VDAC1 selectively transfers apoptotic Ca2+ signals to mitochondria.

Voltage-dependent anion channels (VDACs) are expressed in three isoforms, with common channeling properties and different roles in cell survival. We show that VDAC1 silencing potentiates apoptotic challenges, whereas VDAC2 has the opposite effect. Although all three VDAC isoforms are equivalent in allowing mitochondrial Ca(2+) loading upon agonist stimulation, VDAC1 silencing selectively impairs the transfer of the low-amplitude apoptotic Ca(2+) signals. Co-immunoprecipitation experiments show that VDAC1, but not VDAC2 and VDAC3, forms complexes with IP(3) receptors, an interaction that is further strengthened by apoptotic stimuli. These data highlight a non-redundant molecular route for transferring Ca(2+) signals to mitochondria in apoptosis.

A genetic analysis of the porin gene encoding a voltage-dependent anion channel protein in Drosophila melanogaster.

The voltage-dependent anion channel (VDAC, also known as porin) is an abundant protein in the outer mitochondrial membrane that forms transmembrane channels permeable to solutes. While in mammals at least three different porin genes have been found, only one VDAC-encoding gene, porin, has been described so far in Drosophila melanogaster. It produces transcripts with alternative untranslated sequences. Here we report the identification of two PlacW insertions in the porin gene among a set of P-element insertions that have been mapped to the 32B3-4 region on the second chromosome. Homozygotes, as well as trans-heterozygotes for these insertions, lack VDAC, and die during the late pupal stage. Function can be restored by precise excision of the P transposon, while most deletions in the porin locus, produced by imprecise excisions, display the recessive lethal effect of the original mutant alleles. However, one of the deletions was found to be a hypomorphic male-sterile allele producing low levels of the VDAC protein, indicating that the product of the porin gene is also essential for male fertility. Analysis of the new mutant alleles also showed that the untranslated exon 1B of the porin gene is not required for VDAC synthesis. In the course of our investigation, we found that immediately adjacent to the porin gene are three more genes encoding proteins that share homology with the VDAC protein. The possible evolutionary and functional relationships of the porin-like genes at 32B3-4 are discussed.

Prediction of the transmembrane regions of beta-barrel membrane proteins with a neural network-based predictor.

A method based on neural networks is trained and tested on a nonredundant set of beta-barrel membrane proteins known at atomic resolution with a jackknife procedure. The method predicts the topography of transmembrane beta strands with residue accuracy as high as 78% when evolutionary information is used as input to the network. Of the transmembrane beta-strands included in the training set, 93% are correctly assigned. The predictor includes an algorithm of model optimization, based on dynamic programming, that correctly models eight out of the 11 proteins present in the training/testing set. In addition, protein topology is assigned on the basis of the location of the longest loops in the models. We propose this as a general method to fill the gap of the prediction of beta-barrel membrane proteins.

Intracellular localization and isoform expression of the voltage-dependent anion channel (VDAC) in normal and dystrophic skeletal muscle.

Voltage-dependent anion channels (VDACs) are a family of pore-forming proteins encoded by different genes, with at least three protein products expressed in mammalian tissues. The major recognized functional role of VDACs is to permit the almost free permeability of the outer mitochondrial membrane (OMM). Although VDAC1 is the best known among VDAC isoforms, its exclusively mitochondrial location is still debated. Therefore, we have measured its co-localization with markers of cellular organelles or compartments in skeletal muscle fibers by single or double immunofluorescence and traditional as well as confocal microscopy. Our results show that VDAC1 immunoreactivity corresponds to mitochondria and sarcoplasmic reticulum, while sarcolemmal reactivity, previously reported, was not observed. Since VDAC1 has been suggested to be involved in the control of oxidative phosphorylation, we sought for possible gene regulation of VDAC1, VDAC2 and VDAC3 in skeletal muscle of the dystrophin-deficient mdx mouse, which suffers of an impaired control of energy metabolism. Our results show that, while VDAC1 mRNA and protein and VDAC2 mRNA are normally expressed. VDAC3 mRNA is markedly down-regulated in mdx mouse muscle at different ages (before, during and after the outburst of myofiber necrosis). This finding suggests a possible involvement of VDAC3 expression in the early pathogenic events of the mdx muscular dystrophy.

Characterization of the human porin isoform 1 (HVDAC1) gene by amplification on the whole human genome: A tool for porin deficiency analysis.

The deficiency of porin isoform 1 (HVDAC1) in human skeletal muscle has been associated with a pathological phenotype related to defects in the bioenergetic metabolism. In the best studied case, porin deficiency was not apparent in cultured fibroblasts: this observation raised the conclusion that no molecular defect was in the cDNA sequence coding for the protein. To get more insight in the pathogenetic mechanism that is involved in porin isoform 1 deficiency, we have determined the whole structure of the corresponding human gene. On the basis of the corresponding mouse gene structure and the human cDNA sequence, we designed long extension PCR amplifications using the whole genomic DNA as a template. Exonic/intronic regions were isolated and the exons and surrounding introns sequenced. The 5' and 3' extremities of the gene were determined by genome walking. The porin isoform 1 human gene is made up of 9 exons and spans about 33 kbp. A whole panel of PCR parameters was set and is now ready to be used for specific amplification upon patients' genomic DNA. The analysis of the putative promoter sequence was performed. It revealed the presence of a sterol Repressor element (SRE), an SRY, the testis-determining factor, and a nuclear respiratory factor 2 (NRF-2) binding site. These sites, according to results from literature, could be involved in the functional modulation of the gene expression.

Characterization of channel-forming activity in muscle biopsy from a porin-deficient human patient.

A bioptic specimen from the muscles of a patient suffering from severe myopathy was inspected for the presence of human porin 31HL. Western blotting suggested that the specimen was free of the most abundant eukaryotic porin 31HL (HVDAC1). The specimen was treated with detergent and the soluble protein fraction was passed through a dry hydroxyapatite column. The passthrough of this column was inspected for channel formation in artificial lipid-bilayer membranes. The channel observed under these conditions had a single-channel conductance of about 2.5 nS in 1 M KCl, was cation selective, and was found to be virtually voltage independent. Experiments with a control specimen from a healthy human being, without any indication for muscle myopathy, revealed the presence of the voltage-dependent porin 31HL in the sample. It is discussed whether the patient's bioptic specimen contained another human porin, which has not been studied to date in its natural environment.

Extramitochondrial porin: facts and hypotheses.

Mitochondrial porin, or VDAC, is a pore-forming protein abundant in the outer mitochondrial membrane. Several publications have reported extramitochondrial localizations as well, but the evidence was considered insufficient by many, and the presence of porin in nonmitochondrial cellular compartments has remained in doubt for a long time. We have now obtained new data indicating that the plasma membrane of hematopoietic cells contains porin, probably located mostly in caveolae or caveolae-like domains. Porin was purified from the plasma membrane of intact cells by a procedure utilizing the membrane-impermeable labeling reagent NH-SS-biotin and streptavidin affinity chromatography, and shown to have the same properties as mitochondrial porin. A channel with properties similar to that of isolated VDAC was observed by patch-clamping intact cells. This review discusses the evidence supporting extramitochondrial localization, the putative identification of the plasma membrane porin with the "maxi" chloride channel, the hypothetical mechanisms of sorting porin to various cellular membrane structures, and its possible functions.

Porin is present in the plasma membrane where it is concentrated in caveolae and caveolae-related domains.

Mitochondrial porin, or voltage-dependent anion channel, is a pore-forming protein first discovered in the outer mitochondrial membrane. Later investigations have provided indications for its presence also in other cellular membranes, including the plasma membrane, and in caveolae. This extra-mitochondrial localization is debated and no clear-cut conclusion has been reached up to now. In this work, we used biochemical and electrophysiological techniques to detect and characterize porin within isolated caveolae and caveolae-like domains (low density Triton-insoluble fractions). A new procedure was used to isolate porin from plasma membrane. The outer surface of cultured CEM cells was biotinylated by an impermeable reagent. Low density Triton-insoluble fractions were prepared from the labeled cells and used as starting material to purify a biotinylated protein with the same electrophoretic mobility and immunoreactivity of mitochondrial porin. In planar bilayers, the porin from these sources formed slightly anion-selective pores with properties indistinguishable from those of mitochondrial porin. This work thus provides a strong indication of the presence of porin in the plasma membrane, and specifically in caveolae and caveolae-like domains.

The Drosophila melanogaster gene for the NADH:ubiquinone oxidoreductase acyl carrier protein: developmental expression analysis and evidence for alternatively spliced forms.

We have isolated the Drosophila melanogaster gene encoding the mitochondrial acyl carrier protein (mtACP), a subunit of NADH:ubiquinone oxidoreductase involved in de novo fatty acid synthesis in the mitochondrion. This gene expresses two distinct mature transcripts by alternative splicing, which encode mature polypeptides of 86 (mtACP1A) and 88 (mtACP1B) amino acids, respectively. Drosophila mtACP1 is 72% identical to mammalian mtACP, 47% identical to Arabidopsis thaliana mtACP, and 46% identical to Neurospora crassa mtACP. The most highly conserved region encompasses the site that binds pantetheine-4'-phosphate in all known ACPs. Southern analysis of genomic DNA and in situ hybridization to salivary gland chromosomes indicate that a single gene (mtacp1), located at 61F6-8, encodes the two isoforms of D. melanogaster mtACP1. Sequence analysis revealed that the gene contains four exons and that exons IIIA and IIIB are alternatively spliced. A P-element-induced loss-of-function mutation in the mtacp1 gene causes lethality, indicating that the gene is essential for viability. Developmental Northern analysis shows that mtacp1 is expressed at higher levels during late embryogenesis, in the pupa and in the adult. RNA in situ hybridization on embryos indicates that the mtacp1 gene is highly expressed in the tracheal system. Zygotic mtacp1 function is required for both male and female gametogenesis.

Presence of a voltage-dependent anion channel 1 in the rat postsynaptic density fraction.

Little is known about the molecular organization and functions of the postsynaptic density (PSD), a cytoskeletal specialization on the postsynaptic membrane. In an attempt to elucidate the protein composition of PSD, we have sequenced a 35 kDa protein of the rat forebrain PSD fraction. Amino acid sequence information of the tryptic peptides and immunoblot analyses revealed that the protein is a voltage-dependent anion channel 1 (VDAC1). VDAC1 was enriched in the PSD fraction and was partially soluble in 1% n-octyl glucoside (NOG) or Triton X-100. Our data indicate that VDAC1, which is originally found in the outer mitochondrial membrane, is also present in the central nervous system (CNS) synapses in association with the PSD 'core'.

Identification of nuclear genes encoding mitochondrial proteins: isolation of a collection of D. melanogaster cDNAs homologous to sequences in the Human Gene Index database.

As a first step towards using cross-species comparison to complete the inventory of the nuclear genes that encode mitochondrial polypeptides, and ultimately to understand their function through systematic molecular and genetic analysis in a model organism of choice, we report here the characterization of 41 Drosophila melanogaster cDNAs. These cDNAs were isolated by screening an ovarian expression library with antibodies against mitochondrial proteins and identify 17 novel Drosophila genes. The deduced amino acid sequences encoded by the majority of these cDNAs turned out to show significant homology to mitochondrial proteins previously identified in other species. Among others, ORFs putatively encoding six different subunits of ATP synthase and three NADH:ubiquinone reductase subunits were detected. By in situ hybridization, all cDNAs were mapped to single bands on polytene chromosomes, thus identifying candidate Drosophila genes required for mitochondrial biogenesis and maintenance. A search of the Human Gene Index database made it possible in most cases to align the entire Drosophila coding sequence with a human consensus sequence, suggesting that the cDNAs originate from insect counterparts of expressed mammalian genes. Our experimental strategy represents an efficient approach to the identification and interspecies comparison of genes encoding products targeted to the mitochondrion.

Mapping of the human Voltage-Dependent Anion Channel isoforms 1 and 2 reconsidered.

Eukaryotic porins or VDACs (Voltage-Dependent Anion-selective Channels) are integral membrane proteins forming large hydrophilic pores. Three functioning genes for VDAC isoforms have been detected in mouse and the corresponding cDNAs are known also in humans. Tissue-specific VDAC isoform 1 (HVDAC1) deficiency in human skeletal muscle is responsible of a rare mitochondrial encephalomyopathy, fatal in childhood. Since coding sequences are not affected in the patient, we focused our interest in the gene structure. HVDAC1 and 2 have been previously mapped at chromosomes Xq13-21 and 21, respectively. Screening of an human chromosome X cosmid library resulted only in the isolation of processed pseudogenes, finely mapped at Xq22 and Xp11.2. Here, we report the mapping of HVDAC1 to chromosome 5q31 and HVDAC2 to chromosome 10q22 by FISH. Exon/intron probes, designed on the basis of the mouse gene structures, were obtained by long extension PCR amplification using the whole genomic DNA as a template. The sequence of the probe extremities clearly pointed to a genuine VDAC genomic sequence. Human and mouse regions where VDAC 1 and 2 genes were mapped are known to be synthetic, thus reinforcing the mapping of the human homologues.

Sequence and expression pattern of the Drosophila melanogaster mitochondrial porin gene: evidence of a conserved protein domain between fly and mouse.

We have recently cloned a cDNA encoding mitochondrial porin in Drosophila melanogaster and shown its chromosomal localization (Messina et al., FEBS Lett. (1996) 384, 9-13). Such cDNA was used as a probe for screening a genomic library. We thus cloned and sequenced a 4494-bp genomic region which contained the whole gene for the mitochondrial porin or VDAC. It was found that this D. melanogaster porin gene contains five exons, numbered IA (115 bp), IB (123 bp), II (320 bp), III (228 bp) and IV (752 bp). The exons II, III and IV contain the protein coding sequence and the 3' untranslated sequence (3'-UTR). The first base in exon II precisely corresponds to the first base of the starting ATG codon. Exon IA corresponds to the 5'-UTR sequence reported in the published cDNA sequence. Exon IB corresponds to an alternative 5'-UTR sequence, demonstrated to be transcribed by 5'-RACE experiments. The exon-intron splicing borders and the length of the exon III perfectly match a homologous internal exon detected in the mouse genes. Such exon encodes a protein domain predicted by sequence transmembrane arrangement models to contain major hydrophilic loops and it is thus suspected to have a conserved distinct function. In situ hybridization experiments confirmed the localization of the genomic clone on the chromosome 2L at region 32B3-4. Together with genomic Southern blotting at various stringencies, the same experiment did not confirm the presence of a second genetic locus on D. melanogaster chromosomes. Northern blots demonstrated that the porin gene is a housekeeping one: three messages of approx. 1.2-1.6 kbp are transcribed in every fly developmental stage that was studied. They were shown to derive by an alternative usage of different promoters and polyadenylation sites.

Double-stranded DNA can be translocated across a planar membrane containing purified mitochondrial porin.

The transport of genetic material across biomembranes is a process of great relevance for several fields of study. However, much remains to be learned about the mechanisms underlying transport, one of which implies the involvement of proteic DNA-conducting pores. Entry of genetic material into mitochondria has been observed under both physiological and pathological conditions. We report here that double-stranded DNA can move through a planar bilayer membrane containing isolated mitochondrial porin (voltage-dependent anion channel). The transport is driven by the applied electrical field, and the presence of DNA is associated with a decrease of current conduction by the pores. The passage of DNA does not take place if the bilayer has not been doped with any protein or in the presence of both reconstituted porin and anti-porin antibody. Translocation does not occur if the bilayer contains Shigella sonnei maltoporin, gramicidin A channels, or a 30 pS anion-selective channel plus other proteins. These results show that mitochondrial porin is capable of mediating the transport of genetic material, revealing a new property of this molecule and further confirming the idea that DNA can move through proteic pores.

Novel aspects of the electrophysiology of mitochondrial porin.

The recent findings that mitochondrial porin, VDAC, participates in supramolecular complexes and is present in the plasmamembrane need to be reconciled with its biophysical properties. We report here that VDAC often displays previously unobserved or unappreciated behaviors. Reconstituted VDAC can: a) exhibit fast gating when in any of many conductance substates; b) close completely, although briefly, on its own; c) close for a long periods, in the presence of König's polyanion; d) take several milliseconds to re-open when an applied transmembrane potential is switched off; e) be desensitized by prolonged exposure to high voltages, so that it will not re-open to the full conductance state upon subsequent return to zero voltage; f) display polarity-dependent voltage-induced closure. These behaviors are especially noticeable when the observations are conducted on a single reincorporated channel, suggesting that interactions between copies of VDAC may play a role in determining its electrophysiological properties. Any model of VDAC's structure, gating and function should take these observations into account.

Evaluation of biotinylated cells as a source of antigens for characterization of their molecular profile.

Biotinylated lymphoid cells have been suggested as a useful source of antigen for the immunochemical characterization of their molecular profile. Labelling with biotin eliminates the problems associated with the use of radioactivity. However, this method has not been widely used. This reflects: (1) difficulties in optimizing the signal/background ratio because of the lack of a simple method to quantify biotinylated proteins in a cell lysate, (2) the loss of reactivity with monoclonal antibody of antigen following biotinylation, because of steric hindrance, and (3) the lack of information about the utility of other biotinylated cells as an antigen source. To overcome these limitations, we developed an ELISA to quantify biotinylated proteins in cell lysates and optimized the signal/background ratio. The validity of this approach was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a number of cell surface antigens immunoprecipitated from lymphoid cells by an optimal amount of monoclonal antibody. Furthermore, we showed that biotinylated melanoma cells are a useful source of antigen for immunoprecipitation experiments and that ligation of biotin to antigen does not affect reactivity with monoclonal antibody. Lastly, biotinylated antigens in cell lysates stored at -80 degrees C for 6 months maintained their reactivity with monoclonal antibodies. Biotinylated cells thus represent a useful source of antigen for characterizing the immunochemical profile and analyzing the specificity of antibodies with immunochemical methods.

Cloning and chromosomal localization of a cDNA encoding a mitochondrial porin from Drosophila melanogaster.

We have raised polyclonal antibodies against purified the Drosphila melanogaster mitochondrial porin. They showed high titre and specificity and were thus used as a tool for screening an expression library. The isolated clone 1T1 showed 74% sequence identity in the last 19 residues at the C-terminus of human porin. A subclone of 1T1, containing the porin-like sequence, was thus used as a probe for re-screening a cDNA library and several positive clones were plaque-purified. We present here the sequence of a 1363 bp cDNA encoding a protein of 279 amino acids. Its identity with porin was also confirmed by N-terminal Edman degradation of the purified protein. The D. melanogaster porin shows an overall 51.8% identity with human porin isoform 1 (porin 31HL or HVDAC1) and an overall 55.7% identity with human porin isoform 2 (HVDAC2). Hydrophobicity plots and secondary structure predictions showed a very high similarity with data obtained from known porin sequences. The D. melanogaster porin cDNA was used as a probe for in situ hybridization to polytenic salivar gland chromosomes. It hybridizes with different intensities in two sites, in chromosome 2L, at region 31E and in chromosome 3L at region 79D. Thus, also in Drosophila melanogaster porin polypeptide(s) belong(s) to a multigene family.

The mitochondrial permeability transition pore may comprise VDAC molecules. II. The electrophysiological properties of VDAC are compatible with those of the mitochondrial megachannel.

The electrophysiological properties of isolated mitochondrial porin (VDAC), reconstituted in planar bilayers or proteoliposomes, resemble those of the mitochondrial megachannel believed to be the permeability transition pore. In particular, a correspondence was found with regard to the voltage dependence: VDAC was driven to closed states by potentials of either sign, but the effect was not symmetrical; voltages negative in the compartment to which VDAC was added were more effective. The results are consistent with the hypothesis that the PTP may consist of two cooperating VDAC channels, plus presumably an adenine nucleotide carrier dimer and a third component known to be part of the mitochondrial benzodiazepine receptor.

Purification and Characterization of Porin from Corn (Zea mays L.) Mitochondria.

Mitochondrial porin from corn (Zea mays L. B 73) shoots was solubilized with lauryl(dimethyl)-amine oxide and purified by chromatography on a hydroxyapatite:celite column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein had an apparent molecular mass of 35 kD. When reconstituted in planar lipid bilayer membranes the porin formed ion-permeable channels with single-channel conductance of 2.0 and 4.0 nanosiemens in 1 M KCl. At low transmembrane voltages corn porin had the properties of a general diffusion pore with an estimated effective diameter of 1.6 nm and a small selectivity for anions over cations. The primary structure of corn porin seems to be quite different from that of other mitochondrial porins, because it did not cross-react with monoclonal antibodies against human porin and with polyclonal antibodies against yeast porin. Furthermore, the peptide maps of corn and bovine heart porins were very different. A sequence of 21 amino acids obtained by Edman degradation of peptides generated by porin proteolysis with Staphylococcus aureus V8 protease did not show any significant homology with known sequences of mitochondrial porins. Results of our investigation suggest that corn porin possesses functional properties similar to those of other mitochondrial porins, despite major structural differences.

Location of the dicyclohexylcarbodiimide-reactive glutamate residue in the bovine heart mitochondrial porin.

The mitochondrial porin or VDAC (Voltage-Dependent Anion Channel), the pore-forming structure responsible for the high permeability of the outer mitochondrial membrane, was found to be one of only three mitochondrial proteins bound by [14C]dicyclohexylcarbodiimide (DCCD) at low dosages (1.5 nmol/mg of mitochondrial porin) (De Pinto, V., Tommasino, M., Benz, R., and Palmieri, F. (1985) Biochim. Biophys. Acta 813, 230-242). Treatment of intact mitochondria with DCCD results in the inhibition of their ability to binding hexokinase (Nakashima, R. A., Mangan, P. S., Colombini, M., and Pedersen, P. L. (1986) Biochemistry 25, 1015-1021). In the present study, mitochondrial porin was purified from [14C]DCCD-labeled mitochondria. The purified labeled porin was treated with the cleavage reagent CNBr and with the endoproteases trypsin and V8 from Staphylococcus aureus and blotted to polyvinylidene difluoride membrane. The transferred peptides were detected with Coomassie Blue dye, excised, and sequenced. The sequences of several labeled and unlabeled peptides were obtained and then overlapped. The region containing the [14C]DCCD radioactivity was limited to 50 amino acid residues and completely sequenced. Covalently incorporated [14C]DCCD was exclusively released at the position corresponding to glutamate 72. The DCCD-reactive residue is located in the 4th of 16 predicted transmembrane amphipathic beta-strands. When the sequence surrounding the DCCD site was compared to those surrounding the DCCD-reactive residue of other membrane proteins, no homology was apparent.