Le VDAC (Voltage-Dependent Anion-selective Channel) est un canal ionique de la membrane externe de la mitochondrie. Il est caractérisé par une haute conductance (4nS dans du KCl 1M) à des faibles différences de potentiel et des fermetures vers des niveaux de conductance inférieurs suite à l’application de voltages élevés. La selectivité de l’état de haute conductance est anionique alors que celle du principal état de faible conductance est fortement cationique. La structure secondaire et la fonction est bien conservée chez les animaux, les plantes et les champignons alors que les séquences sont très différentes (moins de 30% d’identités). Au sein des différents groupes, quelques isoformes coexistent. La première partie du travail consiste à étudier l’évolution des isoformes de VDAC, à déterminer le nombre et les propriétés des isoformes. Nous montrons que les végétaux possèdent plus d’isoformes que les mammifères et que celles-ci sont également moins bien conservées. Nous avons défini trois classes d’isoformes sur base de leur charge nette : faiblement, moyennement ou fortement chargées. Les VDAC fortement exprimés et très bien conservés chez les champignons, les plantes et les animaux sont moyennement chargés. A l’opposé, les isoformes portant les plus hautes charges nettes sont très divergentes et peu exprimées. Ces particularités permettent pour la première fois de comparer les isoformes de groupes évolutivement distants. Ensuite, pour relier la structure et la topologie du VDAC à sa fonction, nous avons construit un modèle topologique. Il consiste en feuillet bêta de 18 brins antiparallèles reployés de manière à former un tonneau transmembranaire présentant une courte hélice alpha à son extrémité aminoterminale. Le modèle est compatible avec les séquences de VDAC fongiques, végétaux et animaux. Il rend compte des résultats expérimentaux obtenus par spectroscopie infrarouge et par microscopie électronique de cristaux 2D. Enfin, les résultats d’études topologiques et fonctionnelles publiées dans la littérature supportent également notre modèle. Nos travaux concernant le VDAC32 de Phaseolus coccineus a permis d’améliorer le protocole de purification et d’en obtenir la séquence. La structure et la stabilité du VDAC32 a été étudiée par spectroscopie infrarouge. L’étude de l’orientation du VDAC à l’aide de lumière infrarouge polarisée se base sur des modèles définissant deux angles, alpha et bêta correspondant à l’inclinaison de l’axe du tonneau par rapport à la normale à la membrane et l’inclinaison des brins par rapport à l’axe du tonneau. Nous proposons que l’angle alpha dépend également de l’asymétrie de la protéine. Nos mesures en spectroscopie infrarouge indiquent que la présence du VDAC diminue la température de transition de la membrane et que la structure protéique est sensible à la transition de phase des lipides membranaires. Enfin, nous montrons que la structure du VDAC est modifiée quand le rapport lipides/protéines diminue. L’orientation des brins bêta se rapproche de l’axe et les chaînes latérales des tyrosines deviennent moins ordonnées. Les VDAC sont insérés dans la membrane mitochondriale qui contient environ 5% de stérols. Certains auteurs ont détecté le VDAC dans les membranes plasmiques. Ces dernières sont beaucoup plus riches en stérols. Nous montrons dans ce travail que le VDAC possède la même fonction dans des membranes contenant 0% ou 5% de stérols alors que sa structure est légèrement modifiée. Par contre, en présence de hautes concentrations en stérols, la fonction du VDAC est sensiblement altérée. Ces résultats suggèrent que le VDAC a des propriétés différentes dans la membrane plasmique et dans l’enveloppe de la mitochondrie.

Mitochondria are essential organelles in the cell. They are enveloped by a two membrane system, an inner and outer membrane. Residing in the outer membrane is the voltage-dependant anion-selective channel (VDAC) which is in very high abundance in the outer membrane. Previously, when VDAC was deleted in N. crassa the mitochondria developed unusual morphology and the fungus became temperature sensitive. This was suspected to be caused by alterations in the membrane architecture. It was found that deletion of VDAC causes a reduction in mitochondrial outer membrane fluidity leading to a fragile outer membrane. In the current study it was also found that loss of VDAC significantly impacts the mitochondrial proteome. These results taken together suggest that VDAC is not only a channel for traversing the outer membrane but appears to be important for the structural integrity of the mitochondria. / October 2014

Os canais seletivos a ânions dependente de voltagem (VDACs) são um grupo de proteínas, primeiramente identificadas na membrana mitocondrial externa, capazes de formar estruturas de poros hidrofílicos em membranas. As VDACs são conhecidas pela sua função essencial no metabolismo celular e nos estágios recentes de apoptose. Em mamíferos, foram identificadas três isoformas de VDACs (VDAC1, 2 e 3). Uma pesquisa proteômica, consistindo de eletroforese bi-dimensional seguida por western blotting com anticorpos anti-VDAC 1, anti-VDAC 2 e anti-VDAC 3 e espectrometria de massas com fonte de ionização/desorção à laser assistido por matriz e tempo de vôo foi utilizada para estudar a expressão das isoformas de VDAC em mitocôndrias cerebrais de aves, ratos e bois. Foi estudada a possibilidade que diferenças na expressão relativa das isoformas de VDAC possam ser um fator determinante da proporção espécie-dependente dos sítios de ligação da hexoquinase tipo A: tipo B nas mitocôndrias cerebrais. Os spots foram caracterizados, e a intensidade de sinal foi comparada entre os spots. VDAC1 e VDAC2 foram divididas dentro de múltiplos spots. A VDAC1 foi dividida em dois spots nos géis bi-dimensionais realizados com amostras de cérebros de ratos e bois, e três spots para cérebros de aves. A VDAC2 foi separada em três, cinco e dois spots para cérebros de ratos, bois e aves, respectivamente. Os resultados reportam uma heterogeneidade de carga das VDACs 1 e 2 nos cérebros analisados. A VDAC1 foi a mais expressa das três isoformas. Além disso, a expressão da VDAC1 mais VDAC2 foi muito maior em cérebros de aves e bois do que em cérebros de ratos. Mitocôndrias de cérebro de aves mostraram uma maior expressão de VDAC1 e menor de VDAC2. As mitocôndrias de cérebro bovino apresentaram os níveis mais altos de VDAC2. A VDAC3 não foi detectada nos cérebros das espécies estudadas. / The voltage dependent anion selective channels (VDACs) are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures in membranes. VDAC are known to play an essential role in cellular metabolism and in the early stages of apoptosis. In mammals, three VDACs isoforms (VDAC1, 2, 3) have been identified. A proteomic approach, consisting of two dimensional electrophoresis, followed by western blotting with anti-VDAC 1, anti-VDAC 2 and anti-VDAC 3 and by matrix assisted laser desorption/ionization time of flight mass spectrometry was used to study the expression of VDAC isoforms in rat, bovine and avian brain mitochondria. We were studying the possibility that differences in the relative expression of VDAC isoforms may be a factor in determining the species-dependent ratio of type A: type B hexokinase binding sites on brain mitochondria. The spots were characterized, and the signal intensities among spots were compared. VDAC1 and VDAC2 were divided into multiple spots. VDAC1 was divided in two spots in two dimensional gels of rat and bovine brains and three spots in avian brains. VDAC2 was separated into three, five and two spots in rat, bovine and avian brains, respectively. The results report charge heterogeneity of VDACs 1 and 2 in the analyzed brains. VDAC1 was the most abundantly expressed of the three isoforms. Moreover the expression of VDAC1 plus VDAC2 was much higher in avian and bovine brains than in rat brains. Avian brain mitochondria showed the highest expression of VDAC1 and the lowest of VDAC2. Bovine brain mitochondria had the highest levels of VDAC2. No VDAC 3 was detected in studied species brains.

Os canais seletivos a ânions dependente de voltagem (VDACs) são um grupo de proteínas, primeiramente identificadas na membrana mitocondrial externa, capazes de formar estruturas de poros hidrofílicos em membranas. As VDACs são conhecidas pela sua função essencial no metabolismo celular e nos estágios recentes de apoptose. Em mamíferos, foram identificadas três isoformas de VDACs (VDAC1, 2 e 3). Uma pesquisa proteômica, consistindo de eletroforese bi-dimensional seguida por western blotting com anticorpos anti-VDAC 1, anti-VDAC 2 e anti-VDAC 3 e espectrometria de massas com fonte de ionização/desorção à laser assistido por matriz e tempo de vôo foi utilizada para estudar a expressão das isoformas de VDAC em mitocôndrias cerebrais de aves, ratos e bois. Foi estudada a possibilidade que diferenças na expressão relativa das isoformas de VDAC possam ser um fator determinante da proporção espécie-dependente dos sítios de ligação da hexoquinase tipo A: tipo B nas mitocôndrias cerebrais. Os spots foram caracterizados, e a intensidade de sinal foi comparada entre os spots. VDAC1 e VDAC2 foram divididas dentro de múltiplos spots. A VDAC1 foi dividida em dois spots nos géis bi-dimensionais realizados com amostras de cérebros de ratos e bois, e três spots para cérebros de aves. A VDAC2 foi separada em três, cinco e dois spots para cérebros de ratos, bois e aves, respectivamente. Os resultados reportam uma heterogeneidade de carga das VDACs 1 e 2 nos cérebros analisados. A VDAC1 foi a mais expressa das três isoformas. Além disso, a expressão da VDAC1 mais VDAC2 foi muito maior em cérebros de aves e bois do que em cérebros de ratos. Mitocôndrias de cérebro de aves mostraram uma maior expressão de VDAC1 e menor de VDAC2. As mitocôndrias de cérebro bovino apresentaram os níveis mais altos de VDAC2. A VDAC3 não foi detectada nos cérebros das espécies estudadas. / The voltage dependent anion selective channels (VDACs) are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures in membranes. VDAC are known to play an essential role in cellular metabolism and in the early stages of apoptosis. In mammals, three VDACs isoforms (VDAC1, 2, 3) have been identified. A proteomic approach, consisting of two dimensional electrophoresis, followed by western blotting with anti-VDAC 1, anti-VDAC 2 and anti-VDAC 3 and by matrix assisted laser desorption/ionization time of flight mass spectrometry was used to study the expression of VDAC isoforms in rat, bovine and avian brain mitochondria. We were studying the possibility that differences in the relative expression of VDAC isoforms may be a factor in determining the species-dependent ratio of type A: type B hexokinase binding sites on brain mitochondria. The spots were characterized, and the signal intensities among spots were compared. VDAC1 and VDAC2 were divided into multiple spots. VDAC1 was divided in two spots in two dimensional gels of rat and bovine brains and three spots in avian brains. VDAC2 was separated into three, five and two spots in rat, bovine and avian brains, respectively. The results report charge heterogeneity of VDACs 1 and 2 in the analyzed brains. VDAC1 was the most abundantly expressed of the three isoforms. Moreover the expression of VDAC1 plus VDAC2 was much higher in avian and bovine brains than in rat brains. Avian brain mitochondria showed the highest expression of VDAC1 and the lowest of VDAC2. Bovine brain mitochondria had the highest levels of VDAC2. No VDAC 3 was detected in studied species brains.

The mitochondria are the primary energy providers for most eukaryotic cells. The substrate and products of the mitochondria need to be translocated across the semi-permeable mitochondrial outer membrane (MOM). Mitochondrial porin is an aqueous channel in the MOM thought to provide the primary pathway for metabolite translocation. Porin is a nuclear encoded protein and therefore needs to be transported to the mitochondria, translocated across and assembled within the MOM. Of all the recognition signals required for successful transport, import and assembly, only the β-sorting signal used in assembly is known. In addition, this protein possesses the ability to gate, and in doing so can preferentially allow the passage of anions in the open state and cations in the closed state. However, the precise mechanism by which gating of porin occurs and a complete understanding of porin’s function in vivo remains elusive. The essentiality of porin was examined by constructing a strain of Neurospora crassa deficient for porin. This strain, denoted as WS004, exists as evidence that porin is non-essential for the survival of Neurospora crassa. However, the loss of porin results in a reduction in growth rate due to the dysfunction of the cytochrome mediated respiratory pathway, which was made evident by the reduction of cytochrome b and almost complete lack of cytochrome aa3. WS004 survives by inducing the expression of alternative oxidase, which funnels the electrons from the Q pool directly to oxygen, bypassing the cytochrome b and aa3 containing complexes III and IV respectively. Additional phenotypic differences observed included loss in ability to produce aerial hyphae, reduced amount of conidia produced and strains that were female sterile. It was determined, that additional genetic factors influenced the resulting phenotype due to the loss of porin. LC-MS/MS, in combination with iTRAQ labelling, was utilized to examine changes in the proteome profiles of porin containing and porin lacking mitochondria and showed several different proteins as significantly up- or down-regulated which lend to an explanation to some of the phenotypes observed. Taken together, these results demonstrate the central role of porin in regulating both mitochondrial and cellular processes.

The mitochondria are the primary energy providers for most eukaryotic cells. The substrate and products of the mitochondria need to be translocated across the semi-permeable mitochondrial outer membrane (MOM). Mitochondrial porin is an aqueous channel in the MOM thought to provide the primary pathway for metabolite translocation. Porin is a nuclear encoded protein and therefore needs to be transported to the mitochondria, translocated across and assembled within the MOM. Of all the recognition signals required for successful transport, import and assembly, only the β-sorting signal used in assembly is known. In addition, this protein possesses the ability to gate, and in doing so can preferentially allow the passage of anions in the open state and cations in the closed state. However, the precise mechanism by which gating of porin occurs and a complete understanding of porin’s function in vivo remains elusive. The essentiality of porin was examined by constructing a strain of Neurospora crassa deficient for porin. This strain, denoted as WS004, exists as evidence that porin is non-essential for the survival of Neurospora crassa. However, the loss of porin results in a reduction in growth rate due to the dysfunction of the cytochrome mediated respiratory pathway, which was made evident by the reduction of cytochrome b and almost complete lack of cytochrome aa3. WS004 survives by inducing the expression of alternative oxidase, which funnels the electrons from the Q pool directly to oxygen, bypassing the cytochrome b and aa3 containing complexes III and IV respectively. Additional phenotypic differences observed included loss in ability to produce aerial hyphae, reduced amount of conidia produced and strains that were female sterile. It was determined, that additional genetic factors influenced the resulting phenotype due to the loss of porin. LC-MS/MS, in combination with iTRAQ labelling, was utilized to examine changes in the proteome profiles of porin containing and porin lacking mitochondria and showed several different proteins as significantly up- or down-regulated which lend to an explanation to some of the phenotypes observed. Taken together, these results demonstrate the central role of porin in regulating both mitochondrial and cellular processes.

La stéatose hépatique non-alcoolique consiste en une accumulation de lipides dans le cytoplasme des hépatocytes. Longtemps considérée comme une pathologie bénigne, elle peut être à l’origine du développement d’un stade plus sévère : la stéatohépatite non alcoolique (NASH). La NASH s’accompagne de lésions sévères du foie liées à la genèse d’un stress oxydant, d’une inflammation et de la mort cellulaire. Le rôle de la mitochondrie est au centre de cette maladie, bien que les connaissances sur la dysfonction mitochondriale et ses conséquences sur l’apoptose soient encore insuffisantes. En effet, la mitochondrie est responsable de la dégradation des lipides par -oxydation et elle agit comme un centre intégrateur des signaux apoptotiques en déclenchant une perméabilisation des membranes mitochondriales (PMM) aboutissant à la libération de facteurs apoptogènes. Ce processus est considéré comme le point de non-retour de la voie mitochondriale de l’apoptose. Nos travaux ont porté sur la compréhension des mécanismes moléculaires liant l’apoptose hépatocytaire mitochondriale et la stéatose. La combinaison de quatre modèles expérimentaux de stéatose (biopsies de patients, mitochondries isolées de souris obèses ob/ob ou recevant un régime hypercalorique, et lignées cellulaires) a permis de montrer, dans le foie stéatosique, une sensibilité accrue à l’induction de la PMM et une augmentation de la perméabilité de VDAC (voltage-dependent anion channel), protéine formant un canal dans la membrane externe mitochondriale. Ces observations sont associées à une diminution de la phosphorylation de VDAC sur un résidu thréonine et sa perte d’interaction avec la protéine anti-apoptotique Bcl-XL et la kinase GSK3, révélant ainsi une nouvelle voie de signalisation par les lipides. Cette découverte s’est notamment appuyée sur l’utilisation de tests fonctionnels en mitochondries isolées que nous avions développés et validés dans plusieurs études aux stratégies expérimentales variées. En conclusion, notre étude permet de mieux comprendre la fragilité mitochondriale lipo-induite, stade précédant l’apoptose hépatocytaire, et ouvre des perspectives à visée biomédicale. / Non-alcoholic steatosis is a liver disease characterized by lipid accumulation in the cytoplasm of hepatocytes. For a long time, it has been considered as a benign condition. Now it is known that it can precede the development of a severe stage, non-alcoholic steatohepatitis (NASH). NASH is accompanied by severe dammages of the liver linked to the genesis of oxidative stress, inflammation and cell death. Mitochondrion is a central player of this disease; however, the knowledge of mitochondrial dysfunction and its consequences on apoptosis is still insufficient. Indeed, mitochondria are responsible for lipid degradation by -oxidation. Mitochondria act as a central integrator of apoptotic signals by triggering the mitochondrial membrane permeabilization (MMP) leading to the release of apoptogenic factors. This process is considered as the point of no return of the mitochondrial pathway of apoptosis. We aimed to better understand the molecular mechanisms linking mitochondrial liver apoptosis and steatosis. Combination of four experimental models of steatosis (human biopsies, isolated mitochondria from ob/ob obese mice, high fat diet-fed mice or hepatic cell lines) displayed, in steatotic livers, increased sensitivity to MMP induction and permeability of VDAC (Voltage dependent anion channel), a protein which forms a channel in the outer mitochondrial membrane. These findings are associated with the hypo-phosphorylation of VDAC on a threonine residue and the loss of its interaction with the anti-apoptotic Bcl-XL and GSK3 kinase, thus revealing a new lipid-induced signaling pathway. Our work is based on the use of functional assays on isolated mitochondria that we have developed and validated in several studies involving various strategies. To conclude, our study increases the knowledge on the lipid-induced mitochondrial weakness preceding hepatic apoptosis and opens perspectives in biomedical applications

A VDAC é a proteína mais abundante da membrana mitocondrial externa. Possui diversas funções, tais como o controle da troca de metabólitos, através da membrana, e a participação no maquinário apoptótico. Estudamos o interactoma da VDAC com as proteínas mitocondriais neuronais do cérebro bovino e murino, a fim de compreender se a expressão diferenciada da VDAC1 e VDAC2 verificada entre essas células estão associadas às diferenças nas interações da VDAC. Os complexos proteicos foram analisados por 2D Blue Native SDS-PAGE e identificados via MALDI-TOF TOF usando o software Mascot e o banco de dados NCBInr. Foram identificados 27 e 46 spots em murino e bovino, respectivamente. Nós identificamos proteínas solúveis e incorporadas na membrana que não são participantes da fosforilação oxidativa, dentre elas a aldeido deidrogenase e muitas outras constituintes de complexos mitocondriais já conhecidos tão bem como novos, tais como a putative stomatin-like protein 2 complex e a switch-associated protein 70. Nossos resultados mostraram que os neurônios bovinos possuem mais complexos (5) contendo a VDAC do que em ratos (1), os quais indicam uma cinética diferencial de acoplamento e desacoplamento. Interessantemente, a lista contendo as proteínas identificadas inclui algumas proteínas conhecidas ou supostamente localizadas em compartimentos não-mitocondriais, por exemplo, a myc-induced nuclear antigen. O interactoma diferencial da VDAC entre as espécies bovina e murina, evidencia a presença de uma base comum, porém com diferentes ambientes estruturais, as quais podem ser a base da diferença entre os sítios de ligação A e B observados nas diferentes espécies. / The voltage dependent anion channel (VDAC) is the most abundant protein of outer mitochondrial membrane. VDAC controls metabolite exchange through this membrane and the apoptosis machinery. We studied the interactome of VDAC with mitochondrial proteins of neuronal cells from rat and bovine brain. We wished to understand if the differential expression of VDAC1 and VDAC2 verified between these cells was linked to differences in the VDAC interactions. Protein complexes were analyzed by 2D Blue Native SDS-PAGE and were identified by MALDI-TOF TOF using Mascot software against the NCBInr database. Number of 27 e 46 spots were identified from rat and bovine brain, respectively. We identified soluble and membrane-embedded non-OXPHOS proteins, among them aldehyde dehydrogenase, and many as constituents of known mitochondrial complexes as well as novel ones such as putative stomatin-like protein 2 complex and switchassociated protein 70. Our results showed that bovine neurons had more protein complexes (5) containing VDAC than rat cells (1), which indicates a differential kinetics of assembly or disassembly. Interestingly, the identification list included some proteins known or presumed to be localized to nonmitochondrial compartments, for example, myc-induced nuclear antigen. Our results support evidences of differential apoptotic and energetic mechanisms verified in these brains. The differential VDAC interactome between bovine and murine, support evidences of a common base, but whith different structural environment, which may be the basis of the difference between the binding sites A and B observed in these brains.

A VDAC é a proteína mais abundante da membrana mitocondrial externa. Possui diversas funções, tais como o controle da troca de metabólitos, através da membrana, e a participação no maquinário apoptótico. Estudamos o interactoma da VDAC com as proteínas mitocondriais neuronais do cérebro bovino e murino, a fim de compreender se a expressão diferenciada da VDAC1 e VDAC2 verificada entre essas células estão associadas às diferenças nas interações da VDAC. Os complexos proteicos foram analisados por 2D Blue Native SDS-PAGE e identificados via MALDI-TOF TOF usando o software Mascot e o banco de dados NCBInr. Foram identificados 27 e 46 spots em murino e bovino, respectivamente. Nós identificamos proteínas solúveis e incorporadas na membrana que não são participantes da fosforilação oxidativa, dentre elas a aldeido deidrogenase e muitas outras constituintes de complexos mitocondriais já conhecidos tão bem como novos, tais como a putative stomatin-like protein 2 complex e a switch-associated protein 70. Nossos resultados mostraram que os neurônios bovinos possuem mais complexos (5) contendo a VDAC do que em ratos (1), os quais indicam uma cinética diferencial de acoplamento e desacoplamento. Interessantemente, a lista contendo as proteínas identificadas inclui algumas proteínas conhecidas ou supostamente localizadas em compartimentos não-mitocondriais, por exemplo, a myc-induced nuclear antigen. O interactoma diferencial da VDAC entre as espécies bovina e murina, evidencia a presença de uma base comum, porém com diferentes ambientes estruturais, as quais podem ser a base da diferença entre os sítios de ligação A e B observados nas diferentes espécies. / The voltage dependent anion channel (VDAC) is the most abundant protein of outer mitochondrial membrane. VDAC controls metabolite exchange through this membrane and the apoptosis machinery. We studied the interactome of VDAC with mitochondrial proteins of neuronal cells from rat and bovine brain. We wished to understand if the differential expression of VDAC1 and VDAC2 verified between these cells was linked to differences in the VDAC interactions. Protein complexes were analyzed by 2D Blue Native SDS-PAGE and were identified by MALDI-TOF TOF using Mascot software against the NCBInr database. Number of 27 e 46 spots were identified from rat and bovine brain, respectively. We identified soluble and membrane-embedded non-OXPHOS proteins, among them aldehyde dehydrogenase, and many as constituents of known mitochondrial complexes as well as novel ones such as putative stomatin-like protein 2 complex and switchassociated protein 70. Our results showed that bovine neurons had more protein complexes (5) containing VDAC than rat cells (1), which indicates a differential kinetics of assembly or disassembly. Interestingly, the identification list included some proteins known or presumed to be localized to nonmitochondrial compartments, for example, myc-induced nuclear antigen. Our results support evidences of differential apoptotic and energetic mechanisms verified in these brains. The differential VDAC interactome between bovine and murine, support evidences of a common base, but whith different structural environment, which may be the basis of the difference between the binding sites A and B observed in these brains.

Evidence has accumulated that the voltage dependent anion channel (VDAC), located on the outer membrane of mitochondria, plays a central role in apoptosis. The involvement of VDAC oligomerization in apoptosis has been suggested in various studies. However, it still remains unknown how exactly VDAC supra-molecular assembly can be regulated in the membrane. Previous studies suggested the possible influence of various proteins on the formation of VDAC oligomers, but the important issue of the VDAC oligomeric state regulation by lipids has not been studied so far. Nevertheless, the effect of lipids on the oligomerization of several membrane proteins has been mentioned in the literature and in general, protein-lipid interactions are under extensive investigation. In the present work, I addressed the influence of lipids on VDAC oligomerization experimentally by reconstituting the fluorescently labelled VDAC in giant unilamellar vesicles (GUVs)—a chemically well defined, cell-free minimal model system. Fluorescence cross-correlation spectroscopy was performed to determine the oligomeric state of VDAC. I investigated the effect of important for apoptosis anionic lipids, phosphatidylglycerol and cardiolipin, on VDAC oligomerization. I demonstrated that phosphatidylglycerol significantly enhances VDAC oligomerization in the membrane, whereas cardiolipin disrupts VDAC oligomers. These results suggest that up- or down- regulation of these lipids in mitochondria during apoptosis can tune VDAC oligomerization in the membrane. Thus, this study sheds light on the role played by the above-mentioned lipids in the regulation of VDAC oligomerization during apoptosis and provides additional information on the molecular mechanisms of the programmed cell death. Another objective of this work was to investigate the partitioning of VDAC into liquid disordered or liquid ordered lipid phases. The existence of lipid domains or the lipid rafts in mitochondria and VDAC enrichment in these rafts is still under debate. Additionally, mitochondrial VDAC was recently found in the plasma membrane. The role of this VDAC is not known, however, it was shown to be located in caveolae (specialized lipid rafts) and play an important role in neuronal apotosis and Alzheimer’s disease. Therefore, VDAC partitioning to the lipid rafts is an interesting question for investigation. The possibility to reconstitute VDAC into minimal model systems–GUVs with phase separation, allowed to reveal the preferential partitioning of VDAC into liquid disordered lipid domain, which suggests either non-raft localization of VDAC or the requirement of the other factors for the recruitment of VDAC into lipid rafts.