Search

Mitochondria are most likely descendants of strictly aerobic prokaryotes from the class Alphaproteobacteria. The mitochondrial matrix is surrounded by two membranes according to its relationship with Gram-negative bacteria. Similar to the bacterial outer membrane, the mitochondrial outer membrane acts as a molecular sieve because it also contains diffusion pores. However, it is more actively involved in mitochondrial metabolism because it plays a functional role, whereas the bacterial outer membrane has only passive sieving properties. Mitochondrial porins, also known as eukaryotic porins or voltage-dependent anion-selective channels (VDACs) control the permeability properties of the mitochondrial outer membrane. They contrast with most bacterial porins because they are voltage-dependent. They switch at relatively small transmembrane potentials of 20 to 30 mV in closed states that exhibit different permeability properties than the open state. Whereas the open state is preferentially permeable to anionic metabolites of mitochondrial metabolism, the closed states prefer cationic solutes, in particular, calcium ions. Mitochondrial porins are encoded in the nucleus, synthesized at cytoplasmatic ribosomes, and post-translationally imported through special transport systems into mitochondria. Nineteen beta strands form the beta-barrel cylinders of mitochondrial and related porins. The pores contain in addition an α-helical structure at the N-terminal end of the protein that serves as a gate for the voltage-dependence. Similarly, they bind peripheral proteins that are involved in mitochondrial function and compartment formation. This means that mitochondrial porins are localized in a strategic position to control mitochondrial metabolism. The special features of the role of mitochondrial porins in apoptosis and cancer will also be discussed in this article. [ABSTRACT FROM AUTHOR]

The voltage-dependent anion-selective channel (VDAC) is a porin in the mitochondrial outer membrane (MOM). Unlike bacterial porins, several mitochondrial β-barrels comprise an odd number of β-strands, as is the case for the 19-β-stranded VDAC. Previously, a variant of a VDAC from Neurospora crassa, VDAC-ΔC, lacking the predicted 19th β-strand, was found to form gated, anion-selective channels in artificial membranes. In vivo, the two C-terminal β-strands (β18 and β19) in VDAC form a β-hairpin necessary for import from the cytoplasm into mitochondria and the β-signal required for assembly in the mitochondrial outer membrane resides in β19. The current study demonstrated that the putative 18-stranded β-barrel formed by VDAC-ΔC can be imported and assembled in the MOM in vivo and can also partially rescue the phenotype associated with the deletion of VDAC from a strain of N. crassa. Furthermore, when expressed and purified from Escherichia coli, VDAC-ΔC can be folded into a β-strand-rich form in decyl-maltoside. Size exclusion chromatography (SEC) alone or combined with multi-angle light scattering (SEC-MALS) and analytical ultracentrifugation revealed that, unlike full-length VDACs, VDAC-ΔC can self-organize into dimers and higher order oligomers in the absence of sterol.

The voltage-dependent anion-selective channel (VDAC) is a porin in the mitochondrial outer membrane (MOM). Unlike bacterial porins, several mitochondrial β-barrels comprise an odd number of β-strands, as is the case for the 19-β-stranded VDAC. Previously, a variant of a VDAC from Neurospora crassa , VDAC-ΔC, lacking the predicted 19th β-strand, was found to form gated, anion-selective channels in artificial membranes. In vivo , the two C-terminal β-strands (β18 and β19) in VDAC form a β-hairpin necessary for import from the cytoplasm into mitochondria and the β-signal required for assembly in the mitochondrial outer membrane resides in β19. The current study demonstrated that the putative 18-stranded β-barrel formed by VDAC-ΔC can be imported and assembled in the MOM in vivo and can also partially rescue the phenotype associated with the deletion of VDAC from a strain of N. crassa. Furthermore, when expressed and purified from Escherichia coli , VDAC-ΔC can be folded into a β-strand-rich form in decyl-maltoside. Size exclusion chromatography (SEC) alone or combined with multi-angle light scattering (SEC-MALS) and analytical ultracentrifugation revealed that, unlike full-length VDACs, VDAC-ΔC can self-organize into dimers and higher order oligomers in the absence of sterol. [ABSTRACT FROM AUTHOR]

The highly abundant voltage-dependent anion-selective channel (VDAC) allows transit of metabolites across the mitochondrial outer membrane. Previous studies in Neurospora crassa showed that the LoPo strain, expressing 50% of normal VDAC levels, is indistinguishable from wild-type (WT). In contrast, the absence of VDAC (ΔPor-1), or the expression of an N-terminally truncated variant VDAC (ΔN2-12porin), is associated with deficiencies in cytochromes b and aa3 of complexes III and IV and concomitantly increased alternative oxidase (AOX) activity. These observations led us to investigate complex I and complex II activities in these strains, and to explore their mitochondrial bioenergetics. The current study reveals that the total NADH dehydrogenase activity is similar in mitochondria from WT, LoPo, ΔPor-1 and ΔN2-12porin strains; however, in ΔPor-1 most of this activity is the product of rotenone-insensitive alternative NADH dehydrogenases. Unexpectedly, LoPo mitochondria have increased complex II activity. In all mitochondrial types analyzed, oxygen consumption is higher in the presence of the complex II substrate succinate, than with the NADH-linked (complex I) substrates glutamate and malate. When driven by a combination of complex I and II substrates, membrane potentials (Δψ) and oxygen consumption rates (OCR) under non-phosphorylating conditions are similar in all mitochondria. However, as expected, the induction of state 3 (phosphorylating) conditions in ΔPor-1 mitochondria is associated with smaller but significant increases in OCR and smaller decreases in Δψ than those seen in wild-type mitochondria. High ROS production, particularly in the presence of rotenone, was observed under non-phosphorylating conditions in the ΔPor-1 mitochondria. Thus, the absence of VDAC is associated with increased ROS production, in spite of AOX activity and wild-type OCR in ΔPor-1 mitochondria. [ABSTRACT FROM AUTHOR]

Mitochondrial porin, which forms voltage-dependent anion-selective channels (VDAC) in the outer membrane, can be folded into a 19-β-stranded barrel. The N terminus of the protein is external to the barrel and contains α-helical structure. Targeted modifications of the N-terminal region have been assessed in artificial membranes, leading to different models for gating in vitro. However, the in vivo requirements for gating and the N-terminal segment of porin are less well-understood. Using Neurospora crassa porin as a model, the effects of a partial deletion of the N-terminal segment were investigated. The protein, ΔN2-12porin, is assembled into the outer membrane, albeit at lower levels than the wild-type protein. The resulting strain displays electron transport chain deficiencies, concomitant expression of alternative oxidase, and decreased growth rates. Nonetheless, its mitochondrial genome does not contain any significant mutations. Most of the genes that are expressed in high levels in porin-less N. crassa are expressed at levels similar to that of wild type or are slightly increased in ΔN2-12porin strains. Thus, although the N-terminal segment of VDAC is required for complete function in vivo, low levels of a protein lacking part of the N terminus are able to rescue some of the defects associated with the absence of porin. [ABSTRACT FROM AUTHOR]

Mitochondrial porin, the voltage-dependent anion channel, plays an important role in metabolism and other cellular functions within eukaryotic cells. To further the understanding of porin structure and function, Neurospora crassa wild-type porin was replaced with a deletion variant lacking residues 238-242 (238porin). 238porin was assembled in the mitochondrial outer membrane, but the steady state levels were only about 3% of those of the wild-type protein. The strain harbouring 238porin displayed cytochrome deficiencies and expressed alternative oxidase. Nonetheless, it exhibited an almost normal linear growth rate. Analysis of mitochondrial proteomes from a wild-type strain FGSC9718, a strain lacking porin (ΔPor-1), and one expressing only 238porin, revealed that the major differences between the variant strains were in the levels of subunits of the NADH:ubiquinone oxidoreductase (complex I) of the electron transport chain, which were reduced only in the ΔPor-1 strain. These, and other proteins related to electron flow and mitochondrial biogenesis, are differentially affected by relative porin levels. [ABSTRACT FROM AUTHOR]

A novel feature of the voltage-dependent anion channel (VDAC, mitochondrial porin), is the barrel, comprising an odd number of β-strands and closed by parallel strands. Recent research has focused on the N-terminal segment, which in the available structures, resides in the lumen and is not part of the barrel. In this review, the structural data obtained from vertebrate VDAC are integrated with those from VDAC in artificial bilayers, emphasizing the array of native and tagged versions of VDAC used. The data are discussed with respect to a recent gating model (Zachariae et al. (2012) Structure 20:1–10), in which the N-terminus acts not as a gate on a stable barrel, but rather stabilizes the barrel, preventing its shift into a partially collapsed, low-conductance, closed state. Additionally, the role of the N-terminus in VDAC oligomerization, apoptosis through interactions with hexokinase and its interaction with ATP are discussed briefly. [ABSTRACT FROM AUTHOR]

Here, we review current evidence pointing to the function of VDAC1 in cell life and death, and highlight these functions in relation to cancer. Found at the outer mitochondrial membrane, VDAC1 assumes a crucial position in the cell, controlling the metabolic cross-talk between mitochondria and the rest of the cell. Moreover, its location at the boundary between the mitochondria and the cytosol enables VDAC1 to interact with proteins that mediate and regulate the integration of mitochondrial functions with other cellular activities. As a metabolite transporter, VDAC1 contributes to the metabolic phenotype of cancer cells. This is reflected by VDAC1 over-expression in many cancer types, and by inhibition of tumor development upon silencing VDAC1 expression. Along with regulating cellular energy production and metabolism, VDAC1 is also a key protein in mitochondria-mediated apoptosis, participating in the release of apoptotic proteins and interacting with anti-apoptotic proteins. The involvement of VDAC1 in the release of apoptotic proteins located in the inter-membranal space is discussed, as is VDAC1 oligomerization as an important step in apoptosis induction. VDAC also serves as an anchor point for mitochondria-interacting proteins, some of which are also highly expressed in many cancers, such as hexoki-nase (HK), Bcl2, and Bcl-xL. By binding to VDAC, HK provides both metabolic benefit and apoptosis-suppressive capacity that offers the cell a proliferative advantage and increases its resistance to chemotherapy. VDAC1-based peptides that bind specifically to HK, Bcl2, or Bcl-xL abolished the cell's abilities to bypass the apoptotic pathway. Moreover, these peptides promote cell death in a panel of genetically characterized cell lines derived from different human cancers. These and other functions point to VDAC1 as a rational target for the development of a new generation of therapeutics. [ABSTRACT FROM AUTHOR]

Abstract: Beta-barrel proteins are the main transit points across the mitochondrial outer membrane. Mitochondrial porin, the voltage-dependent, anion-selective channel (VDAC), is responsible for the passage of small molecules between the mitochondrion and the cytosol. Through interactions with other mitochondrial and cellular proteins, it is involved in regulating organellar and cellular metabolism and likely contributes to mitochondrial structure. Tom40 is part of the translocase of the outer membrane, and acts as the channel for passage of preproteins during their import into the organelle. These proteins appear to share a common evolutionary origin and structure. In the current study, the evolutionary relationships between and within both proteins were investigated through phylogenetic analysis. The two groups have a common origin and have followed independent, complex evolutionary pathways, leading to the generation of paralogues in animals and plants. Structures of diverse representatives were modeled, revealing common themes rather than sites of high identity in both groups. Within each group, intramolecular coevolution was assessed, revealing a new set of sites potentially involved in structure–function relationships in these molecules. A weak link between Tom40 and proteins related to the mitochondrial distribution and morphology protein, Mdm10, was identified. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism. [Copyright &y& Elsevier]

Abstract: Porin, the voltage-dependent anion-selective channel (VDAC) in the mitochondrial outer membrane, contributes to metabolism and apoptosis. VDAC function was investigated in Neurospora, an obligate aerobe with a single porin. Porinless strains are viable, with cold-sensitive growth, cytochrome deficiencies and overexpression of alternative oxidase. iTRAQ labeling of mitochondria from a porinless strain and its progenitor revealed a small group of proteins with altered expression levels in the mutant organelles. Porinless Neurospora appears to compensate not by inducing alternative pores, but by altering electron flow and nucleotide metabolism. Transcriptional and post-transcriptional mechanisms contribute to the response, reflecting the extent of porin influence. [Copyright &y& Elsevier]

Mitochondrial porin forms an aqueous pore in the outer membrane, through which selective passage of small metabolites and ions occurs, thereby regulating both mitochondrial function and cellular respiration. Investigations of the structure and function of porin have been performed with whole mitochondria, membrane vesicles, artificial membranes, and in detergent solutions, resulting in numerous models of porin structure. The mechanisms by which this protein functions are undoubtedly linked to its structure, which remained elusive until 2008, with reports of 3 high-resolution structures of this voltage-dependent, anion-selective channel (VDAC). The barrel structure is relatively simple yet unique: it is arranged as 19 anti-parallel β-strands, with β-strands 1 and 19 aligned parallel to each other to close the barrel. The N-terminal helical component is located within the lumen of the channel, although its precise structure and location in the lumen varies. With the basic barrel structure in hand, the data obtained in attempts to model the structure and understand porin over the past 20 years can be re-evaluated. Herein, using the mammalian VDAC structures as templates, the amassed electrophysiological and biochemical information has been reassessed with respect to the functional mechanisms of VDAC activity, with a focus on voltage-dependent gating. La porine mitochondriale forme un pore aqueux dans la membrane externe, à travers lequel des petits métabolites et des ions passent sélectivement, régulant ainsi la fonction mitochondriale et la respiration cellulaire. Des recherches sur la structure et la fonction de la porine ont été réalisées sur des mitochondries entières, des vésicules membranaires, des membranes artificielles et dans des solutions de détergent, résultant en plusieurs modèles de structure de la porine. Les mécanismes par lesquels cette protéine fonctionne sont sans aucun doute liés à sa structure, qui est demeurée floue jusqu’en 2008, quand trois structures à haute résolution de ce canal dépendant du voltage, sélecteur d’anions, ont été rapportées. La structure en tonneau est relativement simple et pourtant unique: elle est arrangée en 19 feuillets β anti-parallèles, les feuillets β 1 et 19 étant alignés parallèlement l’un à l’autre pour fermer le tonneau. La composante hélicoïdale N-terminale est localisée à l’intérieur de la lumière du canal, quoique sa structure précise et sa localisation dans la lumière peuvent varier. Ayant en main cette structure de base en tonneau, les données obtenues en tentant de modéliser la structure et comprendre la porine au cours des 20 dernières années peuvent être réévaluées. En utilisant les structures de VDAC de mammifère comme gabarit, l’information électrophysiologique et biochimique amassée a été reconsidérée relativement aux mécanismes fonctionnels de l’activité de VDAC, en mettant l’emphase sur la sélection dépendante du voltage. [ABSTRACT FROM AUTHOR]

Mitochondrial porins (also known as voltage-dependent anion-selective channels (VDACs)) regulate and contribute to cellular metabolism. These proteins copurify with sterols, and some purified forms of the protein require sterol for insertion into planar artificial membranes. Recently, interactions between detergent-solubilized mitochondrial porins and sterols have been detected by NMR and spectroscopic methods, but the effects of sterols on pore function remained to be assessed. Therefore, in this work, a freeze-thaw technique was used to introduce recombinant Neurospora porin into liposomes containing, or lacking, the native fungal sterol ergosterol. In both types of liposomes, insertion of the protein converts it to a protease-resistant state and low levels of dimeric and trimeric forms are observed. There are only minor differences between the secondary structural components of the protein in the presence or absence of sterol. Ergosterol in proteoliposomes alters their osmotic responses to sucrose, possibly due to increased membrane rigidity or interactions with the protein that were not revealed by the methods used in this study. The presence of ergosterol is associated with an increased change in conformation and loss of function of liposome-embedded porin at high temperature. Taken with other evidence for direct interactions of sterols with porins, these results support a link between these two molecules in mitochondrial membrane activity. [ABSTRACT FROM AUTHOR]

We have recently shown that Rickettsia prowazekii (typhus group rickettsiae) cells incorporate human mitochondrial porin VDAC1. Here, I report on the import of porin by rickettsiae of spotted fever group. It was shown that rickettsial cells of heavy band of Renografin density gradient, known as permeabilized rickettsiae, contain much more VDAC 1 compared to the cells of light band, that is, non-permeabilized rickettsiae. These data hint at a functionality of mitochondrial porin in rickettsial cells. The cells of Rickettsia canadensis were broken in French-press, and total membranes were fractionated on linear sucrose density gradient. In this way, previous data were confirmed that an outer rickettsial membrane does not contain porin, while fraction of intermediate density, which likely represents Bayer's adhesion zones, is enriched in VDAC 1. This is consistent with earlier results and their interpretation that imported porin seems to localize to contact sites between inner and outer rickettsial membranes. Based on well-known phylogenetic relationship of Rickettsiales and mitochondria, an evolutionary scenario for the origin of protein import machinery is proposed. A dependence of rickettsiae upon essential protein(s), destined to organelle, is also viewed as a nature of their obligate endosymbiotic lifestyle. [ABSTRACT FROM AUTHOR]

Abstract: Voltage dependent anion channel (VDAC) is a vital ion channel in mitochondrial outer membranes and its structure was recently shown to be a 19 stranded β-barrel. However the orientation of VDAC in the membrane remains unclear. We probe here the topology and membrane orientation of yeast Saccharomyces cerevisiae in vivo. Five FLAG-epitopes were independently inserted into scVDAC1 and their surface exposure in intact and disrupted mitochondria detected by immunoprecipitation. Functionality was confirmed by measurements of respiration. Two epitopes suggest that VDAC (scVDAC) has its C-terminus exposed to the cytoplasm whilst two others are more equivocal and, when combined with published data, suggest a dynamic behavior. [Copyright &y& Elsevier]

Mitochondrial porins (voltage-dependent anion-selective channels, VDAC) are key contributors to cellular metabolism. When isolated from mitochondria porins copurify with sterols, and some isolated forms of the protein require sterol for insertion into artificial membranes. Nonetheless, the contributions of sterols to the folded state of mitochondrial porin are not understood. Recently, with the goal of high-resolution structural studies, several laboratories have developed methods for folding recombinant porins at high concentration in detergent. In the present study, recombinant Neurospora crassa porin solubilized in detergent-sterol mixtures was examined. Sterols do not significantly alter the secondary structure of porin in lauryl dimethylamine oxide, nor in a mixture of sodium dodecylsulfate and dodecylmaltopyranoside. However, as detected by near-UV circular dichroism spectropolarimetry and fluorescence spectroscopy, the environments surrounding the aromatic amino acids in the detergent-sterol solubilized protein are measurably different from those in detergent alone. Furthermore, the effects are different in the presence of ergosterol, the native sterol in fungal mitochondria, and cholesterol. While these influences on the tertiary arrangement of detergent-solubilized porin are subtle, they may contribute to the generation of a form of the protein competent for insertion into the artificial bilayers used for electrophysiological analyses, and should be considered in future structural studies of porin. Les porines mitochondriales (VDAC) sont des acteurs clés du métabolisme cellulaire. Lorsque isolées à partir des mitochondries, les porines sont purifiées avec les stérols et quelques formes isolées de protéines nécessitent la présence de stérols afin d’être insérées dans des membranes artificielles. Néanmoins, la contribution des stérols à l’état replié des porines mitochondriales n’est pas comprise. Récemment, dans le cadre d’études structurales à haute résolution, plusieurs laboratoires ont développé des méthodes pour replier des porines recombinantes à haute concentration dans un détergent. Dans la présente étude, une porine recombinante de Neurospora crassa solubilisée dans des mélanges de stérols et de détergents a été examinée. Les stérols ne modifient pas de façon significative la structure secondaire de la porine dans l’oxyde de lauryl diméthylamine, ni dans un mélange de sodium dodécylsulfate et de dodécylmaltopyranoside. Cependant, tel que détecté par spectropolarimétrie en dichroïsme circulaire dans le proche UV et par spectroscopie à fluorescence, l’environnement des acides aminés aromatiques de la protéine solubilisée dans un mélange stérols-détergents est différent de manière mesurable à celui des protéines solubilisées dans un détergent seul. De plus, les effets sont différents en présence d’ergostérol, un stérol naturel des mitochondries fongiques, et de cholestérol. Alors que ces influences sur l’arrangement tertiaire des porines solubilisées dans un détergent sont subtiles, elles peuvent contribuer à l’obtention d’une protéine capable d’être insérée dans une bicouche artificielle pour des analyses électrophysiologiques et devraient être prises en compte lors d’études structurales futures des porines. [ABSTRACT FROM AUTHOR]

The voltage-dependent anion channel (VDAC) is the major protein found in the outer membrane of mitochondria. The channel is responsible for the exchange of ATP/ADP and the translocation of ions and other small metabolites over the membrane. In order to obtain large amounts of pure and suitably folded human VDAC for functional and structural studies, the genes of the human isoforms I and II (HVDAC1 and HVDAC2) were cloned in Escherichia coli. High-level expression led to inclusion body formation. Both proteins could be refolded in vitro by adding denatured protein to a solution of zwitterionic or nonionic detergents. A highly efficient and fast protocol for refolding was developed that yielded more than 50 mg of pure human VDACs per liter of cell culture. The native and functional state of the refolded porins was probed by Fourier transform infrared spectroscopy to determine the secondary structure composition and by electrophysiological measurements, demonstrating the pore-forming activity of HVDAC1. Furthermore, binding of HVDAC1 to immobilized ATP was demonstrated. Limited proteolysis of HVDAC1 protein embedded in detergent micelles in combination with matrix-assisted laser desorption ionization mass spectrometric analysis was applied to identify micelle-exposed regions of the protein and to develop an improved topology model. Our analysis strongly suggests a 16-stranded, antiparallel β-barrel with one large and seven short loops and turns. Initial crystallization trials of the protein yielded crystals diffracting to 8 Å resolution. [ABSTRACT FROM AUTHOR]

In plants, as in most eukaryotic cells, import of nuclear-encoded cytosolic tRNAs is an essential process for mitochondrial biogenesis. Despite its broad occurrence, the mechanisms governing RNA transport into mitochondria are far less understood than protein import. This article demonstrates by Northwestern and gel-shift experiments that the plant mitochondrial voltage-dependent anion channel (VDAC) protein interacts with tRNA in vitro. It shows also that this porin, known to play a key role in metabolite transport, is a major component of the channel involved in the tRNA translocation step through the plant mitochondrial outer membrane, as supported by inhibition of tRNA import into isolated mitochondria by VDAC antibodies and Ruthenium red. However VDAC is not a tRNA receptor on the outer membrane. Rather, two major components from the TOM (translocase of the outer mitochondrial membrane) complex, namely TOM20 and TOM40, are important for tRNA binding at the surface of mitochondria, suggesting that they are also involved in tRNA import. Finally, we show that proteins and tRNAs are translocated into plant mitochondria by different pathways. Together, these findings identify unexpected components of the tRNA import machinery and suggest that the plant tRNA import pathway has evolved by recruiting multifunctional proteins. [ABSTRACT FROM AUTHOR]

Abstract: The mitochondrial channel VDAC is known to have two major classes of functional states, a large conductance “open” state that is anion selective, and lower conductance substates that are cation selective. The channel can reversibly switch between open and half-open states, with the latter predominant at increasing membrane voltages of either polarity. We report the presence of a new functional state of VDAC, a cation-selective state with conductance approximately equal to that of the canonical open state. This newly described state of VDAC can be reached from either the half-open cation-selective state or from the open anion-selective state. The latter transition implies that a mechanism exists for selectivity gating in VDAC that is separate from partial closure, which may be relevant to the physiological regulation of this channel and mitochondrial outer membrane permeability. [Copyright &y& Elsevier]

In recent years, it has been recognized that there is a metabolic coupling between the cytosol, ER/SR and mitochondria. In this cross-talk, mitochondrial Ca2+ homeostasis and ATP production and supply play a major role. The primary transporter of adenine nucleotides, Ca2+and other metabolites into and out of mitochondria is the voltage-dependent anion channel (VDAC) located at the outer mitochondrial membrane, at a crucial position in the cell. VDAC has been established as a key player in mitochondrial metabolite and ion signaling and it has also been proposed that VDAC is present in extramitochondrial membranes. Thus, regulation of VDAC, as the main interface between mitochondrial and cellular metabolism, by other molecules is of utmost importance. This article reviews localization and function of VDAC, and focuses on VDAC as a skeletal muscle sarcoplasmic reticulum channel. The regulation of VDAC activity by associated proteins and by inhibitors is also presented. Several aspects of the physiological relevance of VDAC to Ca2+ homeostasis and mitochondria-mediated apoptosis will be discussed. [ABSTRACT FROM AUTHOR]

Voltage-dependent anion-selective channels (VDAC), also known as mitochondrial porins, are key regulators of metabolite flow across the mitochondrial outer membrane. Porins from a wide variety of organisms share remarkably similar electrophysiological properties, in spite of considerable sequence dissimilarity, indicating that they share a common structure. Based on primary sequence considerations, analogy with bacterial porins, and circular dichroism analysis, it is agreed that VDAC spans the outer membrane as a β-barrel. However, the residues that form the antiparallel β-strands comprising this barrel remain unknown. Various predictive methods, largely based on the known structures of bacterial β-barrels, have been applied to the primary sequences of VDAC. Refinement and confirmation of these predictions have developed through numerous investigations of wild-type and variant porins, both in mitochondria and in artificial membranes. These experiments have involved VDAC from several sources, precluding the generation of a unified model. Herein, using the Neurospora VDAC sequence as a template, the published structural information and predictions have been reassessed to delineate a model that satisfies most of the available data.Key words: VDAC, mitochondrial porin, β-barrel.Les canaux anioniques voltage-dépendants (CAVD), ou porines mitochondriales, sont d'importants régulateurs du flux des métabolites au travers de la membrane externe mitochondriale. Les porines d'une grande variété d'organismes ont des propriétés électrophysiologiques remarquablement similaires, malgré que leurs séquences diffèrent considérablement, ce qui montre qu'elles ont une structure commune. En se basant sur la séquence primaire, des analogies aux porines bactériennes et des études de dichroïsme circulaire, il est reconnu que les CAVD forment un cylindre β traversant la membrane externe. Cependant, les résidus d'acides aminés formant les feuillets β antiparallèles de ce cylindre demeurent inconnus. Diverses méthodes prédictives, principalement basées sur les structures connues des cylindres β bactériens, ont été appliquées aux séquences primaires des CAVD. Ces prédictions ont été raffinées et confirmées à la suite de nombreuses études des porines de type sauvage et de leurs variantes dans des mitochondries et des membranes artificielles. Comme ces expériences ont été réalisées en utilisant des CAVD de plusieurs sources, cela exclut la production d'un modèle unifié. Dans cet article, les informations structurales publiées et les prédictions sont réévaluées en se basant sur la séquence du CAVD de Neurospora comme modèle, afin d'esquisser un modèle pouvant s'accorder avec la plupart des données disponibles.Mots clés : CAVD, canal anionique voltage-dépendant, porine mitochondriale, cylindre β.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

Mitochondrial porin facilitates the diffusion of small hydrophilic molecules across the mitochondrial outer membrane. Despite low sequence similarity among porins from different species, a “glycine-leucine-lysine” (GLK) motif is conserved in mitochondrial and Neisseria porins. To investigate the possible roles of these conserved residues, including their hypothesized participation in ATP binding by the protein, we replaced the lysine residue of the GLK motif of Neurospora crassa porin with glutamic acid through site-directed mutagenesis of the corresponding gene. Although the pores formed by this protein have size and gating characteristics similar to those of the wild-type protein, the channels formed by GLEporin are less anion selective than the wild-type pores. The GLEporin retains the ability to be cross linked to [α-32P]ATP, indicating that the GLK sequence is not essential for ATP binding. Furthermore, the pores formed by both GLEporin and the wild-type protein become more cation selective in the presence of ATP. Taken together, these results support structural models that place the GLK motif in a part of the ion-selective β-barrel that is not directly involved in ATP binding. [ABSTRACT FROM AUTHOR]

The association in vitro of rat brain hexokinase to mitochondria from rat liver or yeast (wildtype, porinless, or expressing recombinant human porin) was studied in an effort to identifyminimal requirements for each component. A short hydrophobic N-terminal peptide ofhexokinase, readily cleavable by proteases, is absolutely required for its binding to all mitochondria.Mammalian porins are significantly cleaved at two positions in putative cytoplasmic loopsaround residues 110 and 200, as determined by proteolytic-fragment identification usingantibodies. Recombinant human porin in yeast mitochondria is more sensitive to proteolysisthan wild-type porin in rat liver mitochondria. Recombinant yeast mitochondria, harboringseveral natural or engineered porins from various sources, bind hexokinase to variable extentwith marked preference for the mammalian porin1 isoform. Genetic alteration of this isoformat the C-, but not the N-terminal, results in a significant reduction of hexokinase bindingability. Macromolecular crowding (dextran) promotes a stronger association of the enzyme toall recombinant mitochondria, as well as to proteolytically digested organelles. Consequently,brain hexokinase association with heterologous mitochondria (yeast) in these conditions occursto an extent comparable to that with homologous (rat) mitochondria. The study, also pertinentto the topology and organization of porin in the membrane, represents a necessary first stepin the functional investigation of the physiological role of mammalian hexokinase binding tomitochondria in reconstituted heterologous recombinant systems, as models to cellularmetabolism. [ABSTRACT FROM AUTHOR]