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The Role of the Bcl-2 Family in the Regulation of Outer Mitochondrial Membrane Permeability

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The Role of the Bcl-2 Family in the Regulation of Outer Mitochondrial Membrane Permeability Cell Death and Differentiation (2000) 7, 1182 ± 1191 ã 2000 Macmillan Publishers Ltd All rights reserved 1350-9047/00 $15.00 www.nature.com/cdd Review The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability 1 ,1 MH Harris and CB Thompson* Introduction 1 Abramson Family Cancer Research Institute, University of Pennsylvania, Mitochondria became the subject of intensive scientific Philadelphia, PA 19104, USA investigation in the mid-twentieth century when their role as * Corresponding author: CB Thompson, Abramson Family Cancer Research cellular energy producers was discovered. This culminated in Institute, 450 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160, USA. the discovery and characterization of oxidative phosphoryla- E-mail: [email protected] tion as the mechanism by which most cellular ATP is Received 29.6.00; accepted 14.9.00 produced. Free exchange of substrate and ATP/ADP Edited by G Kroemer between mitochondria and the cytosol was proposed to provide the homeostatic mechanism by which glycolysis and oxidative phosphorylation are coupled to maintain the Abstract intracellular ATP/ADP ratio.1,2 In the last few years, there has been a resurgence of interest in mitochondria following Mitochondria are well known as sites of electron transport and the discovery that mitochondria play a crucial role in the generators of cellular ATP. Mitochondria also appear to be regulation of programmed cell death, or apoptosis.3±9 A sites of cell survival regulation. In the process of programmed number of molecules involved in the execution of apoptosis cell death, mediators of apoptosis can be released from normally reside in mitochondria, safely sequestered from their mitochondria through disruptions in the outer mitochondrial targets and co-factors.10 ± 16 Following an apoptotic stimulus, membrane; these mediators then participate in the activation these proteins can be released from mitochondria and initiate of caspases and of DNA degradation. Thus the regulation of the activation of the downstream effectors of apoptosis, outer mitochondrial membrane integrity is an important caspases. The mechanisms by which these apoptosis control point for apoptosis. The Bcl-2 family is made up of initiators are released from mitochondria remain controver- outer mitochondrial membrane proteins that can regulate cell sial. Elucidating these mechanisms, however, is crucial to the understanding of how and why a cell makes the decision to survival, but the mechanisms by which Bcl-2 family proteins undergo apoptosis. act remaincontroversial.Mostmetabolitesare permeanttothe The mitochondrion is an organelle encompassed by two outer membrane through the voltage dependent anion membranes (Figure 1). The inner membrane, which channel (VDAC), and Bcl-2 family proteins appear to be able surrounds the mitochondrial matrix and is usually tightly to regulate VDAC function. In addition, many Bcl-2 family folded in cristae, holds the molecular complexes of the proteins can form channels in vitro, and some pro-apoptotic electron transport chain. It is across the inner membrane members may form multimeric channels large enough to that the electron transport chain generates the hydrogen release apoptosis promoting proteins from the intermem- ion gradient necessary to make ATP. In order to maintain brane space. Alternatively, Bcl-2 family proteins have been this crucial gradient, transport across the inner membrane hypothesized to coordinate the permeability of both the outer is tightly regulated by many different, specific transporters and inner mitochondrial membranes through the permeability for the metabolites that need to cross the inner membrane, such as the phosphate carrier, the dicarboxylate carrier and transition (PT) pore. Increasing evidence suggests that the tricarboxylate carrier.17 The inner membrane also alterations in cellular metabolism can lead to pro-apoptotic contains protein import channels, as well as the adenine changes, including changes in intracellular pH, redox nucleotide translocator (ANT) that exchanges ADP and potential and ion transport. By regulating mitochondrial ATP between the mitochondrial matrix and the intermem- membrane physiology, Bcl-2 proteins also affect mitochon- brane space. The outer mitochondrial membrane (OMM) drial energy generation, and thus influence cellular bioener- surrounds the inner membrane, creating the intermembrane getics. Cell Death and Differentiation (2000) 7, 1182 ± 1191. space where many of the characterized apoptosis promoting proteins reside, including cytochrome c, apopto- sis inducing factor (AIF) and some pro-caspases. Transport Keywords: apoptosis; Bcl-xL; Bcl-2; Bax; mitochondria; outer mitochondrial membrane across the outer mitochondrial membrane is thought to be less tightly regulated than transport across the inner Abbreviations: ANT, adenine nucleotide translocator; OMM, outer membrane. The high permeability of the outer membrane mitochondrial membrane; ROS, reactive oxygen species; VDAC, is mediated by VDAC, which is the most common protein in voltage dependent anion channel the outer membrane and is permeable to molecules of up to 5000 daltons in its open configuration.18 Nevertheless, the outer membrane is impermeable to holo-cytochrome c and other apoptosis promoting proteins in the intermem- The Bcl-2 family in the regulation of OMM permeability MH Harris and CB Thompson 1183 apoptotic members, such as Bcl-2 and Bcl-xL, typically have BH1 through BH4 domains. The pro-apoptotic members can be divided into two groups: those with BH1, BH2 and BH3 domains, such as Bax and Bak, and those with only BH3 domains, such as Bad, and Bim.24 ± 26 An exception to these categories may be Bcl-xS, a pro-apoptotic protein, which is an alternate splice form of Bcl-xL and contains BH3 and BH4 domains.27 These domains have functional and structural significance. The BH3 domain of pro-apoptotic family members can interact with the hydrophobic cleft formed by the BH1, BH2 and BH3 domains of anti-apoptotic proteins, and this interaction may be an important regulatory 28,29 mechanism. Structural studies of Bcl-xL have shown a striking similarity between Bcl-xL and the colicin family of pore forming proteins,30 and functional studies have shown that Bcl-xL, as well as Bcl-2, Bax, and Bak, are capable of forming channels in synthetic lipid bilayers.31 ± 34 This functional property of Bcl-2 family proteins may be even more significant than originally appreciated, since it was recently shown that cleaved Bid, which shares primary sequence Figure 1 Mitochondria are producers of ATP and regulators of apoptosis. homology with Bcl-xL only in the BH3 domain, has a similar Oxidative phosphorylation occurs across the mitochondrial inner membrane three-dimensional structure to Bcl-x and can also form via the electron transport chain, including cytochrome c (c), and the F F - L 1 0 channels in synthetic lipid bilayers.35 ± 37 The ability of Bcl-2 ATPase (F1F0). ATP made in the matrix is exchanged for cytosolic ADP through the adenine nucleotide translocator (ANT) in the inner membrane and family members to form channels in association with the voltage dependent anion channel (VDAC) in the outer membrane. Bcl-2 intracellular membranes appears to be important to their family proteins regulate mitochondrial physiology and apoptosis. Bcl-xL is function in apoptosis, as does their ability to localize to the constitutively localized to the outer membrane and may be inhibited through OMM.22,29 Thus a number of groups have sought to interactions with BH3-only proteins including Bad, Bim, Bik and Hrk. Bax translocates to the outer membrane after an apoptotic signal and can be toxic determine whether Bcl-2 family proteins may perform their in the absence of a physical interaction with Bcl-xL functions by regulating the permeability of the outer mitochondrial membrane. Furthermore, by regulating the permeability of the outer mitochondrial membrane, Bcl-2 brane space and this impermeability is crucial to the family members could potentially be regulating mitochondrial regulation of apoptosis. For example, if holo-cytochrome c homeostasis in general, rather than being solely involved in is released into the cytosol, it can complex with Apaf-1 and apoptosis regulation. ATP or dATP to activate pro-caspase 9, thus beginning the The mechanisms by which the outer mitochondrial proteolytic cascade that results in the morphological membrane increases in permeability during apoptosis, and features of apoptosis.19 the mechanisms by which Bcl-2 proteins regulate this Maintenance of the permeability of the outer membrane process have remained controversial. A number of models to metabolic anions is crucial for cell survival. Free ATP/ for outer membrane permeabilization have been proposed, ADP exchange, as well as the movement of other such as the non-specific rupture of the outer membrane or metabolic anions such as creatine phosphate, succinate the formation of a specific channel. Bcl-2 family proteins and pyruvate between the cytosol and the matrix, is may regulate both of these processes, via both physical dependent on passage through the outer membrane, interactions and channel forming capabilities. In any case, it primarily through VDAC.20,21 Paradoxically, the continued is clear that the disruption in the OMM during apoptosis exchange of metabolic anions across the outer membrane must be large and freely permeable, since every is required to maintain the integrity of this membrane
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