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.     

Mitochondrial VDAC2 and cell homeostasis: highlighting hidden structural features and unique functionalities

Voltage‐dependent anion channels (VDACs) are the gateway to mitochondrial processes, interlinking the cytosolic and mitochondrial compartments. The mitochondrion acts as a storehouse for cytochrome c, the effector of apoptosis, and hence VDACs become intricately involved in the apoptotic pathway. Isoform 1 of VDAC is abundant in the outer mitochondrial membrane of many cell types, while isoform 2 is the preferred channel in specialized cells including brain and some cancer cells. The primary role of VDACs is metabolite flux. The pro‐ and anti‐apoptotic role of VDAC1 and VDAC2, respectively, are secondary, and are influenced by external factors and interacting proteins. Herein, we focus on the less‐studied VDAC2, and shed light on its unique functions and features. VDAC2, along with sharing many of its functions with VDAC1, such as metabolite and Ca²⁺ transport, also has many delineating functions. VDAC2 is closely engaged in the gametogenesis and steroidogenesis pathways and in protection from oxidative stress as well as in neurodegenerative diseases like Alzheimer's and epilepsy. A closer examination of the functional pathways of VDACs indicates that the unique functions of VDAC2 are a result of the different interactome of this isoform. We couple functional differences to the structural and biophysical evidence obtained for the VDACs, and present a testament of why the two VDAC isoforms with >90% sequence similarity, are functionally diverse. Based on these differences, we suggest that the VDAC isoforms now be considered as paralogs. An in‐depth understanding of VDAC2 will help us to design better biomolecule targets for cancer and neurodegenerative diseases.     

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 supramolecular assemblies of voltage-dependent anion channels in the native membrane

Voltage-dependent anion channels (VDACs) are major constituents of the outer mitochondrial membrane (OMM). These primary transporters of nucleotides, ions and metabolites mediate a substantial portion of the OMM molecular traffic. To study the native supramolecular organization of the VDAC, we have isolated, characterized and imaged OMMs from potato tubers. SDS-PAGE and mass spectrometry of OMMs revealed the presence of the VDAC isoforms POM34 and POM36, as well as the translocase of the OMM complex. Tubular two-dimensional crystals of the VDAC spontaneously formed after incubation of OMMs for two to three months at 4 degrees C. Transmission electron microscopy revealed an oblique lattice and unit cells housing six circular depressions arranged in a hexagon. Atomic force microscopy of freshly isolated OMMs demonstrated (i) the existence of monomers to tetramers, hexamers and higher oligomers of the VDAC and (ii) its spatial arrangement within the oligomers in the native membrane. We discuss the importance of the observed oligomerization for modulation of the VDAC function, for the binding of hexokinase and creatine kinase to the OMM and for mitochondria-mediated apoptosis.     

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.     

Molecular and cell biology of a family of voltage-dependent anion channel porins in Lotus japonicus

Voltage-dependent anion channels (VDACs) are generally considered as the main pathway for metabolite transport across the mitochondrial outer membrane. Recent proteomic studies on isolated symbiosome membranes from legume nodules indicated that VDACs might also be involved in transport of nutrients between plants and rhizobia. In an attempt to substantiate this, we carried out a detailed molecular and cellular characterization of VDACs in Lotus japonicus and soybean (Glycine max). Database searches revealed at least five genes encoding putative VDACs in each of the legumes L. japonicus, Medicago truncatula, and soybean. We obtained and sequenced cDNA clones from L. japonicus encoding five full-length VDAC proteins (LjVDAC1.1-1.3, LjVDAC2.1, and LjVDAC3.1). Complementation of a yeast (Saccharomyces cerevisiae) mutant impaired in VDAC1, a porin of the mitochondrial outer membrane, showed that LjVDAC1.1, LjVDAC1.2, LjVDAC2.1, and LjVDAC3.1, but not LjVDAC1.3, are functional and targeted to the mitochondrial outer membrane in yeast. Studies of the expression pattern of the five L. japonicus VDAC genes revealed largely constitutive expression of each throughout the plant, including nodules. Antibodies to LjVDAC1.1 of L. japonicus and the related POM36 protein of potato (Solanum tuberosum) recognized several proteins between 30 and 36 kD on western blots, including LjVDAC1.1, LjVDAC1.2, LjVDAC1.3, and LjVDAC2.1. Immunolocalization of VDACs in L. japonicus and soybean root nodules demonstrated their presence on not only mitochondria but also on numerous, small vesicles at the cell periphery. No evidence was found for the presence of VDACs on the symbiosome membrane. Nonetheless, the data indicate that VDACs may play more diverse roles in plants than suspected previously.     

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.     

Assessing the role of residue E73 and lipid headgroup charge in VDAC1 voltage gating

The voltage-dependent anion channel (VDAC) is the most abundant protein of the mitochondrial outer membrane (MOM) where it regulates transport of ions and metabolites in and out of the organelle. VDAC function is extensively studied in a lipid bilayer system that allows conductance monitoring of reconstituted channels under applied voltage. The process of switching from a high-conductance state, open to metabolites, to a variety of low-conducting states, which excludes metabolite transport, is termed voltage gating and the mechanism remains poorly understood. Recent studies have implicated the involvement of the membrane-solvated residue E73 in the gating process through β-barrel destabilization. However, there has been no direct experimental evidence of E73 involvement in VDAC1 voltage gating. Here, using electrophysiology measurements, we exclude the involvement of E73 in murine VDAC1 (mVDAC1) voltage gating process. With an established protocol of assessing voltage gating of VDACs reconstituted into planar lipid membranes, we definitively show that mVDAC1 gating properties do not change when E73 is replaced by either a glutamine or an alanine. We further demonstrate that cholesterol has no effect on mVDAC1 gating characteristics, though it was shown that E73 is coordinating residue in the cholesterol binding site. In contrast, we found a pronounced gating effect based on the charge of the phospholipid headgroup, where the positive charge stimulates and negative charge suppresses gating. These findings call for critical evaluation of the existing models of VDAC gating and contribute to our understanding of VDAC's role in control of MOM permeability and regulation of mitochondrial respiration and metabolism.     

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.     

Voltage-dependent anion channels: their roles in plant defense and cell death

The voltage-dependent anion channels (VDACs), mitochondrial outer membrane components, are present in organisms from fungi to animals and plants. They are thought to function in the regulation of metabolite transport between mitochondria and the cytoplasm. Sufficient knowledge on plant VDACs has been accumulated, so that we can here summarize the current information. Then, the involvement of mitochondria in plant defense and cell death is overviewed. While, in mammals, it is suggested that VDAC, also known as a component of the permeability transition pore (PTP) complex formed in the junction site of mitochondrial outer and inner membrane, is a key player in mitochondria-mediated cell death, little is known about the role of plant VDACs in this process. We have shown that plant VDACs are involved in mitochondria-mediated cell death and in defense against a non-host pathogen. In light of the current findings, we discuss the role of the PTP complex and VDAC as its component in plant pathogen defense and cell death.     

Supramolecular assembly of VDAC in native mitochondrial outer membranes

The voltage-dependent anion channel (VDAC) is the most abundant protein in the mitochondrial outer membrane (MOM). Due to its localization, VDAC is involved in a wide range of processes, such as passage of ATP out of mitochondria, and particularly plays a central role in apoptosis. Importantly, the assembly of VDAC provides interaction with a wide range of proteins, some implying oligomerization. However, many questions remain as to the VDAC structure, its supramolecular assembly, packing density, and oligomerization in the MOM is unknown. Here we report the so far highest resolution view of VDAC and its native supramolecular assembly. We have studied yeast MOM by high-resolution atomic force microscopy (AFM) in physiological buffer and found VDAC in two distinct types of membrane domains. We found regions where VDAC was packed at high density (approximately 80%), rendering the membrane a voltage-dependent molecular sieve. In other domains, VDAC has a low surface density (approximately 20%) and the pore assembly ranges from single molecules to groups of up to 20. We assume that these groups are mobile in the lipid bilayer and allow association and dissociation with the large assemblies. VDAC has no preferred oligomeric state and no long-range order was observed in densely packed domains. High-resolution topographs show an eye-shaped VDAC with 3.8 nm x 2.7 nm pore dimensions. Based on the observed VDAC structure and the pair correlation function (PCF) analysis of the domain architectures, we propose a simple model that could explain the phase behavior of VDAC, and illustrates the sensitivity of the molecular organization to conditions in the cell, and the possibility for modulation of its assembly. The implication of VDAC in cytochrome c release from the mitochondria during cell apoptosis has made it a target in cancer research.     

Plant VDAC: Facts and speculations

The voltage-dependent anion-selective channel (VDAC) is the most abundant protein in the mitochondrial outer membrane and the major transport pathway for a large variety of compounds ranging from ions to large polymeric molecules such as DNA and tRNA. Plant VDACs feature a secondary structure content and electrophysiological properties akin to those of VDACs from other organisms. They however undergo a specific regulation. The general importance of VDAC in plant physiology has only recently emerged. Besides their role in metabolite transport, plant VDACs are also involved in the programmed cell death triggered in response to biotic and abiotic stresses. Moreover, their colocalization in non-mitochondrial membranes suggests a diversity of function. This review summarizes our current understanding of the structure and function of plant VDACs. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Olesoxime,a cholesterol-like neuroprotectant restrains synaptic vesicle exocytosis in the mice motor nerve terminals: Possible role of VDACs

Olesoxime is a cholesterol-like neuroprotective compound that targets to mitochondrial voltage dependent anion channels (VDACs). VDACs were also found in the plasma membrane and highly expressed in the presynaptic compartment. Here, we studied the effects of olesoxime and VDAC inhibitors on neurotransmission in the mouse neuromuscular junction. Electrophysiological analysis revealed that olesoxime suppressed selectively evoked neurotransmitter release in response to a single stimulus and 20 Hz activity. Also olesoxime decreased the rate of FM1–43 dye loss (an indicator of synaptic vesicle exocytosis) at low frequency stimulation and 20 Hz. Furthermore, an increase in extracellular Cl⁻ enhanced the action of olesoxime on the exocytosis and olesoxime increased intracellular Cl⁻ levels. The effects of olesoxime on the evoked synaptic vesicle exocytosis and [Cl⁻]_i were blocked by membrane-permeable and impermeable VDAC inhibitors. Immunofluorescent labeling pointed on the presence of VDACs on the synaptic membranes. Rotenone-induced mitochondrial dysfunction perturbed the exocytotic release of FM1–43 and cell-permeable VDAC inhibitor (but not olesoxime or impermeable VDAC inhibitor) partially mitigated the rotenone-driven alterations in the FM1–43 unloading and mitochondrial superoxide production. Thus, olesoxime restrains neurotransmission by acting on plasmalemmal VDACs whose activation can limit synaptic vesicle exocytosis probably via increasing anion flux into the nerve terminals.     

Effects of VDAC isoforms on CuZn-superoxide dismutase activity in the intermembrane space of Saccharomyces cerevisiae mitochondria

Copper and zinc containing superoxide dismutase (CuZnSOD) is located primarily in the cytosol but a small amount of the enzyme has also been identified in the intermembrane space of mitochondria (termed here IMS CuZnSOD). Using Saccharomyces cerevisiae mutants depleted of either isoform of VDAC (voltage-dependent anion-selective channel), we have shown that the activity of IMS CuZnSOD coincides with the presence of a given VDAC isoform and changes in a growth phase dependent way. Moreover, the IMS CuZnSOD activity correlates with the levels of O2*- release from mitochondria and the cytosol redox state. The latter in turn seems to influence the levels of the mitochondrial outer membrane channel protein other than VDAC. Thus, we conclude that in the case of S. cerevisiae both VDAC isoforms influence the IMS CuZnSOD activity and subsequently the expression levels of some mitochondrial proteins.     

Voltage-Dependent Anion Channel Proteins in Synaptosomes of the Torpedo Electric Organ: Immunolocalization, Purification, and Characterization

In this study, we purified and characterized the voltage-dependent anion channel (VDAC) from the Torpedo electric organ. Using immunogold labeling, VDAC was colocalized with the voltage-gated Ca²⁺ channel in the synaptic plasma membrane. By immunoblot analysis, five protein bands in synaptosomes isolated from the Torpedo electric organ cross reacted with two monoclonal anti-VDAC antibody. No more than about 7 to 10% mitochondrial contains could be detected in any synaptosomal membrane preparation tested. This was estimated by comparing the specific activity in mitochondria and synaptosomes of succinate–cytochrome-c oxidoreductase and antimycin-insensitive NADH–cytochrome-c oxidoreductase activities; mitochondrial inner and outer membrane marker enzymes, respectively. [¹⁴C]DCCD (dicyclohexylcarbodiimide), which specifically label mitochondrial VDAC, labeled four 30–35 kDa protein bands that were found to interact with the anti-VDAC antibody. The distribution of the Torpedo VDAC protein bands was different among membranes isolated from various tissues. VDAC was purified from synaptosomes and a separation between two of the proteins was obtained. The two purified proteins were characterized by their single channel activity and partial amino acid sequences. Upon reconstitution into a planar lipid bilayer, the purified VDACs showed voltage-dependent channel activity with properties similar to those of purified mitochondrial VDAC. Amino acid sequence of four peptides, derived from VDAC band II, exhibited high homology to sequences present in human VDAC1 (98%), VDAC2 (91.8%), and VDAC3 (90%), while another peptide, derived from VDAC band III, showed lower homology to either VDAC1 (88.4%) or VDAC2 (79%). Two more peptides show high homology to the sequence present in mouse brain VDAC3 (100 and 78%). In addition, we demonstrate the translocation of ATP into synaptosomes, which is inhibited by DCCD and by the anion transport inhibitor DIDS. The possible function of VDAC in the synaptic plasma membrane is discussed.     

VDAC1 serves as a mitochondrial binding site for hexokinase in oxidative muscles

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are the main pathway for metabolites across the mitochondrial outer membrane and may serve as binding sites for kinases, including hexokinase. We determined that mitochondria-bound hexokinase activity is significantly reduced in oxidative muscles (heart and soleus) in vdac1^−/− mice. The activity data were supported by western blot analysis using HK2 specific antibody. To gain more insight into the physiologic mean of the results with the activity data, VDAC deficient mice were subjected to glucose tolerance testing and exercise-induced stress, each of which involves tissue glucose uptake via different mechanisms. vdac1^−/− mice exhibit impaired glucose tolerance whereas vdac3^−/− mice have normal glucose tolerance and exercise capacity. Mice lacking both VDAC1 and VDAC3 (vdac1^−/−/vdac3^−/−) have reduced exercise capacity together with impaired glucose tolerance. Therefore, we demonstrated a link between VDAC1 mediated mitochondria-bound hexokinase activity and the capacity for glucose clearance.     

The voltage-dependent anion channel as a biological transistor: theoretical considerations

The voltage-dependent anion channel (VDAC) is a porin of the mitochondrial outer membrane with a bell-shaped permeability-voltage characteristic. This porin restricts the flow of negatively charged metabolites at certain non-zero voltages, and thus might regulate their flux across the mitochondrial outer membrane. Here, we have developed a mathematical model illustrating the possibility of interaction between two steady-state fluxes of negatively charged metabolites circulating across the VDAC in a membrane. The fluxes interact by contributing to generation of the membrane electrical potential with subsequent closure of the VDAC. The model predicts that the VDAC might function as a single-molecule biological transistor and amplifier, because according to the obtained calculations a small change in the flux of one pair of different negatively charged metabolites causes a significant modulation of a more powerful flux of another pair of negatively charged metabolites circulating across the same membrane with the VDAC. Such transistor-like behavior of the VDAC in the mitochondrial outer membrane might be an important principle of the cell energy metabolism regulation under some physiological conditions.     

VDAC electronics: 2. A new,anaerobic mechanism of generation of the membrane potentials in mitochondria

Mitochondrial hexokinase (HK) and creatine kinase (CK) known to form complexes with a voltage dependent anion channel (VDAC) have been reported to increase cell death resistance under hypoxia/anoxia. In this work we propose a new, non-Mitchell mechanism of generation of the inner and outer membrane potentials at anaerobic conditions. The driving force is provided by the Gibbs free energy of the HK and CK reactions associated with the VDAC–HK and the ANT (adenine nucleotide translocator)–CK–VDAC complexes, respectively, both functioning as voltage generators. In the absence of oxygen, the cytosolic creatine phosphate can be directly used by the ANT–CK–VDAC contact sites to produce ATP from ADP in the mitochondrial matrix. After that, ATP released through the fraction of unbound ANTs in exchange for ADP is used in the mitochondrial intermembrane space by the outer membrane VDAC–HK electrogenic complexes to convert cytosolic glucose into glucose-6-phosphate. A simple computational model based on the application of Ohm's law to an equivalent electrical circuit showed a possibility of generation of the inner membrane potential up to − 160 mV, under certain conditions, and of relatively high outer membrane potential without wasting of ATP that normally leads to cell death. The calculated membrane potentials depended on the restriction of ATP/ADP diffusion in narrow cristae and through the cristae junctions. We suggest that high inner membrane potential and calcium extrusion from the mitochondrial intermembrane space by generated positive outer membrane potential prevent mitochondrial permeability transition, thus allowing the maintenance of mitochondrial integrity and cell survival in the absence of oxygen.