Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations

Recent evidence suggests that the ability of ceramides to induce apoptosis is due to a direct action on mitochondria. Mitochondria are known to contain enzymes responsible for ceramide synthesis and hydrolysis and mitochondrial ceramide levels have been shown to be elevated prior to the mitochondrial phase of apoptosis. Ceramides have been reported to induce the release of intermembrane space proteins from mitochondria, which has been linked to their ability to form large channels in membranes. The aim of this study was to determine if the membrane concentration of ceramide required for the formation of protein permeable channels is within the range that is present in mitochondria during the induction phase of apoptosis. Only a very small percentage of the ceramide actually inserts into the mitochondrial membranes. The permeability of the mitochondrial outer membrane correlates directly with the level of ceramide in the membrane. Importantly, the concentration of ceramide at which significant channel formation occurs is consistent with the level of mitochondrial ceramide that occurs during the induction phase of apoptosis (4 pmol ceramide/nanomole phospholipid). Similar results were obtained with short- and long-chain ceramide. Ceramide channel formation is specific to mitochondrial membranes in that no channel formation occurs in the plasma membranes of erythrocytes even at concentrations 20 times higher than those required for channel formation in mitochondrial outer membranes. Thus, ceramide channels are good candidates for the pathway by which proapoptotic proteins are released from mitochondria during the induction phase of apoptosis.     

Sphingosine, a product of ceramide hydrolysis, influences the formation of ceramide channels

Ceramides are known to have a regulatory function in apoptosis, including the release of cytochrome c and other proapoptotic factors from the mitochondrial intermembrane space. Ceramides can form large, stable channels in the outer mitochondrial membrane, leading to the proposal that ceramide channels are the pathway through which these proteins are released. Here, we report that sphingosine, a product of ceramide hydrolysis by ceramidase, is capable of destabilizing ceramide channels, leading to their disassembly. Sphingosine is directly responsible for the disassembly of ceramide channels in planar membrane experiments and markedly reduces the ability of ceramide to induce the release of intermembrane space proteins from mitochondria in vitro. Low concentrations of both L and D sphingosine potentiate the release of intermembrane space proteins by long-chain ceramide and channel formation in liposomes. These results provide evidence for a mechanism by which the disassembly of ceramide channels, as initiated by ceramidase, could be accelerated by the direct interaction of the hydrolysis product with the ceramide channels themselves. This mechanism therefore could form a positive feedback loop for rapid shut-down of ceramide channels. However, potentiation of ceramide channel formation is also possible and thus both effects could influence the propensity for mitochondria-mediated apoptosis.     

线粒体神经酰胺对细胞凋亡的诱导作用

凋亡诱导期,线粒体内神经酰胺水平升高,当每纳摩尔线粒体膜磷脂内含4～6皮摩尔神经酰胺时,神经酰胺即在线粒体外膜形成稳定的跨膜通道,从而使外膜通透性增加,线粒体膜间蛋白释放,启动细胞凋亡.神经酰胺通道只能在线粒体外膜形成,它是由神经酰胺柱组成的桶装结构,神经酰胺的反式双键具有增加通道的稳定性的作用.     

Ceramide channels and their role in mitochondria-mediated apoptosis

A key, decision-making step in apoptosis is the release of proteins from the mitochondrial intermembrane space. Ceramide can self-assemble in the mitochondrial outer membrane to form large stable channels capable of releasing said proteins. Ceramide levels measured in mitochondria early in apoptosis are sufficient to form ceramide channels in the outer membrane. The channels are in dynamic equilibrium with non-conducting forms of ceramide in the membrane. This equilibrium can be strongly influenced by other sphingolipids and Bcl-2 family proteins. The properties of ceramide channels formed in a defined system, planar phospholipid membranes, demonstrate that proteins are not required for channel formation. In addition, experiments in the defined system reveal structural information. The results indicated that the channels are barrel-like structures whose staves are ceramide columns that span the membrane. Ceramide channels are good candidates for the protein release pathway that initiates the execution phase of apoptosis.     

Ceramide channels increase the permeability of the mitochondrial outer membrane to small proteins

Ceramides are known to play a major regulatory role in apoptosis by inducing cytochrome c release from mitochondria. We have previously reported that C(2)- and C(16)-ceramide, but not dihydroceramide, form large channels in planar membranes (Siskind, L. J., and Colombini, M. (2001) J. Biol. Chem. 275, 38640-38644). Here we show that ceramides do not trigger a cytochrome c secretion or release mechanism, but simply raise the permeability of the mitochondrial outer membrane, via ceramide channel formation, to include small proteins. Exogenously added reduced cytochrome c was able to freely permeate the mitochondrial outer membrane with entry to and exit from the intermembrane space facilitated by ceramides in a dose- and time-dependent manner. The permeability pathways were eliminated upon removal of C(2)-ceramide by bovine serum albumin, thus ruling out a detergent-like effect of C(2)-ceramide on membranes. Ceramide channels were not specific to cytochrome c, as ceramides induced release of adenylate kinase, but not fumerase from isolated mitochondria, showing some specificity of these channels for the outer mitochondrial membrane. SDS-PAGE results show that ceramides allow release of intermembrane space proteins with a molecular weight cut-off of about 60,000. These results indicate that the ceramide-induced membrane permeability increases in isolated mitochondria are via ceramide channel formation and not a release mechanism, as the channels that allow cytochrome c to freely permeate are reversible, and are not specific to cytochrome c.     

Anti-apoptotic Bcl-2 Family Proteins Disassemble Ceramide Channels

Early in mitochondria-mediated apoptosis, the mitochondrial outer membrane becomes permeable to proteins that, when released into the cytosol, initiate the execution phase of apoptosis. Proteins in the Bcl-2 family regulate this permeabilization, but the molecular composition of the mitochondrial outer membrane pore is under debate. We reported previously that at physiologically relevant levels, ceramides form stable channels in mitochondrial outer membranes capable of passing the largest proteins known to exit mitochondria during apoptosis (Siskind, L. J., Kolesnick, R. N., and Colombini, M. (2006) Mitochondrion 6, 118-125). Here we show that Bcl-2 proteins are not required for ceramide to form protein-permeable channels in mitochondrial outer membranes. However, both recombinant human Bcl-x(L) and CED-9, the Caenorhabditis elegans Bcl-2 homologue, disassemble ceramide channels in the mitochondrial outer membranes of isolated mitochondria from rat liver and yeast. Importantly, Bcl-x L and CED-9 disassemble ceramide channels in the defined system of solvent-free planar phospholipid membranes. Thus, ceramide channel disassembly likely results from direct interaction with these anti-apoptotic proteins. Mutants of Bcl-x L act on ceramide channels as expected from their ability to be anti-apoptotic. Thus, ceramide channels may be one mechanism for releasing pro-apoptotic proteins from mitochondria during the induction phase of apoptosis.     

Sphingosine Forms Channels in Membranes That Differ Greatly from Those Formed by Ceramide

Ceramide channels formed in the outer membrane of mitochondria have been proposed to be the pathways by which proapoptotic proteins are released from mitochondria during the early stages of apoptosis. We report that sphingosine also forms channels in membranes, but these differ greatly from the large oligomeric barrel-stave channels formed by ceramide. Sphingosine channels have short open lifetimes and have diameters less than 2 nm, whereas ceramide channels have long open lifetimes, enlarge in size reaching diameters in excess of 10 nm. Unlike ceramide, sphingosine forms channels in erythrocyte plasma membranes that vary in size with concentration, but with a maximum possible channel diameter of 2 nm. In isolated mitochondria, a large proportion of the added sphingosine was rapidly metabolized to ceramide in the absence of externally added fatty acids or fatty-acyl-CoAs. The ceramide synthase inhibitor, fumonisin B1 failed to prevent sphingosine metabolism to ceramide and actually increased it. However, partial inhibition of conversion to ceramide was achieved in the presence of ceramidase inhibitors, indicating that reverse ceramidase activity is at least partially responsible for sphingosine metabolism to ceramide. A small amount of cytochrome c release was detected. It correlated with the level of ceramide converted from sphingosine. Thus, sphingosine channels, unlike ceramide channels, are not large enough to allow the passage of proapoptotic proteins from the intermembrane space of mitochondria to the cytoplasm.     

Dihydroceramide hinders ceramide channel formation: Implications on apoptosis

Early in apoptosis, ceramide levels rise and the mitochondrial outer membrane becomes permeable to small proteins. The self-assembly of ceramide to form channels could be the means by which intermembrane space proteins are released to induce apoptosis. Dihydroceramide desaturase converts dihydroceramide to ceramide. This conversion may be removing an inhibitor as well as generating a pro-apoptotic agent. We report that both long and short chain dihydroceramides inhibit ceramide channel formation in mitochondria. One tenth as much dihydroceramide was sufficient to inhibit the permeabilization of the outer membrane by about 95% (C₂) and 51% (C₁₆). Similar quantities inhibited the release of carboxyfluorescein from liposomes indicating that other mitochondrial components are not necessary for the inhibition. The apoptogenic activity of ceramide may thus depend on the ceramide to dihydroceramide ratio resulting in a more abrupt transition from the normal to the apoptotic state when the de novo pathway is used in mitochondria.     

Ceramide channels and mitochondrial outer membrane permeability

Among the permeability pathways in the mitochondrial outer membrane (MOM), whose elucidation was pioneered by Kathleen Kinnally, there is one formed by the lipid, ceramide. Electron microscopic visualization shows that ceramide channels are large cylindrical structures of varying pore size, with a most frequent size of 10 nm in diameter, large enough to allow all soluble proteins to translocate between the cytosol and the mitochondrial intermembrane space. Similar results were obtained with electrophysiological measurements. Studies of the dynamics of the channels are consistent with a right cylinder. Ceramide channels form at mole fractions of ceramide that are found in the MOM early in the apoptotic process, before or at the time of protein release from mitochondria. That these channels are good candidates for the protein release pathway is supported by the fact that channel formation is inhibited by anti-apoptotic proteins and favored by Bax. Bcl-xL inhibits ceramide channel formation by binding to the apolar ceramide tails using its hydrophobic grove. Bax interaction with the polar regions of ceramide results in MOM permeabilization through synergy with ceramide. Evidence that ceramide channels actually function to favor apoptosis in vivo is supported by the expression of Bcl-xL containing point mutations in cells induced to undergo apoptosis. The Bcl-xL mutants inhibit differentially Bax and ceramide channels and thus tease apart, to some extent, these two modes of MOM permeabilization. Ceramide channels have the right properties and appropriate regulation to be key players in the induction of apoptosis.     

Alteration of mitochondrial function and cell sensitization to death

Stimulation of cell death is a powerful instrument in the organism’s struggle with cancer. Apoptosis represents one mode of cell death. However, in a variety of tumor cells proapoptotic mechanisms are downregulated, or not properly activated, whereas antiapoptotic mechanisms are upregulated. Mitochondria are known as key players in the regulation of apoptotic pathways. Specifically, permeabilization of the mitochondrial outer membrane and subsequent release of proapoptotic proteins from the intermembrane space are viewed as decisive events in the initiation and/or execution of apoptosis. Disruption of mitochondrial functions by anticancer drugs, which induce oxidative stress, inhibit mitochondrial respiration, or uncouple oxidative phosphorylation, can sensitize mitochondria in these cells and facilitate outer membrane permeabilization.     

Polycations induce the release of soluble intermembrane mitochondrial proteins

The release of proapoptotic proteins from the intermembrane space of mitochondria is an early critical step in many pathways to apoptosis. Induction of the mitochondrial permeability transition pore (PTP) was suggested to be the mechanism of the release of soluble mitochondrial intermembrane proteins (SIMP) in apoptosis. However, several studies suggested that proapoptotic proteins (e.g. Bax and Bid) can induce the release of SIMP (e.g. cytochrome c (cyt c) and adenylate kinase 2 (AK2)) in vivo and in vitro independent of PTP. We have found that a number of structurally diverse polycations, such as aliphatic polyamines (e.g. spermine and to a lesser extent spermidine), aminoglycosides (e.g. streptomycin, gentamicin and neomycin), and cytotoxic peptides (e.g. melittin), induce the release of SIMP from liver mitochondria, in vitro. All the polycations released AK2 together with cyt c, suggesting that rupture of the outer membrane is a common mechanism of cyt c release by these polycations. Several polycations (e.g. spermine, spermidine and neomycin) induced SIMP release without inducing significant swelling, and this release was not inhibited significantly by the PTP inhibitor cyclosporin. In contrast, under the same conditions, streptomycin and melittin induced swelling and SIMP release that was inhibited strongly by cyclosporin. Gentamicin-induced swelling and release of SIMP were partially inhibited by cyclosporin. The affinity of polyamines to the anionic phospholipids of the mitochondrial membranes (spermine=neomycin>gentamicin>streptomycin=spermidine) correlated roughly with their ability to induce PTP-independent release of SIMP, which suggests that the binding of polycations to the anionic phospholipids of the outer mitochondrial membrane facilitates the rupture of this membrane. However, some polycations facilitated the induction of PTP, possibly by binding to cardiolipin on the inner membrane. This dual mechanism may be relevant to the induction of SIMP release in apoptosis.     

A tale of two mitochondrial channels,MAC and PTP,in apoptosis

The crucial step in the intrinsic, or mitochondrial, apoptotic pathway is permeabilization of the mitochondrial outer membrane. Permeabilization triggers release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space into the cytosol where these factors ensure propagation of the apoptotic cascade and execution of cell death. However, the mechanism(s) underlying permeabilization of the outer membrane remain controversial. Two mechanisms, involving opening of two different mitochondrial channels, have been proposed to be responsible for the permeabilization; the permeability transition pore (PTP) in the inner membrane and the mitochondrial apoptosis-induced channel (MAC) in the outer membrane. Opening of PTP would lead to matrix swelling, subsequent rupture of the outer membrane, and an unspecific release of intermembrane proteins into the cytosol. However, many believe PTP opening is a consequence of apoptosis and this channel is thought to principally play a role in necrosis, not apoptosis. Activation of MAC is exquisitely regulated by Bcl-2 family proteins, which are the sentinels of apoptosis. MAC provides specific pores in the outer membrane for the passage of intermembrane proteins, in particular cytochrome c, to the cytosol. The electrophysiological characteristics of MAC are very similar to Bax channels and depletion of Bax significantly diminishes MAC activity, suggesting that Bax is an essential constituent of MAC in some systems. The characteristics of various mitochondrial channels and Bax are compared. The involvement of MAC and PTP activities in apoptosis of disease and their pharmacology are discussed.     

The mitochondrial TOM complex is required for tBid/Bax-induced cytochrome c release

Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.     

Ceramide and activated Bax act synergistically to permeabilize the mitochondrial outer membrane

A critical step in apoptosis is mitochondrial outer membrane permeabilization (MOMP), releasing proteins critical to downstream events. While the regulation of this process by Bcl-2 family proteins is known, the role of ceramide, which is known to be involved at the mitochondrial level, is not well-understood. Here, we demonstrate that Bax and ceramide induce MOMP synergistically. Experiments were performed on mitochondria isolated from both rat liver and yeast (lack mammalian apoptotic machinery) using both a protein release assay and real-time measurements of MOMP. The interaction between activated Bax and ceramide was also studied in a defined isolated system: planar phospholipid membranes. At concentrations where ceramide and activated Bax have little effects on their own, the combination induces substantial MOMP. Direct interaction between ceramide and activated Bax was demonstrated both by using yeast mitochondria and phospholipid membranes. The apparent affinity of activated Bax for ceramide increases with ceramide content indicating that activated Bax shows enhanced propensity to permeabilize in the presence of ceramide. An agent that inhibits ceramide-induced but not activated Bax induced permeabilization blocked the enhanced MOMP, suggesting that ceramide is the key permeabilizing entity, at least when ceramide is present. These and previous findings that anti-apoptotic proteins disassemble ceramide channels suggest that ceramide channels, regulated by Bcl-2-family proteins, may be responsible for the MOMP during apoptosis.     

Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space

Apoptosis-inducing factor (AIF) is a mitochondrial intermembrane flavoprotein that is translocated to the nucleus in response to proapoptotic stimuli, where it induces nuclear apoptosis. Here we show that AIF is synthesized as an approximately 67-kDa preprotein with an N-terminal extension and imported into mitochondria, where it is processed to the approximately 62-kDa mature form. Topology analysis revealed that mature AIF is a type-I inner membrane protein with the N-terminus exposed to the matrix and the C-terminal portion to the intermembrane space. Upon induction of apoptosis, processing of mature AIF to an approximately 57-kDa form occurred caspase-independently in the intermembrane space, releasing the processed form into the cytoplasm. Bcl-2 or Bcl-XL inhibited both these events. These findings indicate that AIF release from mitochondria occurs by a two-step process: detachment from the inner membrane by apoptosis-induced processing in the intermembrane space and translocation into the cytoplasm. The results also suggest the presence of a unique protease that is regulated by proapoptotic stimuli in caspase-independent cell death.     

The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability

Mitochondria are well known as sites of electron transport and generators of cellular ATP. Mitochondria also appear to be sites of cell survival regulation. In the process of programmed cell death, mediators of apoptosis can be released from mitochondria through disruptions in the outer mitochondrial membrane; these mediators then participate in the activation of caspases and of DNA degradation. Thus the regulation of outer mitochondrial membrane integrity is an important control point for apoptosis. The Bcl-2 family is made up of outer mitochondrial membrane proteins that can regulate cell survival, but the mechanisms by which Bcl-2 family proteins act remain controversial. Most metabolites are permeant to the outer membrane through the voltage dependent anion channel (VDAC), and Bcl-2 family proteins appear to be able to regulate VDAC function. In addition, many Bcl-2 family proteins can form channels in vitro, and some pro-apoptotic members may form multimeric channels large enough to release apoptosis promoting proteins from the intermembrane space. Alternatively, Bcl-2 family proteins have been hypothesized to coordinate the permeability of both the outer and inner mitochondrial membranes through the permeability transition (PT) pore. Increasing evidence suggests that alterations in cellular metabolism can lead to pro-apoptotic changes, including changes in intracellular pH, redox potential and ion transport. By regulating mitochondrial membrane physiology, Bcl-2 proteins also affect mitochondrial energy generation, and thus influence cellular bioenergetics. Cell Death and Differentiation (2000) 7, 1182 - 1191     

Mitochondria frozen with trehalose retain a number of biological functions and preserve outer membrane integrity

In apoptosis, Bcl-2-family proteins regulate the barrier function of the mitochondrial outer membrane (MOM), controlling the release of proapoptotic proteins from the intermembrane space into the cytoplasm. This process can be studied in vitro with freshly isolated mouse liver mitochondria. Unfortunately, mitochondria frozen/thawed in standard sucrose-mannitol buffers become leaky and useless for apoptosis research. However, here we show that mitochondria frozen in buffer containing the sugar, trehalose, maintained MOM integrity and responsiveness to Bcl-2-family proteins, much like fresh mitochondria. Trehalose also preserved ultrastructure, as well as biological functions such as ATP synthesis, calcium-induced swelling, transmembrane potential, and the import and processing of protein precursors. However, bioenergetic function was somewhat reduced. Thus, trehalose-frozen mitochondria retained most of the biological features of mitochondria including MOM integrity. Although not ideal for studies involving bioenergetics, this method will facilitate research on apoptosis and other mitochondrial functions that rely on an intact MOM.     

Is mPTP the gatekeeper for necrosis, apoptosis, or both?

Permeabilization of the mitochondrial membranes is a crucial step in apoptosis and necrosis. This phenomenon allows the release of mitochondrial death factors, which trigger or facilitate different signaling cascades ultimately causing the execution of the cell. The mitochondrial permeability transition pore (mPTP) has long been known as one of the main regulators of mitochondria during cell death. mPTP opening can lead to matrix swelling, subsequent rupture of the outer membrane, and a nonspecific release of intermembrane space proteins into the cytosol. While mPTP was purportedly associated with early apoptosis, recent observations suggest that mitochondrial permeabilization mediated by mPTP is generally more closely linked to events of late apoptosis and necrosis. Mechanisms of mitochondrial membrane permeabilization during cell death, involving three different mitochondrial channels, have been postulated. These include the mPTP in the inner membrane, and the mitochondrial apoptosis-induced channel (MAC) and voltage-dependent anion-selective channel (VDAC) in the outer membrane. New developments on mPTP structure and function, and the involvement of mPTP, MAC, and VDAC in permeabilization of mitochondrial membranes during cell death are explored. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

The Role of the Mitochondrial Apoptosis Induced Channel MAC in Cytochrome <Emphasis Type="Italic">c</Emphasis> Release

Permeabilization of the mitochondrial outer membrane is a crucial event during apoptosis. It allows the release of proapoptotic factors, like cytochrome c, from the intermembrane space, and represents the commitment step in apoptosis. The mitochondrial apoptosis-induced channel, MAC, is a high-conductance channel that forms during early apoptosis and is the putative cytochrome c release channel. Unlike activation of the permeability transition pore, MAC formation occurs without loss of outer membrane integrity and depolarization. The single channel behavior and pharmacology of reconstituted MAC has been characterized with patch-clamp techniques. Furthermore, MAC’s activity is compared to that detected in mitochondria inside the cells at the time cytochrome c is released. Finally, the regulation of MAC by the Bcl-2 family proteins and insights concerning its molecular composition are also discussed.     

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.