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.     

Role of phosphorylation of porin (VDAC) in regulation of mitochondrial outer membrane under normal conditions and alcohol intoxication

The present work is an overview of the factors regulating permeability of the outer membrane of mitochondria and the state of the channels formed by porin (voltage-dependent anion channels, VDAC). According to the accumulated data, modulation of the outer membrane permeability can be induced by endogenous phosphorylation of VDAC channels. Different protein kinases, such as protein kinase A, protein kinase C, tyrosine protein kinase, hexokinase, glycogen synthetase kinase-3β (GSK-3β), Akt and p38 kinases, were shown to be involved in VDAC phosphorylation. Among these protein kinases, alcohol-induced stress-kinases, GSK-3β, Akt, and p38 identified in mitochondria may participate in phosphorylation of porin, modulation of VDAC conductance, and regulation of the outer membrane permeability.     

Reflections on VDAC as a voltage-gated channel and a mitochondrial regulator

There is excellent agreement between the electrophysiological properties and the structure of the mitochondrial outer membrane protein, VDAC, ex vivo. However, the inference that the well-defined canonical “open” state of the VDAC pore is the normal physiological state of the channel in vivo is being challenged by several lines of evidence. Knowing the atomic structure of the detergent solubilized protein, a long sought after goal, will not be sufficient to understand the functioning of this channel protein. In addition, detailed information about VDAC’s topology in the outer membrane of intact mitochondria, and the structural changes that it undergoes in response to different stimuli in the cell will be needed to define its physiological functions and regulation.     

Role of voltage-dependent anion channels in glutathione transport into yeast mitochondria

Glutathione (GSH) is imported into mitochondria from the extra-mitochondrial cytoplasm. Translocation across the inner membrane of mitochondria is thought to occur via the dicarboxylate and 2-oxoglutarate carriers; however, the means by which GSH passes through the outer membrane is unknown. Disruption of the outer membrane of yeast mitochondria using either digitonin or osmotic shock did not alter GSH accumulation as compared with accumulation in intact mitochondria. These results suggested that passage across the outer membrane was not the rate-limiting step in GSH accumulation. Mitochondria isolated from yeast strains with a disruption in the major pore-forming protein of the outer membrane, VDAC1, accumulated GSH to a greater extent than mitochondria isolated from a wild-type strain. Disruption of the gene for VDAC2 did not affect GSH import. Thus, neither VDAC form is essential for GSH translocation into mitochondria, and the participation of another outer membrane channel in GSH import is possible.     

Approaching the structure of human VDAC1, a key molecule in mitochondrial cross-talk

The voltage dependent anion-channel, VDAC, is the major constitutive protein of the outer membrane of mitochondria. Functionally, VDAC is involved in the exchange of small metabolites over the mitochondrial outer membrane and supports enzymes of the cytoplasm with energy precursors i.e. ATP. Moreover, the channel alone or in complex with proteins of the inner mitochondrial membrane or the intermembrane space provides a basis for docking of cytosolic proteins which can regulate outer membrane permeability in several ways. Structurally, this channel has a bacterial origin by evolution and partly resembles bacterial porin functions. However, the structure seems more complex as a variety of interactions on both channel sides can occur. Therefore, our work described is aiming to determine the structure of VDAC at atomic resolution and together with functional data to understand better how this channel can carry out such a variety of differing functions.     

The 'ins' and 'outs' of mitochondrial membrane channels.

The outer membrane of the mitochondrion contains thousands of copies of a pore-forming protein called VDAC or porin. Considerable progress has been made towards elucidating the molecular structure of this channel. Moreover, mounting evidence that the permeability of VDAC may be regulated is challenging the textbook notion of the outer membrane as a simple sieve. Numerous other channel activities have been detected by electrophysiol approaches in both the outer and inner mitochondrial membranes. The inner-membrane channels do not appear to be open under normal physiological conditions and so should not dissipate energy-transducing ion gradients. The biological functions of the different classes of mitochondrial channels are uncertain, but several possibilities (including protein translocation) are being explored.     

Mdm10 is an ancient eukaryotic porin co-occurring with the ERMES complex

Mitochondrial β-barrel proteins fulfill central functions in the outer membrane like metabolite exchange catalyzed by the voltage-dependent anion channel (VDAC) and protein biogenesis by the central components of the preprotein translocase of the outer membrane (Tom40) or of the sorting and assembly machinery (Sam50). The mitochondrial division and morphology protein Mdm10 is another essential outer membrane protein with proposed β-barrel fold, which has so far only been found in Fungi. Mdm10 is part of the endoplasmic reticulum mitochondria encounter structure (ERMES), which tethers the ER to mitochondria and associates with the SAM complex. In here, we provide evidence that Mdm10 phylogenetically belongs to the VDAC/Tom40 superfamily. Contrary to Tom40 and VDAC, Mdm10 exposes long loops towards both sides of the membrane. Analyses of single loop deletion mutants of Mdm10 in the yeast Saccharomyces cerevisiae reveal that the loops are dispensable for Mdm10 function. Sequences similar to fungal Mdm10 can be found in species from Excavates to Fungi, but neither in Metazoa nor in plants. Strikingly, the presence of Mdm10 coincides with the appearance of the other ERMES components. Mdm10's presence in both unikonts and bikonts indicates an introduction at an early time point in eukaryotic evolution.     

Regulation of the mitochondrial outer membrane channel,VDAC

The channel-forming protein, VDAC, located in the mitochondrial outer membrane, is probably responsible for the high permeability of the outer membrane to small molecules. The ability to regulate this channelin vitro raises the possibility that VDAC may perform a regulatory rolein vivo. VDAC exists in multiple, quasi-degenerate conformations with different permeability properties. Therefore a modest input of energy can change VDAC's conformation. The ability to use a membrane potential to convert VDAC from a high (open) to a low (closed) conducting form indicates the presence of a sensor in the protein that allows it to respond to the electric field. Titration and modification experiments point to a polyvalent, positively charged sensor. Soluble, polyvalent anions such as dextran sulfate and Konig's polyanion seem to be able to interact with the sensor to induce channel closure. Thus there are multiple ways of applying a force on the sensor so as to induce a conformational change in VDAC. Perhaps cells use one or more of these methods.     

Does the voltage dependent anion channel modulate cardiac ischemia–reperfusion injury?

The voltage dependent anion channel (VDAC) provides exchange of metabolites, anions, and cations across the outer mitochondrial membrane. VDAC provides substrates and adenine nucleotides necessary for electron transport and therefore plays a key role in regulating mitochondrial bioenergetics. VDAC has also been suggested to regulate the response to cell death signaling. Emerging data show that VDAC is regulated by protein–protein interactions as well as by post-translational modifications. This review will focus on the regulation of VDAC and its potential role in regulating cell death in cardiac ischemia–reperfusion. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

A 3D model of the voltage-dependent anion channel (VDAC)

Eukaryotic porins are a group of membrane proteins whose best known role is to form an aqueous pore channel in the mitochondrial outer membrane. As opposed to the bacterial porins (a large family of protein whose 3D structure has been determined by X-ray diffraction), the structure of eukaryotic porins (also termed VDACs, voltage-dependent anion-selective channels) is still a matter of debate. We analysed the secondary structure of VDAC from the yeast Saccharomyces cerevisiae, the fungus Neurospora crassa and the mouse with different types of neural network-based predictors. The predictors were able to discriminate membrane β-strands, globular -helices and membrane -helices and localised, in all three VDAC sequences, 16 β-strands along the chain. For all three sequences the N-terminal region showed a high propensity to form a globular -helix. The 16 β-strand VDAC motif was thus aligned to a bacterial porin-derived template containing a similar 16 β-strand motif. The alignment of the VDAC sequence with the bacterial porin sequence was used to compute a set of 3D coordinates, which constitutes the first 3D prediction of a eukaryotic porin. All the predicted structures assume a β-barrel structure composed of 16 β-strands with the N-terminus outside the membrane. Loops are shorter in this side of the membrane than in the other, where two long loops are protruding. The shape of the pore varies between almost circular for Neurospora and mouse and slightly oval for yeast. Average values between 3 and 2.5 nm at the C-carbon backbone are found for the diameter of the channels. In this model VDAC shows large portions of the structure exposed on both sides of the membrane. The architecture we determine allows speculation about the mechanism of possible interactions between VDAC and other proteins on both sides of the mitochondrial outer membrane. The computed 3D model is consistent with most of the experimental results so far reported.     

Mouse VDAC Isoforms Expressed in Yeast: Channel Properties and Their Roles in Mitochondrial Outer Membrane Permeability

The channel-forming protein called VDAC forms the major pathway in the mitochondrial outer membrane and controls metabolite flux across that membrane. The different VDAC isoforms of a species may play different roles in the regulation of mitochondrial functions. The mouse has three VDAC isoforms (VDAC1, VDAC2 and VDAC3). These proteins and different versions of VDAC3 were expressed in yeast cells (S. cerevisiae) missing the major yeast VDAC gene and studied using different approaches. When reconstituted into liposomes, each isoform induced a permeability in the liposomes with a similar molecular weight cutoff (between 3,400 and 6,800 daltons based on permeability to polyethylene glycol). In contrast, electrophysiological studies on purified proteins showed very different channel properties. VDAC1 is the prototypic version whose properties are highly conserved among other species. VDAC2 also has normal gating activity but may exist in 2 forms, one with a lower conductance and selectivity. VDAC3 can also form channels in planar phospholipid membranes. It does not insert readily into membranes and generally does not gate well even at high membrane potentials (up to 80 mV). Isolated mitochondria exhibit large differences in their outer membrane permeability to NADH depending on which of the mouse VDAC proteins was expressed. These differences in permeability could not simply be attributed to different amounts of each protein present in the isolated mitochondria. The roles of these different VDAC proteins are discussed. Received: 19 June 1998/Revised: 1 April 1999     

A soluble mitochondrial protein increases the voltage dependence of the mitochondrial channel,VDAC

A soluble protein isolated from mitochondria has been found to modulate the voltage-dependent properties of the mitochondrial outer membrane channel, VDAC. This protein, called the VDAC modulator, was first found inNeurospora crassa and then discovered in species from other eukaryotic kingdoms. The modulator-containing fraction (at a crude protein concentration of 20 µg/ml) increases the voltage dependence of VDAC channels over 2–3-fold. At higher protein concentrations (50–100 µg/ml), some channels seem to remain in a closed state or be blocked while others display the higher voltage dependence and are able to close at low membrane potentials. By increasing the steepness of the voltage-dependent properties of VDAC channels, this modulator may serve as an amplifierin vivo to increase the sensitivity of the channels in response to changes in the cell's microenvironment, and consequently, regulate the metabolic flux across the outer mitochondrial membrane by controlling the gating of VDAC channels.     

线粒体电压依赖性阴离子通道及其调控功能

电压依赖性阴离子通道(voltage-dependent anion channel,VDAC)是存在于线粒体外膜上的31kDa膜蛋白,能在膜上形成亲水性通道,调控阴离子、阳离子、ATP以及其他代谢物进出线粒体,在调节细胞代谢、维持胞内钙稳态,调节细胞凋亡和坏死等过程中发挥重要功能。现就VDAC的结构、特性、活性调节及对细胞功能的调控作一综述。     

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.     

Direct membrane insertion of voltage-dependent anion-selective channel protein catalyzed by mitochondrial Tom20.

Insertion of newly synthesized proteins into or across the mitochondrial outer membrane is initiated by import receptors at the surface of the organelle. Typically, this interaction directs the precursor protein into a preprotein translocation pore, comprised of Tom40. Here, we show that a prominent beta-barrel channel protein spanning the outer membrane, human voltage- dependent anion-selective channel (VDAC), bypasses the requirement for the Tom40 translocation pore during biogenesis. Insertion of VDAC into the outer membrane is unaffected by plugging the translocation pore with a partially translocated matrix preprotein, and mitochondria containing a temperature-sensitive mutant of Tom40 insert VDAC at the nonpermissive temperature. Synthetic liposomes harboring the cytosolic domain of the human import receptor Tom20 efficiently insert newly synthesized VDAC, resulting in transbilayer transport of ATP. Therefore, Tom20 transforms newly synthesized cytosolic VDAC into a transmembrane channel that is fully integrated into the lipid bilayer.     

Inhibition of Bak Activation by VDAC2 Is Dependent on the Bak Transmembrane Anchor

Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death.     

Bcl-2 family of proteins: life-or-death switch in mitochondria

An increase in the permeability of outer mitochondrial membrane is central to apoptotic cell death, and results in the release of several apoptogenic factors such as cytochrome c into the cytoplasm to activate downstream destructive programs. The voltage-dependent anion channel (VDAC or mitochondrial porin) plays an essential role in disrupting the mitochondrial membrane barrier and is regulated directly by members of the Bcl-2 family proteins. Anti-apoptotic Bcl-2 family members interact with and close the VDAC, whereas some, but not all, proapoptotic members interact with VDAC to open protein-conducting pore through which apoptogenic factors pass. Although the VDAC is involved directly in breaking the mitochondrial membrane barrier and is a known component of the permeability transition pore complex, VDAC-dependent increase in outer membrane permeability can be independent of the permeability transition event such as mitochondrial swelling followed by rupture of the outer mitochondrial membrane. VDAC interacts not only with Bcl-2 family members but also with proteins such as gelsolin, an actin regulatory protein, and appears to be a convergence point for a variety of cell survival and cell death signals.     

NMR structural investigation of the mitochondrial outer membrane protein VDAC and its interaction with antiapoptotic Bcl-xL

Bcl-2 family proteins are essential regulators of cell death and exert their primary pro- or antiapoptotic roles at the mitochondrial outer membrane. Previously, pro- and antiapoptotic Bcl-2 proteins have been shown to interact with the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. VDAC is a 283-residue integral membrane protein that forms an aqueous pore in the outer mitochondrial membrane, through which metabolites and other small molecules pass between the cytosol and intermembrane space. The essential life-sustaining function of VDAC in metabolite trafficking is believed to be regulated by proteins of the Bcl-2 family. The protective role of antiapoptotic Bcl-xL may be through its interaction with VDAC. Here, VDAC has been expressed, purified, and refolded into a functional form amenable to NMR studies. Various biophysical experiments indicate that micelle-bound VDAC is in intermediate exchange between monomer and trimer. Using NMR spectroscopy, gel filtration, and chemical cross-linking, we obtained direct evidence for binding of Bcl-xL to VDAC in a detergent micelle system. The VDAC-interacting region of Bcl-xL was characterized by NMR with chemical shift perturbation and transferred cross-saturation. The interaction region was mapped to a putative helical hairpin motif of Bcl-xL that was found to insert into detergent micelles. Our results suggest that Bcl-xL can bind to one or two VDAC molecules forming heterodimers and heterotrimers. Our characterization of the VDAC/Bcl-xL complex offers initial structural insight into the role of antiapoptotic Bcl-xL in regulating apoptotic events in the mitochondrial outer membrane.     

VDAC isoforms in mammals

VDACs (Voltage Dependent Anion selective Channels) are a family of pore-forming proteins discovered in the mitochondrial outer membrane. In the animal kingdom, mammals show a conserved genetic organization of the VDAC genes, corresponding to a group of three active genes. Three VDAC protein isoforms thus exist. From a historically point of view most of the data collected about this protein refer to the VDAC1 isoform, the first to be identified and also the most abundant in the organisms. In this work we compare the information available about the three VDAC isoforms, with a special emphasis upon the human proteins, here considered prototypical of the group, and we try to shed some light on specific functional roles of this apparently redundant group of proteins. A new hypothesis about the VDAC(s) involvement in ROS control is proposed. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

VDAC isoforms in mammals

VDACs (Voltage Dependent Anion selective Channels) are a family of pore-forming proteins discovered in the mitochondrial outer membrane. In the animal kingdom, mammals show a conserved genetic organization of the VDAC genes, corresponding to a group of three active genes. Three VDAC protein isoforms thus exist. From a historically point of view most of the data collected about this protein refer to the VDAC1 isoform, the first to be identified and also the most abundant in the organisms. In this work we compare the information available about the three VDAC isoforms, with a special emphasis upon the human proteins, here considered prototypical of the group, and we try to shed some light on specific functional roles of this apparently redundant group of proteins. A new hypothesis about the VDAC(s) involvement in ROS control is proposed. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.