Multicopy suppressors of phenotypes resulting from the absence of yeast VDAC encode a VDAC-like protein.

The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel). Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1. However, cells missing the POR1 gene (delta por1) were able to grow on yeast media containing a nonfermentable carbon source (glycerol) but not on such media at elevated temperature (37 degrees C). If VDAC normally provides the pathway for metabolites to pass through the outer membrane, some other protein(s) must be able to partially substitute for that function. To identify proteins that could functionally substitute for POR1, we have screened a yeast genomic library for genes which, when overexpressed, can correct the growth defect of delta por1 yeast grown on glycerol at 37 degrees C. This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amino acid sequence identity to the previously identified yeast VDAC protein (YVDAC1). YVDAC2 can functionally complement defects present in delta por1 strains only when it is overexpressed. Deletion of the POR2 gene alone had no detectable phenotype, while yeasts with deletions of both the POR1 and POR2 genes were viable and able to grow on glycerol at 30 degrees C, albeit more slowly than delta por1 single mutants. Like delta por1 single mutants, they could not grow on glycerol at 37 degrees C. Subcellular fractionation studies with antibodies which distinguish YVDAC1 and YVDAC2 indicate that YVDAC2 is normally present in the outer mitochondrial membrane. However, no YVDAC2 channels were detected electrophysiologically in reconstituted systems. Therefore, mitochondrial membranes made from wild-type cells, delta por1 cells, delta por1 delta por2 cells, and delta por1 cells overexpressing YVDAC2 were incorporated into liposomes and the permeability of resulting liposomes to nonelectrolytes of different sizes was determined. The results indicate that YVDAC2 does not confer any additional permeability to these liposomes, suggesting that it may not normally form a channel. In contrast, when the VDAC gene from Drosophila melanogaster was expressed in delta por1 yeast cells, VDAC-like channels could be detected in the mitochondria by both bilayer and liposome techniques, yet the cells failed to grow on glycerol at 37 degrees C. Thus, channel-forming activity does not seem to be either necessary or sufficient to restore growth on nonfermentable carbon sources, indicating that VDAC mediates cellular functions that do not depend on the ability to form channels.     

Regulation of Metabolite Flux through Voltage-Gating of VDAC Channels

The mitochondrial outer membrane channel, VDAC, is thought to serve as the major permeability pathway for metabolite flux between the cytoplasm and mitochondria. The permeability of VDAC to citrate, succinate, and phosphate was studied in channels reconstituted into planar phospholipid membranes. All ions showed large changes in permeability depending on whether the channel was in the open or in the low conductance, ``closed' state, with the closed state always more cation selective. This was especially true for the divalent and trivalent anions. Additionally, the anion flux when the voltage was zero was shown to decrease to 5–11% of the open state flux depending on the anion studied. These results give the first rigorous examination of the ability of metabolites to permeate through VDAC channels and indicate that these channels can control the flux of these ions through the outer membrane. This lends more evidence to the growing body of experiments that suggest that the outer mitochondrial membrane has a much more important role in controlling mitochondrial activity than has been thought historically. Received: 4 November 1996/Revised: 8 January 1997     

Actin Modulates the Gating of Neurospora crassa VDAC

VDAC forms the major pathway for metabolites across the mitochondrial outer membrane. The regulation of the gating of VDAC channels is an effective way to control the flow of metabolites into and out of mitochondria. Here we present evidence that actin can modulate the gating process of Neurospora crassa VDAC reconstituted into membranes made with phosphatidylcholine. An actin concentration as low as 50 nm caused the VDAC-mediated membrane conductance to drop by as much as 85% at elevated membrane potentials. Actin's effect could be quickly reversed by adding pronase to digest the protein. α-Actin, from mammalian muscle, has a stronger effect than β- and γ-actin from human platelets. The monomeric form of actin, G-actin, is effective. Stabilization of the fibrous form, F-actin, with the mushroom toxin, phalloidin, blocks the effect of actin on VDAC, indicating that F-actin might be ineffective. Cytochalasin B did not interfere with the ability of actin to favor VDAC closure. DNase-I did effectively block actin's effect on VDAC, and VDAC decreased actin's inhibitory effect on DNase-I activity, indicating that N. crassa VDAC competes with DNase-I for the same binding site on actin. The actin-VDAC interaction might be a mechanism by which actin regulates energy metabolism. Received: 28 August 2000/Revised: 1 December 2000     

Inactivation of the Peptide-Sensitive Channel from the Yeast Mitochondrial Outer Membrane: Properties,Sensitivity to Trypsin and Modulation by a Basic Peptide

The yeast Peptide Sensitive Channel (PSC), a cationic channel of the mitochondrial outer membrane closes with slow kinetics at potentials of either polarity. The properties of this inactivation closely resemble those of the Voltage-Dependent Anion Channel (VDAC) slow kinetics closures. Addition of trypsin to one compartment suppresses the inactivation observed when this compartment is made positive, but does not affect the inactivation observed at potentials of reverse polarity. Both sides of the channel are sensitive. The reduced form of the Mast Cell Degranulating peptide (rMCD) increases the rate of inactivation, but only when the polarity of the compartment to which it is added is positive. The effect is not reversed by washing the peptide out, but is suppressed by trypsin. The peptide can bind to both sides of the membrane. The effect of rMCD on PSC closely resembles that of the ``modulator' on VDAC. The similarities between PSC and VDAC suggest that the former might be a cationic porin of the mitochondrial outer membrane possessing a structure closely related to that of VDAC. Received: 2 February 1996/Revised: 18 October 1996     

Purification and characterization of the voltage-dependent anion channel from the outer mitochondrial membrane of yeast

Summary The outer mitochondrial membranes of all organisms so far examined contain a protein which forms voltage-dependent anion selective channels (VDAC) when incorporated into planar phospholipid membranes. Previous reports have suggested that the yeast (Saccharomyces cerevisiae) outer mitochondrial membrane component responsible for channel formation is a protein of 29,000 daltons which is also the major component of this membrane. In this report, we describe the purification of this 29,000-dalton protein to virtual homogeneity from yeast outer mitochondrial membranes. The purified protein readily incorporates into planar phospholipid membranes to produce ionic channels. Electrophysiological characterization of these channels has demonstrated they have a size, selectivity and voltage dependence similar to VDAC from other organisms. Biochemically, the purified protein has been characterized by determining its amino acid composition and isoelectric point (pI). In addition, we have shown that the purified protein, when reconstituted into liposomes, can bind hexokinase in a glucose-6-phosphate dependent manner, as has been shown for VDAC purified from other sources. Since physiological characterization suggests that the functional parameters of this protein have been conserved, antibodies specific to yeast VDAC have been used to assess antigenic conservation among mitochondrial proteins from a wide number of species. These experiments have shown that yeast VDAC antibodies will recognize single mitochondrial proteins fromDrosophila, Dictyostelium andNeurospora of the appropriate molecular weight to be VDAC from these organisms. No reaction was seen to any mitochondrial protein from rat liver, rainbow trout,Paramecium, or mung bean. In addition, yeast VDAC antibodies will recognize a 50-kDa mol wt protein present in tobacco chloroplasts. These results suggest that there is some antigenic as well as functional conservation among different VDACs.     

The Role of Yeast VDAC Genes on the Permeability of the Mitochondrial Outer Membrane

In addition to the POR1 gene, which encodes the well-characterized voltage dependent anion-selective channel (YVDAC1) of the mitochondrial outer membrane, the yeast Saccharomyces cerevisiae contains a second gene (POR2) encoding a protein (YVDAC2) with 50% sequence identity to YVDAC1. Mitochondria isolated from yeast cells deleted for the POR1 gene (Δpor1) had a profoundly reduced outer membrane permeability as measured by the ability of an intermembrane space dehydrogenase to oxidize exogenously added NADH. Mitochondria missing either YVDAC1 or both YVDAC1 and YVDAC2 showed a 2-fold increase in the rate of NADH oxidation when the outer membrane was deliberately damaged. Mitochondria from parental cells showed only a 10% increase indicating that the outer membrane is highly permeable to NADH. In the absence of YVDAC1, we calculate that the outer membrane permeability to NADH is reduced 20-fold. The low NADH permeability in the presence of YVDAC2 was not due to the low levels of YVDAC2 expression as mitochondria from cells expressing levels of YVDAC2 comparable to those of YVDAC1 in parental cells showed no substantial increase in NADH permeability, indicating a minimal role of YVDAC2 in this permeability. The residual permeability may be due to other pathways because cells missing both genes can still grow on nonfermentable carbon sources. However, YVDAC1 is clearly the major pathway for NADH flux through the outer membrane in these mitochondria. Received: 23 May 1997/Revised: 3 October 1997     

Under Conditions of Insufficient Permeability of VDAC1, External NADH May Use the TOM Complex Channel to Cross the Outer Membrane of Saccharomyces cerevisiae Mitochondria

Thus far, only three channel-forming activities have been identified in the outer membrane of the yeast Saccharomyces cerevisiae mitochondria. Two of them, namely the TOM complex channel (translocase of the outer membrane) and the PSC (peptide-sensitive channel) participate in protein translocation and are probably identical, whereas a channel-forming protein called VDAC (voltage-dependent anion channel) serves as the major pathway for metabolites. The VDAC is present in two isoforms (VDAC1 and VDAC2) of which only VDAC1 has been shown to display channel-forming activity. Moreover, the permeability of VDAC1 has been reported to be limited in uncoupled mitochondria of S. cerevisiae. The presented data indicate that in S. cerevisiae-uncoupled mitochondria, external NADH, applied at higher concentrations (above 50 nmoles per 0.1 mg of mitochondrial protein), may use the TOM complex channel, besides VDAC1, to cross the outer membrane. Thus, the permeability of VDAC1 could be a limiting step in transport of external NADH across the outer membrane and might be supplemented by the TOM complex channel.     

Swapping of the N-terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti-aging features to the cell

Voltage-dependent anion-selective channels (VDACs) are pore-forming proteins allowing the permeability of the mitochondrial outer membrane. The VDAC3 isoform is the least abundant and least active in a complementation assay performed in a yeast strain devoid of porin-1. We swapped the VDAC3 N-terminal 20 amino acids with homologous sequences from the other isoforms. The substitution of the VDAC3 N-terminus with the VDAC1 N-terminus caused the chimaera to become more active than VDAC1. The VDAC2 N-terminus improved VDAC3 activity, though to a lesser extent. The VDAC3 carrying the VDAC1 N-terminus was able to complement the lack of the yeast porin in mitochondrial respiration and in modulation of reactive oxygen species (ROS). This chimaera increased life span, indicating a more efficient bioenergetic metabolism and/or a better protection from ROS.     

Each mammalian mitochondrial outer membrane porin protein is dispensable: effects on cellular respiration.

Voltage-dependent anion channels (VDACs, also known as mitochondrial porins) are small pore-forming proteins of the mitochondrial outer membrane found in all eukaryotes. Mammals harbor three distinct VDAC isoforms, with each protein sharing 65-70% sequence identity. Deletion of the yeast VDAC1 gene leads to conditional lethality that can be partially or completely complemented by the mammalian VDAC genes. In vitro, VDACs conduct a variety of small metabolites and in vivo they serve as a binding site for several cytosolic kinases involved in intermediary metabolism, yet the specific physiologic role of each isoform is unknown. Here we show that mouse embryonic stem cells lacking each isoform are viable but exhibit a 30% reduction in oxygen consumption. VDAC1 and VDAC2 deficient cells exhibit reduced cytochrome c oxidase activity, whereas VDAC3 deficient cells have normal activity. These results indicate that VDACs are not essential for cell viability and we speculate that reduced respiration in part reflects decreased outer membrane permeability for small metabolites necessary for oxidative phosphorylation.     

VDAC1, having a shorter N-terminus than VDAC2 but showing the same migration in an SDS-polyacrylamide gel, is the predominant form expressed in mitochondria of various tissues

The voltage-dependent anion channel (VDAC) is a pore-forming protein expressed in the outer membrane of eukaryotic mitochondria. Three isoforms of it, i.e., VDAC1, VDAC2, and VDAC3, are known to be expressed in mammals; however, the question as to which is the main isoform in mitochondria is still unanswered. To address this question, we first prepared standard VDACs by using a bacterial expression system and raised various antibodies against them by using synthetic peptides as immunogens. Of the three bacterially expressed VDAC isoforms, VDAC3 showed faster migration in SDS-polyacrylamide gels than VDAC1 and VDAC2, although VDAC2 is longer than VDAC1 and VDAC3, due to a 12-amino acid extension of its N-terminal region. Even with careful structural characterization of the expressed VDACs by LC-MS/MS analysis, serious structural modifications of VDACs causing changes in their migration in SDS-polyacrylamide gels were not detected. Next, immunoreactivities of the raised antibodies toward these bacterially expressed VDAC isoforms were evaluated. Trials to prepare specific antibodies against the three individual VDAC isoforms were not successful except in the case of VDAC1. However, using a synthetic peptide corresponding to the highly conserved region among the three VDACs, we were successful in preparing an antibody showing essentially equal immunoreactivities toward all three VDACs. When mitochondrial outer membrane proteins of various rat tissues were subjected to 2-dimensional electrophoresis followed by immunoblotting with this antibody, six immunoreactive protein spots were detected. These spots were characterized by LC-MS/MS analysis, and the signal intensities among the spots were compared. As a result, the signal intensity of the spot representing VDAC1 was the highest, and thus, VDAC1 was concluded to be the most abundantly expressed of the three VDAC isoforms in mammalian mitochondria.     

Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes

Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 μM digitonin. Blockade of VDAC with Koenig’s polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 μM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC.     

On the Role of VDAC in Apoptosis: Fact and Fiction

Research on VDAC has accelerated as evidence grows of its importance in mitochondrial function and in apoptosis. New investigators entering the field are often confounded by the VDAC literature and its many apparent conflicts and contradictions. This review is an effort to shed light on the situation and identify reliable information from more questionable claims. Our views on the most important controversial issues are as follows: VDAC is only present in the mitochondrial outer membrane. VDAC functions as a monomer. VDAC functions normally with or without Ca²⁺. It does not form channels that mediate the flux of proteins through membranes (peptides and unfolded proteins are excluded from this statement). Closure of VDAC, not VDAC opening, leads to mitochondria outer membrane permeabilization and apoptosis.     

Redox regulation of protein expression in Saccharomyces cerevisiae mitochondria: possible role of VDAC

Using Saccharomyces cerevisiae mutants depleted of either isoform of VDAC (voltage dependent anion selective channel) we studied the role of the cytosol and mitochondria redox states in regulation of the expression levels of some mitochondrial proteins. The studied proteins are MnSOD and subunits of the protein import machinery of the mitochondrial outer membrane, i.e. Tom70, Tom40 and Tob55 (Sam50). We have shown that both the cytosol and mitochondria redox states depend on the presence of a given VDAC isoform. The cytosol redox state is mediated by VDAC1, although VDAC2 has a quantitative effect, whereas the mitochondria redox state depends on the presence of both VDAC isoforms. Moreover, we have shown that the cytosol redox status but not the mitochondrial one is decisive for the expression levels of the studied mitochondrial proteins. Thus, expression levels of some mitochondrial proteins is influenced by VDAC and this regulatory process at least partially does not require its channel activity as VDAC2 does not form a channel. Thus, VDAC can be regarded as a participant of signaling pathways in S. cerevisiae cells.     

Isoforms of voltage-dependent anion channel of the outer mitochondrial membrane and experimental models to study their physiological role

The review outlines our current understanding of the role of porins, the proteins forming voltage-dependent anion channels (VDAC), in regulation of permeability of the outer mitochondrial membrane. Recent data on the porin structure, amino acid sequence, and isoforms are discussed. The existence of three different VDAC isoforms in mammalian cells suggests that each isoform may play a specific physiological role that remains unknown so far. Different model systems and methods used for studies of functional differences between VDAC isoforms are overviewed. Particular attention is paid to studies of mammalian VDAC isoforms by means of expression of the corresponding genes in yeast and human cells as well as creation of stem cell clones and animals with genetically deficient isoforms of VDAC. It is concluded that permeability of the outer membrane plays a crucial role in the mechanisms of metabolic regulation and that porins are vitally important in the physiology of mammals. The data on the functional role of the VDAC isoforms can be useful for under-standing the mechanisms of such pathologies as cancer, diabetes, and neuromuscular diseases.     

Post-translational modifications of VDAC1 and VDAC2 cysteines from rat liver mitochondria

VDACs three isoforms (VDAC1, VDAC2, VDAC3) are integral proteins of the outer mitochondrial membrane whose primary function is to permit the communication and exchange of molecules related to the mitochondrial functions. We have recently reported about the peculiar over-oxidation of VDAC3 cysteines. In this work we have extended our analysis, performed by tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS, to the other two isoforms VDAC1 and VDAC2 from rat liver mitochondria, and we have been able to find also in these proteins over-oxidation of cysteines. Further PTM of cysteines as succination has been found, while the presence of selenocysteine was not detected. Unfortunately, a short sequence stretch containing one genetically encoded cysteine was not covered both in VDAC2 and in VDAC3, raising the suspect that more, unknown modifications of these proteins exist. Interestingly, cysteine over-oxidation appears to be an exclusive feature of VDACs, since it is not present in other transmembrane mitochondrial proteins eluted by hydroxyapatite. The assignment of a functional role to these modifications of VDACs will be a further step towards the full understanding of the roles of these proteins in the cell.     

Respiratory State and Phosphatidylserine Import in Brain Mitochondria In Vitro

The mechanism of phosphatidylserine (PS) movement from donor membranes into rat brain mitochondria was investigated. Mitochondria were incubated with liposomes and subjected to density gradient centrifugation. The energized state was monitored by flow cytometry measuring the fluorescence of membrane-potential-sensitive rhodamine-123 dye. Mitochondria density decreased upon increase of the respiratory rate, as a consequence of their association with liposomes. After interaction of mitochondria with ¹⁴C-PS containing liposomes, ¹⁴C-PS became a substrate of PS decarboxylase, as monitored by the formation of ¹⁴C-phosphatidylethanolamine (PE), indicating translocation of ¹⁴C-PS to the inner membrane. The kinetics of ¹⁴C-PE formation showed a high rate upon addition of ADP, malate and pyruvate (state 3) compared to control (state 1). In state 3, ¹⁴C-PE formation decreased in the presence of NaN₃. Mitochondria-associated membranes (MAM) are the major site of PS synthesis. However, their role in the translocation of PS to mitochondria has not been completely elucidated. A crude mitochondrial fraction (P₂) containing MAM, synaptosomes and myelin was prelabeled with ¹⁴C-PS and incubated in different respiratory states. At a high respiratory rate, low-density labeled mitochondria, whose band overlaps that of synaptosomes, were obtained by centrifugation. A parallel decrease of both radioactivity and protein in MAM fraction was observed, indicating that the association of MAM and mitochondria had occurred. Synthesis and translocation of ¹⁴C-PS in P₂ membranes were also studied by incubating P₂ with ¹⁴C-serine. In the resting state ¹⁴C-PS accumulated in MAM, indicating that the transfer to mitochondria was a limiting step. In state 3 both the transfer rate of ¹⁴C-PS and its conversion to ¹⁴C-PE increased. Respiratory mitochondrial activity modulated the association of MAM and mitochondria, triggering a mechanism that allowed the transport of PS across the outer mitochondrial membrane. Received: 7 April 1999/Revised: 21 September 1999     

VDAC as a voltage-dependent mitochondrial gatekeeper under physiological conditions

Mitochondria, composed of two membranes, play a key role in energy production in eukaryotic cells. The main function of the inner membrane is oxidative phosphorylation, while the mitochondrial outer membrane (MOM) seems to control the energy flux and exchange of various charged metabolites between mitochondria and the cytosol. Metabolites cross MOM via the various isoforms of voltage-dependent anion channel (VDAC). In turn, VDACs interact with some enzymes, other proteins and molecules, including drugs. This work aimed to analyze various literature experimental data related to targeting mitochondrial VDACs and VDAC-kinase complexes on the basis of the hypothesis of generation of the outer membrane potential (OMP) and OMP-dependent reprogramming of cell energy metabolism. Our previous model of the VDAC-hexokinase-linked generation of OMP was further complemented in this study with an additional regulation of the MOM permeability by the OMP-dependent docking of cytosolic proteins like tubulin to VDACs. Computational analysis of the model suggests that OMP changes might be involved in the mechanisms of apoptosis promotion through the so-called transient hyperpolarization of mitochondria. The high concordance of the performed computational estimations with many published experimental data allows concluding that OMP generation under physiological conditions is highly probable and VDAC might function as an OMP-dependent gatekeeper of mitochondria, controlling cell life and death. The proposed model of OMP generation allows understanding in more detail the mechanisms of cancer death resistance and anticancer action of various drugs and treatments influencing VDAC voltage-gating properties, VDAC content, mitochondrial hexokinase activity and VDAC-kinase interactions in MOM.     

Voltage-dependent anion-selective channels VDAC2 and VDAC3 are abundant proteins in bovine outer dense fibers, a cytoskeletal component of the sperm flagellum

Outer dense fibers (ODF) are specific subcellular components of the sperm flagellum. The functions of ODF have not yet been clearly elucidated. We have investigated the protein composition of purified ODF from bovine spermatozoa and found that one of the most abundant proteins is a 30-32-kDa polypeptide. This protein was analyzed by sequencing peptides derived following limited proteolysis. Peptide sequences were found to match VDAC2 and VDAC3. VDACs (voltage-dependent, anion-selective channels) or eukaryotic porins are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures in membranes. In mammals, three VDAC isoforms (VDAC1, -2, -3) have been identified by cDNA cloning and sequencing. Antibodies against synthetic peptides specific for the three mammal VDAC isoforms were generated in rabbits. Their specificity was demonstrated by immunoblotting using recombinant VDAC1, -2, and -3. In protein extracts of bovine spermatozoa, VDAC1, -2, and -3 were detected by specific antibodies, while only VDAC2 and -3 were found as solubilized proteins derived from purified bovine ODFs. Immunofluorescence microscopy of spermatozoa revealed that anti-VDAC2 and anti-VDAC3 antibodies clearly bound to the sperm flagellum, in particular to the ODF. Transmission electron immunomicroscopy supported the finding that VDAC2 protein is abundant in the ODF. Since the ODF does not have any known membranous structure, it is tempting to speculate that VDAC2 and VDAC3 might have an alternative structural organization and different functions in ODF than in mitochondria.     

Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane

The diffusion of metabolites across the outer mitochondrial membrane is essential for coupled cellular respiration. The outer membrane of mitochondria isolated from growth factor-deprived cells is impaired in its ability to exchange metabolic anions. When added to mitochondria, recombinant Bcl-x(L) restores metabolite exchange across the outer membrane without inducing the loss of cytochrome c from the intermembrane space. Restoration of outer membrane permeability to anionic metabolites does not occur directly through Bcl-x(L) ion channels. Instead, recombinant Bcl-x(L) maintains the outer mitochondrial membrane channel, VDAC, in an open configuration. Consistent with these findings, when ADP-induced oxidative phosphorylation is limited by exogenous beta-NADH, recombinant Bcl-x(L) can sustain outer mitochondrial membrane permeability to ADP. beta-NADH limits respiration by promoting the closed configuration of VDAC. Together these results demonstrate that following an apoptotic signal, Bcl-x(L) can maintain metabolite exchange across the outer mitochondrial membrane by inhibiting VDAC closure.     

Bid, but not Bax, regulates VDAC channels

During apoptosis, cytochrome c is released from mitochondria into the cytosol, where it participates in caspase activation. Various and often conflicting mechanisms have been proposed to account for the increased permeability of the mitochondrial outer membrane that is responsible for this process. The voltage-dependent anion channel (VDAC) is the major permeability pathway for metabolites in the mitochondrial outer membrane and therefore is a very attractive candidate for cytochrome c translocation. Here, we report that properties of VDAC channels reconstituted into planar phospholipid membranes are unaffected by addition of the pro-apoptotic protein Bax under a variety of conditions. Contrary to other reports (Shimizu, S., Narita, M., and Tsujimoto, Y. (1999) Nature 399, 483-487; Shimizu, S., Ide, T., Yanagida, T., and Tsujimoto, Y. (2000) J. Biol. Chem. 275, 12321-12325; Shimizu, S., Konishi, A., Kodama, T., and Tsujimoto, Y. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3100-3105), we found no electrophysiologically detectable interaction between VDAC channels isolated from mammalian mitochondria and either monomeric or oligomeric forms of Bax. We conclude that Bax does not induce cytochrome c release by acting on VDAC. In contrast to Bax, another pro-apoptotic protein (Bid) proteolytically cleaved with caspase-8 affected the voltage gating of VDAC by inducing channel closure. We speculate that by decreasing the probability of VDAC opening, Bid reduces metabolite exchange between mitochondria and the cytosol, leading to mitochondrial dysfunction.