Properties of channels in the mitochondrial outer membrane

Patch-clamping studies with native outer mitochondrial membranes show a complex behavior. In the range of potentials in which the polarity of the pipette is positive, the behavior resembles that of VDAC incorporated into bilayers. Accordingly, there is a decrease in conductance with voltage. An involvement of VDAC is also supported by responses of the patches to the presence of polyanion or treatment with succinic anhydride, both of which affect VDAC. In contrast, in the negative range of potential, the conductance of the patches generally increases with the magnitude of the voltage. The increase in conductance shows a biphasic time course which is consistent with a model in which channels are first activated (first phase) and then assembled into larger high-conductance channels (second phase). A variety of experiments support the notion that an assembly takes place. The time course of the conductance increase is consistent with formation of the second-phase channels from 6±1 subunits.     

Ionic permeability of the mitochondrial outer membrane

The ionic permeability of the outer mitochondrial membrane (OMM) was studied with the patch clamp technique. Electrical recording of intact mitochondria (hence of the outer membrane (OM)), derived from mouse liver, showed the presence of currents corresponding to low conductances (< 50 pS), as well as of four distinct conductances of 99 pS,152 pS, 220 pS and 307 pS (in 150 mM KCl). The latter were voltage gated, being open preferentially at positive (pipette) potentials. Very similar currents were found by patch clamping liposomes containing the isolated OM derived from rat brain mitochondria. Here a conductance of approximately 530 pS, resembling in its electrical characteristics a conductance already attributed to mitochondrial contact sites (Moran et al. 1990), was also detected. Immunoblot assays of mitochondria and of the isolated OM with antibodies against the outer membrane voltage-dependent anion channel (VDAC) (Colombini 1979), showed the presence of the anion channel in each case. However, the typical electrical behaviour displayed by such a channel in planar bilayers could not be detected under our experimental conditions. From this study, the permeability of the OMM appears different from what has been reported hitherto, yet is more in line with that multifarious and dynamic structure which apparently should belong to it, at least within the framework of mitochondrial biogenesis (Pfanner and Neupert 1990).     

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.     

Importance of the mitochondrial outer membrane channel as a model biological channel

The channels of the mitochondrial outer membrane represent a useful model for studies into the mechanisms underlying phenomena of voltage-dependent gating and ion selectivity.     

The cation-selective substate of the mitochondrial outer membrane pore: Single-channel conductance and influence on intermembrane and peripheral kinases

The polyanion-induced substate of the outer mitochondrial membrane was studiedin vivo andin vitro. Study of the substate in artificial bilayers showed that it is highly cation selective. The induction of the substate in intact mitochondria leads to a complete inhibition of the intermembrane kinases, such as creatine kinase and adenylate kinase, which were excluded from the external ATP pool. Peripheral kinases, such as hexokinase, were blocked when they utilized internal ATP. The results with intact mitochondria suggested the existence of two regions of the outer membrane containing channels of different states, which may be involved in the regulation of intermembrane and peripheral kinases.     

Reconstitution of the native mitochondrial outer membrane in planar bilayers. Comparison with the outer membrane in a patch pipette and effect of aluminum compounds

Summary Detergent-free rat brain outer mitochondrial membranes were incorporated in planar lipid bilayers in the presence of an osmotic gradient, and studied at high (1 m KCl) and low (150 mm KCl) ionic strength solutions. By comparison, the main outer mitochondrial membrane protein, VDAC, extracted from rat liver with Triton X-100, was also studied in 150 mm KCl. In 1 m KCl, brain outer membranes gave rise to electrical patterns which resembled very closely those widely described for detergent-extracted VDAC, with transitions to several subconducting states upon increase of the potential difference, and sensitivity to polyanion. The potential dependence of the conductance of the outer membrane, however, was steeper and the extent of closure higher than that observed previously for rat brain VDAC. In 150 mm KCl, bilayers containing only one channel had a conductance of 700 ± 23 pS for rat brain outer membranes, and 890 ± 29 pS for rat liver VDAC. Use of a fast time resolution setup allowed demonstration of open-close transitions in the millisecond range, which were independent of the salt concentration and of the protein origin. We also found that a potential difference higher than approx. ± 60 mV induced an almost irreversible decrease of the single channel conductance to few percentages of the full open state and a change in the ionic selectivity. These results show that the behavior of the outer mitochondrial membrane in planar bilayers is close to that detected with the patch clamp (Moran et al., 1992, Eur. Biophys. J. 20:311–319).The neurotoxicological action of aluminum was studied in single outer membrane channels from rat brain mitochondria. We found that m concentrations of Al Cl₃ and aluminum lactate decreased the conductance by about 50%, when the applied potential difference was positive relative to the side of the metal addition.The authors thank Dr. O. Moran for helpful discussions, Dr. M. Colombini for a sample of polyanion, and the Sharing Company for financial support to Dr. T. M. This work was partly supported by funds from the Ministero dell' Universitá e della Ricerca Scientifica e Tecnologica of Italy.     

Topological orientation of mitochondrial GDPmannose: dolichyl-monophosphate mannosyltransferase in the outer membrane

Previous studies have shown the existence of an autonomous mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase, located in mitochondiral outer membrane of liver cells. As nothing is known about glycosylation sites in mitochondria, we have investigated the topological orientation of this enzyme in intact mitochondria, using controlled proteolysis with trypsin. Mitochondria were purified sequentially by mild ultrasonic treatment and sucrose density gradient. Purity and homogeneity of mitochondrial fraction were assessed by electron microscopy and specific marker enzymes measures. Our data provide evidence for a mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase facing the cytoplasmic side of the outer membrane. However, the exposure of this enzyme to the water phase has been shown to be dependent on the ionic strength of the environment.     

Characterization and function of the mitochondrial outer membrane peptide-sensitive channel

The PSC (peptide-sensitive Channel), a cationic channel of large conductance, has been characterized in yeast and mammalian mitochondria by three different methods, tip-dip, patch clamp of giant liposomes, and planar bilayers. The yeast and mammalian PSC share the common property to be blocked by basic peptides such as pCyt OX IV (1–12)Y which contains the first 12 residues of the presequence of cytochromec oxidase subunit IV. The electrophysiological data are consistent with a translocation of the peptide through the pore. Analysis of the frequency of observation of the PSC in different fractions indicates that the channel is located in the outer mitochondrial membrane. Uptake measurements of iodinated peptides by intact mitochondria from a porin-less mutant show that the peptides are translocated through the outer membrane, presumably at the level of PSC. Among the peptides active on PSC, several, such as pCyt OX IV (1–22) and the reduced form of the mast cell degranulating peptide, induce an alteration of the voltage dependence or of the inactivation rate subsisting after washing and which is eliminated only by proteolysis of the interacting peptide. These irreversible effects may account for the variability of the properties of the PSC which would interact with cytosolic or intermembrane cations, peptides, or proteins, thus modulating the channel permeability. Finally, several lines of evidence suggest the participation of the PSC in protein translocation and some interaction with the general insertion pore of the outer membrane translocation machinery.     

Structure of the mitochondrial outer membrane channel derived from electron microscopy of 2D crystals

A structural model for the channel in the mitochondrial outer membrane is presented, derived from electron microscopic studies of two-dimensional crystals and inferences from the primary structure of the 30-kDa polypeptide which forms the channel. The channel is represented as a cylindrical beta-barrel, with a carbon backbone diameter of 3.8 nm. The axial projection of the cylinder is divided radially into four sectors by four interchannel contact points. These sectors are characterized in terms of their interactions with lipid and macromolecular ligands, and in terms of the presence or absence of exposed basic amino acids.     

The yeast dynamin-like protein, Mgm1p, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance

The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37 degrees C, while loss of the mitochondrial genome occurred after 4-24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1-null mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.     

Mitochondrial outer membrane permeabilization during apoptosis: The role of mitochondrial fission

Mitochondria continually fuse and divide to yield a dynamic interconnected network throughout the cell. During apoptosis, concomitantly with permeabilization of the mitochondrial outer membrane (MOMP) and cytochrome c release, mitochondria undergo massive fission. This results in the formation of small, round organelles that tend to aggregate around the nucleus. Under some circumstances, preceding their fission, mitochondria tend to elongate and to hyperfuse, a process that is interpreted as a cell defense mechanism. Since many years, there is a controversy surrounding the physiological relevance of mitochondrial fragmentation in apoptosis. In this review, we present recent advances in this field, describe the mechanisms that underlie this process, and discuss how they could cooperate with Bax to trigger MOMP and cytochrome c release. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Mmm2p, a mitochondrial outer membrane protein required for yeast mitochondrial shape and maintenance of mtDNA nucleoids

The mitochondrial outer membrane protein, Mmm1p, is required for normal mitochondrial shape in yeast. To identify new morphology proteins, we isolated mutations incompatible with the mmm1-1 mutant. One of these mutants, mmm2-1, is defective in a novel outer membrane protein. Lack of Mmm2p causes a defect in mitochondrial shape and loss of mitochondrial DNA (mtDNA) nucleoids. Like the Mmm1 protein (Aiken Hobbs, A.E., M. Srinivasan, J.M. McCaffery, and R.E. Jensen. 2001. J. Cell Biol. 152:401-410.), Mmm2p is located in dot-like particles on the mitochondrial surface, many of which are adjacent to mtDNA nucleoids. While some of the Mmm2p-containing spots colocalize with those containing Mmm1p, at least some of Mmm2p is separate from Mmm1p. Moreover, while Mmm2p and Mmm1p both appear to be part of large complexes, we find that Mmm2p and Mmm1p do not stably interact and appear to be members of two different structures. We speculate that Mmm2p and Mmm1p are components of independent machinery, whose dynamic interactions are required to maintain mitochondrial shape and mtDNA structure.     

Interaction of hexokinase with the outer mitochondrial membrane and a hydrophobic matrix

The major portion of rat brain hexokinase (HK type I) is bound to the outer membrane of mitochondria and glucose-6-phosphate (G6P) can release the bound enzyme. In an attempt to look at the hydrophobic component of binding, interaction of the enzyme with a purely hydrophobic matrix, palmityl-substituted Sepharose-4B (Sepharose-lipid) was investigated. Hexokinase readily bound to this matrix with retention of its catalytic activity. Glucose-6-phosphate which has a releasing effect on the mitochondrially bound enzyme, enhanced binding of the enzyme on the hydrophobic matrix. Chymotrypsin treatment of hexokinase which causes loss of binding to mitochondria, also results in loss of adsorption to the hydrophobic matrix, thus demonstrating that the hydrophobic tail present at its N-terminal end is essential for binding in both cases. Data presented provide some new information relevant to understanding how hexokinase interacts with its natural binding matrix, the mitochondrion.     

Evidence for small pores in the outer membrane of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa NCTC6750 and Escherichia coli K12 were used to study permeability of whole, intact cells to a series of labelled oligosaccharides. Stationary phase, oxygen depleted simple salts batch cultures were used. An efflux method was used to compare diffusion from cells of various ³H-labelled sugars (an homologous series based on isomaltitol) with diffusion of [¹⁴C]sucrose. Both plasmolysed and unplasmolysed cell suspensions were used. The data are consistent with an E. coli pore exclusion limit of approx. 833 Da for unplasmolysed cells and of about 670 Da for plasmolysed cells. For P. aeruginosa the data indicated a relatively small pore exclusion limit about the same size as sucrose with plasmolysis having little effect. These findings were confirmed with P. aeruginosa PAO1 grown in nutrient broth.     

Assessing mitochondrial outer membrane permeabilization during apoptosis

Mitochondria play a pivotal role in the regulation of apoptosis. An imbalance in apoptosis can lead to disease. Unscheduled apoptosis has been linked to neurodegeneration while inhibition of apoptosis can cause cancer. An early and key event during apoptosis is the release of factors from mitochondria. In apoptosis the mitochondrial outer membrane becomes permeable, leading to release of apoptogenic factors into the cytosol. One such factor, cytochrome c, is an electron carrier of the respiratory chain normally trapped within the mitochondrial intermembrane space. Many apoptotic studies investigate mitochondrial outer membrane permeabilization (MOMP) by monitoring the release of cytochrome c. Here, we describe three reliable techniques that detect cytochrome c release from mitochondria, through subcellular fractionation or immunocytochemistry and fluorescence microscopy, or isolated mitochondria and recombinant Bax and t-Bid proteins in vitro. These techniques will help to identify mechanisms and characterize factors regulating MOMP.     

Purification and characterization of the pore forming protein of yeast mitochondrial outer membrane

One of the major outer membrane proteins of yeast mitochondria was isolated and purified. It migrated as a single band with an apparent molecular weight of 30 kDa on a SDS-electrophoretogram. When reconstituted in lipid bilayer membranes the protein formed pores with a single channel conductance of 0.45 nS in 0.1 M KCl. The pores had the characteristics of general diffusion pores with an estimated diameter of 1.7 nm. The pore of mitochondrial outer membranes of yeast shared some similarities with the pores formed by mitochondrial and bacterial porins. The pores switched to substates at voltages higher than 20 mV. The possible role of this voltagedependence in the metabolism of mitochondria is discussed.     

Ca-induced release of mitochondrial m-calpain from outer membrane with binding of calpain small subunit and Grp75

Although mitochondrial μ- and m-calpains play significant roles in apoptotic cell death, their activating mechanisms have not been determined. The purpose of this study was to determine the core factors that are involved in activating mitochondrial outer membrane (OM)-bound calpains. To accomplish this, we solubilized OM-bound calpains and separated them by DEAE-Sepharose column chromatography, and identified them by immunoblots. We also determined the core factors that activated the OM-bound calpains and release them from the OM by calpain assays, immunoprecipitations, and immunoblots. The OM-bound m-calpain large subunit was not associated with the small subunit or with Grp75 chaperone. Free calpain small subunit was located in the IMS and caused the release of the OM-bound m-calpain large subunit from the OM together with Grp75, ATP, and Ca²⁺. Our results showed that the activating mechanism of mitochondrial OM-bound m-calpain and the release of mitochondrial m-calpain from the OM have important implications in facilitating apoptotic cell death.     

Pores from mitochondrial outer membranes of yeast and a porin-deficient yeast mutant: A comparison

Reconstitution experiments were performed on lipid bilayer membranes in the presence of purified mitochondrial porin from yeast and of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant. The addition of the porin resulted in a strong increase of the membrane conductance, which was caused by the formation of ion-permeable channels in the membranes. Yeast porin has a single-channel conductance of 4.2 nS in 1 M KCl. In the open state it behaves as a general diffusion pore with an effective diameter of 1.7 nm and possesses properties similar to other mitochondrial porins. Surprisingly, the membrane conductance also increased in the presence of detergent extracts of the mitochondrial outer membrane of the mutant. Single-channel recordings of lipid bilayer membranes in the presence of small concentration of the mutant membranes suggested that this membrane also contained a pore. The reconstituted pores had a single-channel conductance of 2.0 nS in 1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. This means that the pores present in the mitochondrial outer membranes of the yeast mutant have a much smaller effective diameter than normal mitochondrial porins. Zero-current membrane potential measurements suggested that the second mitochondrial porin is slightly cation-selective, while yeast porin is slightly anion-selective in the open state but highly cation-selective in the closed state. The possible role of these pores in the metabolism of mitochondria is discussed.     

Structure of the outer mitochondrial membrane analysis of x-ray diffraction from the plant membrane

X-ray diffraction from centrifugally oriented specimens of plant outer mitochondrial membranes suggests that these membranes contain prominent in-plane subunits. The short lamellar repeat which these specimens display (as low as 5.1 nm) points to a predominantly internal localization of the protein components of these membranes. The simplest model for the putative in-plane subunit consistent with autocorrelation analysis of the normal-incidence diffraction data consists of two concentric rings of electron density with diameters of (approx.) 2 and 4 nm. These rings could represent the planar projections of concentric cylindrical shells, aligned normal to the membrane surface.     

Evidence that the crystalline arrays in the outer membrane of Neurospora mitochondria are composed of the voltage-dependent channel protein

Antibodies were raised in rabbits against the outer membrane of Neurospora mitochondria. Antibodies were obtained that were specific for this membrane's major polypeptide ($\text{M, 31 000}$) and its slower-migrating derivatives on SDS-polyacrylamide gels. These antibodies inhibited the insertion into phospholipid bilayers of voltage-dependent ion channels from detergent extracts of the mitochondrial outer membranes. The same antibodies bound preferentially to membranes containing crystalline surface arrays in outer mitochondrial membrane fractions. These results indicate that the 31 kDa polypeptide is a component both of the ion channels and of the membrane arrays, suggesting identity between the functional and structural entities.