Molecular genetics of the VDAC ion channel: Structural model and sequence analysis

The voltage-dependent anion-selective channel of the outer mitochondrial membrane provides a unique system in which to study the molecular basis of voltage gating of ion flow. We have cloned and sequenced acDNA coding for this protein in yeast. From the derived amino acid sequence, we have generated a preliminary model for the secondary structure of the protein which suggests that the protein forms a -barrel type structure. Comparison of the VDAC amino acid sequence with that of the bacterial porins has indicated that the two classes of molecules appear to be unrelated.     

Indications of a common folding pattern for VDAC channels from all sources

Previous research on the mitochondrial channel VDAC from the yeastS. cerevisiae had identified protein strands forming the wall of VDAC's aqueous pore. Here we report the results of analyzing the primary sequences of VDAC from various sources to see if the transmembrane folding pattern identified from this yeast is conserved for VDAC of different species. We analyzed the primary sequences of VDAC from higher plants, fungi, invertebrates, and vertebrates and found that all have a very similar -partern profile with 12–15 peaks indicating potential sided beta strands that are candidates for protein strands forming the wall of the aqueous pore. All these VDAC sequences can be put into the 13 transmembrane strand folding pattern previously identified for yeast VDAC. These folding patterns agree with available experimental data: both electrophysiological and protease digestion data. Although the primary sequences of VDAC from very diverse organisms show low homology, sequence similarity in the proposed corresponding 13 transmembrane strands is substantial. Competing proposals utilizing 16 transmembrane strands are in conflict with electrophysiological experimental observations and violate the constraints on such strands, such as no charged amino acids facing the phospholipid membrane and sufficient number of residues to span the membrane.     

Solubilization and reconstitution of the mitochondrial peptide-sensitive channel

In addition to the voltage-dependent anion channel (VDAC), mitochondrial outer membranes contain a cationic channel of large conductance, which is blocked by a mitochondrial addressing peptide (peptide-sensitive channel, PSC). Bovine adrenal cortex mitochondria were solubilized in 1.5% octyl -glucoside, and membrane vesicles were reconstituted by slow dilution with a low ionic strength buffer. The reconstituted vesicles contained a functional channel possessing the electrical characteristics of the cationic channel, including its sensitivity to the mitochondrial addressing peptide. Important features of the described protocol are the nature of the detergent, its concentration, and the addition of glycerol during the whole procedure. No solubilization could be observed in the presence of cholate.     

Detection of likely transmembrane β-strand regions in sequences of mitochondrial pore proteins using the Gibbs sampler

The mitochondrial channel VDAC is presumed to fold as a -barrel although the number and identity of transmembrane -strands in the protein are controversial. Previously, a novel multiple alignment algorithm called the Gibbs sampler was used to detect a residue-frequency motif in sequences of bacterial outer-membrane proteins that corresponds to transmembrane -strands in bacterial porins of known structure (Neuwaldet al., 1995,Protein Science,4, 1618. In the present study, this bacterial motif has been used to screen sets of mitochondrial membrane protein sequences, with matches occurring in only two classes of proteins: VDACs and the outer-membrane protein import pore (ISP42, MOM38). These results suggest a structural (and perhaps evolutionary) relatedness between the bacterial and mitochondrial pore proteins, with the mitochondrial subsequences that match the bacterial motif corresponding to transmembrane -strands as in the porins.     

Extramitochondrial Porin: Facts and Hypotheses

Mitochondrial porin, or VDAC, is a pore-forming protein abundant in the outer mitochondrialmembrane. Several publications have reported extramitochondrial localizations as well, butthe evidence was considered insufficient by many, and the presence of porin in nonmitochondrialcellular compartments has remained in doubt for a long time. We have now obtained newdata indicating that the plasma membrane of hematopoietic cells contains porin, probablylocated mostly in caveolae or caveolae-like domains. Porin was purified from the plasmamembrane of intact cells by a procedure utilizing the membrane-impermeable labeling reagentNH-SS-biotin and streptavidin affinity chromatography, and shown to have the same propertiesas mitochondrial porin. A channel with properties similar to that of isolated VDAC wasobserved by patch-clamping intact cells. This review discusses the evidence supportingextramitochondrial localization, the putative identification of the plasma membrane porin with themaxi chloride channel, the hypothetical mechanisms of sorting porin to various cellularmembrane structures, and its possible functions.     

Biogenesis of mitochondrialc-type cytochromes

Cytochromesc andc ₁ are essential components of the mitochondrial respiratory chain. In both cytochromes the heme group is covalently linked to the polypeptide chain via thioether bridges. The location of the two cytochromes is in the intermembrane space; cytochromec is loosely attached to the surface of the inner mitochondrial membrane, whereas cytochromec ₁ is firmly anchored to the inner membrane. Both cytochromec andc ₁ are encoded by nuclear genes, translated on cytoplasmic ribosomes, and are transported into the mitochondria where they become covalently modified and assembled. Despite the many similarities, the import pathways of cytochromec andc ₁ are drastically different. Cytochromec ₁ is made as a precursor with a complex bipartite presequence. In a first step the precursor is directed across outer and inner membranes to the matrix compartment of the mitochondria where cleavage of the first part of the presequence takes place. In a following step the intermediate-size form is redirected across the inner membrane; heme addition then occurs on the surface of the inner membrane followed by the second processing reaction. The import pathway of cytochromec is exceptional in practically all aspects, in comparison with the general import pathway into mitochondria. Cytochromec is synthesized as apocytochromec without any additional sequence. It is translocated selectively across the outer membrane. Addition of the heme group, catalyzed by cytochromec heme lyase, is a requirement for transport. In summary, cytochromec ₁ import appears to follow a conservative pathway reflecting features of cytochromec ₁ sorting in prokaryotic cells. In contrast, cytochromec has invented a rather unique pathway which is essentially non-conservative.     

Oriented channel insertion reveals the motion of a transmembrane beta strand during voltage gating of VDAC

Yeast VDAC channels (isolated from the mitochondrial outer membrane) form large aqueous pores whose walls are believed to consist of 1 a helix and 12 strands. Each channel has two voltage-gating processes: one closes the channels at positive potentials, the other at negative. When VDAC is reconstituted into phospholipid (soybean) membranes, the two gating processes have virtually the same steepness of voltage dependence and the same midpoint voltage. Substituting lysine for glutamate at either end of one putative strand (E145K or E152K) made the channels behave asymmetrically, increasing the voltage dependence of one gating process but not the other. The asymmetry was the same whether 1 or 100 channels were in the membrane, indicating oriented channel insertion. However, the direction of insertion varied from membrane to membrane, indicating that the insertion of the first channel was random and subsequent insertions were directed by the previously inserted channel (s). This raises the prospect of an auto-directed insertion with possible implications to protein targeting in cells. Each of the mutations affected a different gating process because the double mutant increased voltage dependence of both processes. Thus this strand may slide through the membrane in one direction or the other depending on the gating process. We propose that the model of folding for VDAC be altered to move this strand into the sensor region of the protein where it may act as a tether and guide/restrict the motion of the sensor.This work was supported by grants from the Office of Naval Research (N00014-90-J-1024) and the National Institutes of Health (GM 35759). Present address: Department of Physiology, 6811 Med. Sciences Bldg 2, University of Michigan, Ann Arbor, MI 48109 Present address: Department of Physiology, K.U. Leuven Medical School, Gasthuijsberg, 3000 Leuven, Belgium     

Mitochondrial protein import

Most polypeptides of mitochondria are imported from the cytosol. Precursor proteins contain targeting and sorting information, often in the form of amino-terminal presequences. Precursors first bind to receptors in the outer membrane. Two putative import receptors have been identified: a 19-kilodalton protein (MOM19) inNeurospora mitochondria, and a 70-kilodalton protein (MAS70) in yeast. Some precursors integrate directly into the outer membrane, but the majority are translocated through one or both membranes. This process requires an electrochemical potential across the inner membrane. Import appears to occur through a hydrophilic pore, although the inner and outer membranes may contain functionally separate translocation machineries. In yeast, a 42-kilodalton protein (ISP42) probably forms part of the outer membrane channel. After import, precursors interact with chaperonin ATPases in the matrix. Presequences then are removed by the matrix protease. Finally, some proteins are retranslocated across the inner membrane to the intermembrane space.     

Transmembrane arrangement of mitochondrial porin or voltage-dependent anion channel (VDAC)

Porin or voltage-dependent anion-selective channel (VDAC) is the main protein responsible for the high permeability of the outer mitochondrial membrane. The mitochondrial porin is mainly composed of sided -strands, in analogy with bacterial porin, whose structure has been resolved at 1.8 Å resolution. In mitochondrial porins the N-terminal region forms an amphipathic -helix, a structure conserved in organisms very distant from the evolutionary point of view. This part of the protein is exposed to the water phase, as demonstrated by ELISA assays. Various extramembranous loops have been identified by specific proteolytic cleavages. These overall, combined results were used to draw a model of the transmembrane arrangement of mammalian porin. This model is compared to other mitochondrial and bacterial porin models.     

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.     

Probing the structure of the mitochondrial channel,VDAC, by site-directed mutagenesis: A progress report

The voltage-dependent anion-selective channel (VDAC) of the mitochondrial outer membrane is formed by a small ( 30 kDa) polypeptide, but shares with more complex channels the properties of voltage-dependent gating and ion selectivity. Thus, it is a useful model for studying these properties. The molecular biology techniques available in yeast allow us to construct mutant versions of the cloned yeast VDAC genein vitro, using oligonucleotide-directed mutagenesis, and to express the mutant genes in yeast cells in the absence of wild-type VDAC. We find that one substitution mutation (lys 61 to glu) alters the selectivity of VDAC.     

Intracellular localization of VDAC proteins in plants

Voltage-dependent anion channels (VDACs) are porin-type -barrel diffusion pores. They are prominent in the outer membrane of mitochondria and facilitate metabolite exchange between the organelle and the cytosol. Here we studied the subcellular distribution of a plant VDAC-like protein between plastids and mitochondria in green and non-green tissue. Using in vitro studies of dual-import into mitochondria and chloroplasts as well as transient expression of fluorescence-labeled polypeptides, it could be clearly demonstrated that this VDAC isoform targets exclusively to mitochondria and not to plastids. Our results support the idea that plastids evolved a concept of solute exchange with the cytosol different from that of mitochondria.Abbreviations AOX Alternative oxidase - p Precursor form - POM36 Putative outer mitochondrial membrane proteins of 36 kDa - SSU Small subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) - VDAC Voltage-dependent anion channel     

Computational Observation of an Ion Permeation Through a Channel Protein

The ion permeation process, driven by a membrane potential through an outer membrane protein, OmpF porin of Escherichia coli, was simulated by molecular dynamics. A Na⁺ ion, initially placed in the solvent region at the outer side of the porin channel, moved along the electric field passing through the porin channel in a 1.3 nsec simulation; the permeation rate was consistent with the experimentally estimated channel activity (10⁸10⁹/sec). In this simulation, it was indicated that the ion permeation through the porin channel proceeds by a push-out mechanism, and that Asp113 is an important residue for the channel activity.     

Water movement during channel opening and closing

By applying the osmotic stress of a nonpenetrating polymer, we have measured the change v in polymer-inaccessible internal water volume of a voltagegated ionic channel. The voltage-dependent anion channel (VDAC) from mitochondrial outer membranes shows a v comparable in magnitude to the full channel volume estimated from solute penetrability, single-channel conductance, or image reconstruction. It thus appears that channel gating involves significant structure reorganization and water movement rather than the minimal changes caused by a local constriction or blockade. Hydration of the inner channel surface may be an important factor in channel gating as is the hydration of molecular surfaces in controlling macromolecular interaction in solution.     

The precursor of the F1β subunit of the ATP synthase is covalently modified upon binding to plant mitochondria

We present evidence for a unique covalent modification of a nuclear-encoded precursor protein targeted to plant mitochondria. We investigated the early events of in vitro import for the mitochondrial precursor of the ATP synthase F₁ subunit from Nicotiana plumbaginifolia (pF₁) into plant mitochondria. When pF₁ of 59 kDa was incubated with mitochondria isolated from different higher-plant species, a band of 61 kDa was generated. The 61 kDa protein was a covalently modified form of the 59 kDa pF₁. The modification was dependent on the 25 amino acid long N-terminal region of the presequence of pF₁. The modification was catalysed by an enzyme located in the outer mitochondrial membrane which was specific for higher plants and could not be washed off from the membrane by urea, KCl or EDTA. The modification was ATP- and Ca²⁺-dependent, but it was not affected by inhibitors of protein kinases. No inhibition of the modification was observed with phosphatase, methylation or acylation inhibitors. The modification occurs prior to translocation through the mitochondrial outer membrane. Inhibition of the modification process does not affect the import of the precursor protein, hence precursor modification was not a prerequisite for import. Both the modified and the unmodified pF₁ proteins were strongly associated with the mitochondrial outer membrane.     

A method for the investigation of those transformations under which the visual recognition of a given object is invariant

An experiment is described which shows in operation the program set out in Foster (1972a) for the investigation of the invariance transformations of visual recognition. The concern in the present study is with the Lie group of rotations SO(2), and a certain centrally located foveal Landolt ring. By presenting to the visual system this Landolt ring and a rotated image in rapid succession, one attempted to induce a specified rotation-type phi-motion. Two subjects were employed. Both reported the existence of the required type of phi-motion for rotations ₀ of the Landolt ring about the visual axis with -2/72/7. By appealing to the basic Proposition 2 of Foster (1972 a), the conclusion is reached that the visual system appears capable of effecting upon a certain centrally located foveal annulus the local 1-parameter group of rotations about the visual axis ₀, [–2/7,2/7].     

Ultrastructure of a cave-wall cyanophyte-Gloeocapsa NS4

Gloeocapsa strain NS4, a cyanophyte (cyanobacterium) which grows in low light levels inside cave entrances, was studied in the electron microscope by thin sectioning and freeze-etching. The cells are surrounded by a microfibrillar sheath divided by dense lamellae, which are probably an acidic mucopolysaccharide. Inside this is a typical Gramnegative cell wall. Double-replica freeze-fracture showed that the outer envelope of the wall fractures to give two faces each consisting of densely-packed particles; the particles of the outer leaflet seem to consist of subunits arranged in a hollow cylinder. A structural model of the outer envelope is proposed. The plasma membrane fractures to give a PF face with 3000 9 nm particles m^-1 and an EF face with 150–700 11–12 nm particles m^-1. The thylakoids are arranged in a pattern not previously found in a unicellular cyanophyte, parallel arrays which intersect, and may fuse with, the plasma membrane. The thylakoid membranes have 2,850 particles m^-1, mean size 10.9 nm, on the PF face and 560 particles m^-1, mean size 12.3 nm, on the EF face. Phycobilisomes are difficult to see, but may be unusually large. These ultrastructural features may be adaptations to a very low light habitat.     

Existence and uniqueness of a sharp travelling wave in degenerate non-linear diffusion Fisher-KPP equations

In this paper we use a dynamical systems approach to prove the existence of a unique critical value c ^* of the speed c for which the degenerate density-dependent diffusion equation u _ct = [D(u)u _x ] _x + g(u) has: 1. no travelling wave solutions for 0 < c < c ^*, 2. a travelling wave solution u(x, t) = (x - c ^* t) of sharp type satisfying (– ) = 1, () = 0 ^*; '(^*–) = – c ^*/D'(0), '(^*+) = 0 and 3. a continuum of travelling wave solutions of monotone decreasing front type for each c > c ^*. These fronts satisfy the boundary conditions (– ) = 1, '(– ) = (+ ) = '(+ ) = 0. We illustrate our analytical results with some numerical solutions.     

Morphology of freeze-etchedTreponema refringens (Nichols)

The freeze-etch technique was used to study the morphology ofTreponema refringens (Nichols). There is a single band of cytoplasmic fibrils which follows a path in the form of a right-handed helix with a periodicity of 1500 nm around the body of the treponeme just below the cytoplasmic membrane. There are two major fracture planes, one located in the interior of the outer envelope and the second in the interior of the cytoplasmic membrane. The blebs or surface protuberances, which are quite prominent in negativestained preparations, were not evident with freeze-etch preparation, indicating they are not a part of the normal structure of this organism. The outer envelope in untreated cells was observed to closely fit the body of the treponeme, whereas the outer envelope of glutaraldehyde-treated cells had a loose, wrinkled appearance. Thus the loose-fitting outer envelope generally described for treponemes is most likely an artifact of preparation for negative-staining and thinsectioning.     

Hexagonally ordered “rosettes” of particles in the plasma membrane ofMicrasterias denticulata Bréb. and their significance for microfibril formation and orientation

Summary Cells ofMicrasterias denticulata Bréb. at the stage of secondary wall formation have been studied by freeze-etching. It was found that the plasma membrane exhibits oval areas in which arrays of membrane particles occur. These particles form rosettes which are arranged in a hexagonally ordered lattice with a center to center spacing of 25 nm. Nearly the same periodicities can be found between microfibrils. It is concluded that the rosettes probably together with the thickened area of the plasma membrane below them represent the apparatus for the production and orientation of microfibrils. The hypothesis suggesting the incorporation of membrane templates functional in microfibril formation, originally advanced byKiermayer andDobberstein (1973) has received further support.