), VDAC immunoblots of proteins displaced from the GST–Bax matrix (e) and a GST matrix (f). Lanes (g) and (h), ANT immunoblots of proteins displaced from the GST–Bax matrix (g) and a GST matrix (h). Lanes (b–d) and lanes (e–h) were obtained with different membrane extracts. The data show that both GST–CyP-D and GST–Bax selectively ‘pull down’ VDAC/ANT complexes from mitochondrial membrane extracts, whereas GST does not, confirming that the VDAC/ANT complexes bind to CyP-D and to Bax. Further details of protocols are given in [16].
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Cyclophilins are found throughout nature and present a number of isoforms with defined subcellular locations, e.g. CyP-A and CyP-40 (cytosol), CyP-B and CyP-C (endoplasmic reticulum), CyP-D (mitochondria). They catalyse the rotation of Xaa–Pro peptide bonds in proteins. In general most peptide groups assume the trans configuration, which is more stable (about 8 kJ mol–1) because of steric hindrance. The energy difference between the cis and trans configurations of peptidylprolyl bonds is much less, how ever, so that a significant fraction of Pro residues in proteins follow a cis peptide bond. The peptidylprolyl cis–transisomerase (PPIase) activity of CyP facilitates the folding of nascent proteins. For example, the folding of newly imported mitochondrial proteins is impeded in deletion mutants (yeast) lacking mitochondrial cyclophilin [17]. But CyPs also form complexes with mature, fully folded proteins. Examples include CyP-40 and the oestrogen receptor [18], CyP-A and the antioxidant protein, Aop1 [19] and, as described above, CyP-D and VDAC/ANT. In these cases, it seems reasonable to consider that the CyP facilitates conformational change of the complexed protein by catalysing the rotation of surface-exposed peptidyl prolyl bonds. In addition, cyclophilins (at least, CyP-A in E. coli ) preferentially bind cis-Pro isomers. Thus, CyPs may catalyse the formation of, and stabilise, a particular conformation of the target protein [20]. If CyP-D establishes a particular conformation of ANT then, clearly, it would need to bind to ANT via its active (PPIase) site. In this case, CyP-D binding to ANT would be expected to be blocked by CSA, since CSA occludes the active site in CyP. Moreover, CSA occlusion of the active site is accompanied by very little conformational change in CyP so that CSA inhibition of ANT binding to CyP could reasonably be interpreted that the active site is involved. It has been reported that CyP-D binding to ANT alone is reduced by CSA [21]. In our hands, however, CSA produced negligible inhibition of CyP-D binding to the VDAC/ANT complex [16]. In subsequent experiments (unpublished), we have detected some reduction in VDAC/ANT binding to CyP-D when the [CSA] is raised to around 20 µM, which is > 104 times the KD value of free CyP-D and CSA. If this does reflect competition between CSA and VDAC/ANT for the PPIase active site, then it is clear that the VDAC/ANT complex, at least when isolated, must have a very high affinity for CyP-D. Alternatively, the VDAC/ANT complex may bind to a site on CyP-D distal from the active site, which would explain the poor ability of CSA to prevent VDAC/ANT binding. This particular issue has not been settled. The use of a photoactive CSA derivative has allowed the interactions between CyP-D, CSA and competing proteins to be analysed in intact mitochondria [22,23]. Mitochondria were preincubated with a radiolabelled, photoactive CSA in the dark, allowing it to equilibrate with CyP-D, before photactivation and covalent radiolabelling of the CyP-D associated with the CSA (as shown in Fig. 3). We used a
CSA derivative with a photoactive group and spacer at position 8 of the ring; substituents here do not interfere with CSA binding to CyP (involving residues 1–3 and 9–11), and the derivative photolabels CyP quite efficiently (e.g. [22,23]). If CyP were substantially free in the matrix, then its photolabelling would not be expected to be influenced by reagents that interact with ANT. However, we found that photolabelling of intramitochondrial CyP-D by photoactive CSA was markedly enhanced by extramitochondrial ADP [22,23] (Fig. 3). Since the interaction of free CyP-D with CSA is unaffected by ADP, the result means that some CyP-D in mitochondria must be associated with an ADPbinding protein. Moreover, this protein would need to bind ADP on the outside of the inner membrane. The obvious candidate is ANT. Thus, we arrive at the rather important conclusion that a fraction of ANT (presumably VDAC/ANT) is associated with CyP-D in isolated mitochondria. In other words, it appears that the VDAC/ANT/CyP-D complex may form under normal physiological conditions.
Fig. 3. CyP-D associates with ANT in intact mitochondria. Upper panel: a radiolabelled, photoactive CSA derivative was reacted with isolated heart mitochondria in the presence (closed circles) and absence (open circles) of 1 mM ADP. The medium also contained 1 mM EGTA to remove Ca2+. The mitochondrial membranes were extracted with 6% Chaps as detergent, and the extracts were analysed by gel filtration. Lower panel: CyP-D (PPIase) was assayed in the fractions obtained by gel filtration. The data show that ADP promotes CSA interaction (photolabelling) with CyP-D (recovered in fractions 9 and 10), indicating that CyP-D associates with an ADP-binding protein (ANT) in intact mitochondria. Further details of protocols are given in [22,23].
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Other observations made with the isolated VDAC/ANT/CyP-D complex are consistent with the notion that this complex is a physiological entity. In particular, assembly of the complex does not appear to be influenced by factors associated with PT pore opening. Thus the retention of VDAC/ANT complexes by CyP-D (GST fusion; as in Fig. 2) is unaffected by the presence of Ca2+, ADP, ATP, and dithiothreitol to maintain reduced thiols ([16]; unpublished data). Although low ATP, high Ca2+ and oxidative stress (thiol oxidation) trigger PT pore opening, these studies with the isolated complex suggest that these factors act on the preexisting VDAC/ANT/CyP-D complex, rather than drive its assembly from the separate components.
4. VDAC/ANT/CyP-D complexes and the PT pore PT pore activity has been reconstituted from purified VDAC, ANT, and recombinant CyP-D [16] (Fig. 4). VDAC/ANT/CyP-D complexes in Chaps were mixed with phospholipids and incorporated into proteoliposomes by detergent dialysis. Fluorescein sulphonate was introduced into the vesicles (brief sonication) and the vesicles were separated out by gel filtration. PT pore opening was assayed from the loss of fluorescein sulphonate to the medium (increased fluorescence due to decreased quenching on dilution). CSA-sensitive PT pore opening was observed on addition of Ca2+ in the presence of Pi [16] (Fig. 4). These conditions correspond to those that activate the PT pore in isolated mitochondria when both Ca2+ and Pi are required [24]. These results provide good evidence that VDAC, ANT and CyP-D are all that is required to form the PT pore. Of these, CyP-D is clearly a component, since the reconstituted activity is blocked by CSA. But the reconstitutions do not tell us whether the PT pore comprises ANT/CyP-D or VDAC/ANT/CyP-D, or both. PT pore activity has also been reconstituted from protein fractions that contain VDAC, ANT and CyP-D along with a number of other proteins including hexokinase [25]. Hexokinase associates reversibly with VDAC according to its activity. Thus, hexokinase binding to VDAC is increased in the presence of the substrates ATP and glucose and is decreased in the presence of the product inhibitor glucose6-phosphate. ATP and glucose were found to inhibit the reconstituted PT pore activity, and the inhibition was reversed by glucose-6-phosphate [25]. The obvious interpretation is that the reconstituted PT pore contained VDAC as the site of hexokinase attachment, and that attached hexokinase blocked the pore. These findings can be related to earlier observations on the capacity of extramitochondrial adenine nucleotides to prevent pore opening in isolated mitochondria. In the presence of Mg2+, > 1.5 mM ATP (buffered enzymatically) was required to block PT pore opening (inner membrane depolarisation) [26]. ADP (1 mM; buffered enzymatically) produced no inhibition of PT pore opening. These concentrations bear no relation to the binding affinities of
Fig. 4. Reconstitution of PT pore activity from purified VDAC, ANT, and recombinant CyP-D. VDAC, ANT and CyP-D (GST fusion) were purified as in Fig. 2, lane (b), and incorporated into proteoliposomes containing fluorescein sulphonate. The vesicles were incubated in medium containing 1 mM Pi and in the presence (lower trace) and absence of 1 µM CSA. Ca2+ of 100 µM was added at the arrows. Increase in fluorescence indicates release of fluorescein from the vesicles (decreased fluorescence quenching on dilution). The data show that CSA-sensitive PT pore opening can be reconstituted from purified VDAC, ANT and CyP-D. Further details of protocols are given in [16].
ANT for ADP and ATP and suggest that the inhibitory action of ATP may have been mediated by kinase attachment to VDAC. From this it seems that the VDAC/ANT/CyP-D complex can form the PT pore. But it does not rule out the possibility that ANT/CyP-D can do likewise.
5. VDAC/ANT complexes and the action of Bax Mitochondria amplify the apoptotic signalling pathway by releasing apoptogenic proteins from the intermembrane space. These proteins include cytochrome c, AIF (apoptosis inducing factor) and some procaspases. Current indications are that large holes or breaks appear in the mitochondrial
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outer membrane, since the apoptogenic proteins are released along with other intermembrane space proteins, some of which are quite large, e.g. sulphite oxidase [27]. Outer membrane permeabilisation is elicited by proapoptotic Bcl-2 family members, notably Bid, Bak and Bax. In non-apoptosing cells, these proteins reside in the cytosol, but under apoptotic stimuli they migrate to mitochondria leading to outer membrane permeabilisation. Bid is resident in the cytosol as an inactive precurser; this is cleaved by caspase-8 to produce a 15 kDa C-terminal fragment that binds tightly to mitochondria. Bax is retained in the cytosol of healthy cells as an inactive monomer. The mechanisms that trigger Bax translocation to mitochondria are unclear, but it seems that on association with mitochondria it undergoes conformational change [28] and oligomerisation [29]. These changes may be facilitated by Bid on the mitochondrial surface. The question arises whether these proapoptotic Bcl-2 family proteins target specific receptor proteins on the mitochondrial outer membrane. As yet, no such receptor proteins have been unambiguously identified, and the question remains open. But there are indications that the junctional complexes may be involved at some stage in the action of Bax on mitochondria. Shimizu et al. [30] reported various lines of evidence that Bax can interact with VDAC. Thus recombinant Bax coimmunoprecipitates with VDAC. VDAC normally allows free permeation of sucrose, but VDAC proteoliposomes are poorly permeable to sucrose at low pH (pH 5.2) and, under these conditions, sucrose permeability is markedly enhanced by Bax, again indicating Bax–VDAC interactions. The VDAC liposomes also became permeable to cytochrome c on addition of Bax, although larger proteins were retained. Other investigators have used partially purified VDAC/ANT preparations containing Bax. As in isolated mitochondria, the ANT ligand atractylate induces PT pore opening in proteoliposomes containing the VDAC/ANT complex, evidently by establishing an ANT conformation prone to distortion into an open pore state. Immunodepletion of Bax from these complexes led to a loss of atractylate induced pore activity [31], pointing to an interaction , directly or indirectly, of Bax with ANT. Work in our laboratory broadly corroborates the above data. Thus, VDAC/ANT complexes are selectively retained by GST–Bax fusion protein immobilised on glutathione-Ssepharose (Fig. 2). In these experiments, we have detected no retention of CyP-D, suggesting that Bax/VDAC/ANT and VDAC/ANT/CyP-D are alternative complexes of VDAC/ANT. Other studies implicate the VDAC/ANT complex in apoptosis, without indicating the capacity in which it may be involved. Bauer et al. [32] identified ANT-1 as a proapoptotic protein from screening an expression library for dominant apoptosis-inducing genes. Overexpression of ANT-1 led to spontaneous apoptosis in a number of cell lines, whereas transfection of ANT-2 was innocuous. The
apoptogenic action of ANT-1 was unrelated to its basic function in ADP–ATP exchange, since transport-deficient mutants were also proapoptotic. Other indications have been derived from studies of viral and bacterial infected cells. The protein, vMIA, produced by human cytomegalovirus, blocks fas-mediated apoptosis. It inhibits cytochrome c release to the cytosol, but does not inhibit Bid cleavage, indicating that it blocks the action of truncated Bid on mitochondria. In line with this, vMIA locates to mitochondria (Immunogold) of transfected cells, and it coimmunoprecipitates ANT from cell extracts [33]. The proapoptotic HIV 1 protein R also binds ANT (surface plasmon resonance studies) [34]; the binding is inhibited by a peptide corresponding to a segment of ANT (amino acids 104–116) that loops into the intermembrane space. Other apoptoticregulatory, viral and bacterial proteins have been recognised that target VDAC. HBX is produced by hepatitis B virus infected liver cells, leading to apoptotic cell death. When transfected into cells, HBX colocalises with mitochondria (immunofluorescence) and coimmunoprecipitates VDAC from cell extracts [35]. Neisseria mengintidis infection inhibits apoptosis; the major outer membrane protein, Por B, does likewise when transfected into cells. Again, this protein translocates primarily with mitochondria and coimmunoprecipitates with VDAC from cell extracts [36]. In spite of these positive indications for interactions between Bax and VDAC/ANT, it is unclear at what step any such interaction takes place. On binding to mitochondria Bax changes conformation and oligomerises. The conformational changes lead to exposure of the N-terminal region, which becomes accessible to trypsin and to antibodies [28,37]. At the same time, Bax becomes cross-linkable, indicating oligomerisation [38]. This appears to be an essential step in the apoptogenic function of Bax. Cells transfected with a chimeric FK506 binding protein-Bax become apoptotic when dimerisation is promoted with a bivalent ligand [38]. Bax oligomers produced in vitro by the appropriate detergent are better able to release cytochrome c from isolated mitochondria. Antonsson and coworkers [29] have isolated the mitochondrial Bax oligomers from detergent extracts of staurosporine-treated cells. The oligomers/complexes displayed apparent Mr values of 96 and 260 kDa on gel filtration, but contained no ANT or VDAC. Thus, although Bax may well interact with the junctional complexes at some stage in its action on mitochondria, that stage has yet to be identified. The fact that Bax can interact (experimentally, at least) with the VDAC/ANT complex may account for numerous observations that added Bax promotes PT pore activity in isolated mitochondria (e.g. [39]). This behaviour has sometimes been interpreted to indicate that the physiological action of Bax in releasing cytochrome c is mediated via PT pore opening. A more conservative interpretation would be that Bax can bind to components of the PT pore (in line with the data discussed above) and, in so doing, modifies their properties, including ‘deformation’ of the complex into the
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PT pore under pathological conditions. It does not necessarily mean that PT pore opening is part of the normal action of Bax. In line with this, Bax can release cytochrome c from isolated mitochondria in the absence of Ca2+ [40], the basic trigger for PT pore opening, and the release occurs without the increase in matrix volume that accompanies PT pore opening in vitro [27].
6. The VDAC/ANT/CyP-D complex in ischaemic injury In isolated mitochondria, deformation of the VDAC/ANT/Cyp-D complex into the PT pore requires a particularly stringent set of conditions, namely, high intramitochondrial [Ca2+], high [Pi], oxidative stress, and low extramitochondrial [ATP] (reviewed in Ref. [10]). These factors operate synergistically, but in isolated mitochondria, at least, two conditions are paramount, namely high [Ca2+], and low [ATP]. In our experience, PT pore opening (as measured unambiguously by radiolabelled sucrose entry into the matrix space) has an absolute requirement for Ca2+,
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and PT pore opening under any condition is completely blocked by physiological concentrations of ATP [10]. If similar restrictions apply in vivo, and there is no reason to suggest that they do not, then we may anticipate PT pore opening only in extreme pathological states. Such conditions are found in ischaemia/reperfusion-induced injury. Ischaemia leads to ATP dissipation with consequent rises in cell Ca2+ and Pi, and oxidative stress occurs on reperfusion (reviewed in Ref. [10]). This striking correlation between the conditions required for PT pore opening and those that are known to provoke reperfusion injury first led to the hypothesis that PT pore opening may be a key lesion in this form of injury [41,42] (Fig. 5). The hypothesis stands up to fairly close scrutiny regarding the actual changes in ATP and Ca2+ that are required for injury and for PT pore opening. In heart cells irreversible injury follows when about two-thirds of cell ATP have been dissipated [43], which agrees reasonably well with the [ATP] below which PT pore opening can be detected in isolated mitochondria (1.5 mM ATP) [26]. Mitochondrial Ca2+ overload occurs when mitochondria are exposed to
Fig. 5. A model for the involvement of the PT pore in necrotic and apoptotic cell death induced by ischaemia and reperfusion. Ischaemia leads to ATP dissipation and consequent rises in cell Ca2+ and Pi. On reperfusion, the increased cytosolic Ca2+ leads to mitochondrial Ca2+ overload. These factors together with oxidative stress trigger PT pore opening. If widespread, PT pore opening overwhelms the capacity of the cell to maintain ATP and the cells become necrotic. If PT pore opening is limited (localised), allowing maintenance of cell ATP, outer membrane rupture and release of apoptogenic proteins from the intermembrane space may be sufficient to initiate the caspase cascade, leading to apoptotic cell death. The degree of PT pore opening may be a factor determining the proportion of necrotic and apoptotic cell death.
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maintained Ca2+ levels (not transients) exceeding 1–2 µM (discussed in Ref. [10]). This limiting value of resting cytosolic free Ca2+ consistent with mitochondrial viability correlates well with the [Ca2+] required to trigger irreversible cell injury (cardiomyocytes). Thus, when aequorinloaded (Ca2+ indicator) myocytes are subjected to anoxia, there is an eventual progressive rise in resting cytosolic Ca2+ [44]. The rise is reversed on reoxygenation provided that a limit of 1–3 µM resting Ca2+ is not exceeded. If this limiting value is exceeded, however, the myocytes undergo lethal injury. The hypothesis is also supported by various studies of PT pore opening in ischaemia, anoxia, and related conditions. In particular, the PT pore inhibitor. CSA, protects against ischaemia- or anoxia-induced injury in a number of systems (reviewed in Ref. [10]). The groups of Halestrap [45] and Lemasters [46] have demonstrated the movement of otherwise impermeant small molecules (e.g. 2-deoxyglucose, calcein) across the mitochondrial inner membrane in ischaemia/anoxia-stressed cells, consistent with PT pore opening. Ischaemia/reperfusion produces both necrotic and apoptotic cell death. Characteristically, the regions that suffer the most severely ischaemic conditions become necrotic, whereas the less stressed regions that escape necrosis succumb subsequently by apoptosis. Evidently, a period of ischaemia can engage the apoptotic signalling pathway in some way. In seeking to resolve the underlying mechanism it is important to distinguish between the physiological process of programmed cell death, which specifically selects apoptosis (‘authorised’ apoptosis), and the ‘accidental’ apoptosis that accompanies necrosis in ischaemic disease. As discussed above, ‘authorised’ apoptosis might utilise the VDAC/ANT complex in some capacity. But, it seems unlikely that this would involve the complex acting as a PT pore, since this would introduce the possibility of a rapid ATP dissipation, leading to cell necrosis, the undesired outcome. But when apoptosis (‘accidental’) and necrosis coexist, as they do in ischaemic disease, it is conceivable that they are a product of the same mechanism [47]. Extensive PT pore opening, producing rapid ATP dissipation, overwhelming the ATP generating capacity of the cell, would produce a rapid loss of cell viability. But a limited degree of PT pore opening could be tolerated. Cellular energy transduction would be less efficient, but there would be no immediate threat to the life of the cell provided that cellular ATP was maintained. However, any rupture of the mitochondrial outer membrane breakage under such conditions might be sufficient to trigger the caspase cascade (Fig. 5). PT pore opening in isolated mitochondria produces matrix swelling, which can result in physical rupture of the outer membrane [39]. Might limited PT pore opening in vivo do the same? Firstly, limited PT pore opening has been detected in vivo. When heart cells are loaded with positively charged rhodamines, the dyes accumulate electrophoretically according to the magnitude of the mitochondrial inner mem-
brane potential. This allows the potential to be monitored by fluorescence imaging techniques. The dyes also photodecompose, producing reactive oxygen species in the mitochondrial matrix. Under these conditions, a small number of mitochondria (e.g. in cardiomyocytes, astrocytes) are seen to undergo occasional, transient depolarisations. The depolarisations require mitochondrial Ca2+ uptake [48] and are blocked by CSA [49], indicating that they are due to transient PT pore opening. Most probably, the mitochondria which exhibit this behaviour are those located close to the Ca2+ release channels of the sarco(endo)plasmic reticulum, since the depolarisations are also blocked by agents that deplete the reticulum of Ca2+ (thapsigargin) and that inhibit Ca2+ release from the reticulum (ryanodine) [48]. Secondly, there is evidence that transient PT pore opening is sufficient to release cytochrome c. Gogvadze et al. [50] reported that relatively low doses of Ca2+, barely sufficient to cause detectable loss of the inner membrane potential in populations of isolated mitochondria, can induce cytochrome c release via PT pore opening. It was concluded that the behaviour reflected variations in the threshold for PT pore opening from one mitochondrion to the next, such that with limiting Ca2+, PT pore opening and cytochrome c release occurred from those most susceptible, whilst the potential (tetraphenylphosphonium ion uptake) was maintained by the resistant fraction. However, an alternative interpretation is possible based on the previous work in this laboratory and the phenomenon of transient PT pore opening [24]. In essence, by entrapping Ca2+ buffers in the matrix space, and with Ca2+ transport inhibitors present, it can be shown that PT pores open and close continuously at a frequency determined by the matrix [Ca2+], and that there is no critical [Ca2+] threshold. The relation between PT pore opening and matrix free [Ca2+] is linear up to 20 µM Ca2+ [24]. At any matrix [Ca2+] there is a dynamic steady state distribution between those mitochondria that have open pores and those that do not. At low Ca2+ loads, when only a small fraction of mitochondria have open pores at any point in time, the potential is maintained by the remainder. But added radiolabelled sucrose eventually enters all mitochondria (transient PT pore opening) and does so according to a single rate constant, i.e. the mitochondria behave homogeneously. When considered together, therefore, the two pieces of work [24,50], both carried out with liver mitochondria, indicate that transient PT pore opening can release cytochrome c from the intermembrane space of isolated mitochondria. The critical question, as yet unan swered, is whether transient PT pore opening in vivo can do the same.
Acknowledgements The authors’ work described was supported by the British Heart Foundation (awards PG /2000009, PG / 96162, FS /97072).
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