Supramolecular assembly of VDAC in native mitochondrial outer membranes

The voltage-dependent anion channel (VDAC) is the most abundant protein in the mitochondrial outer membrane (MOM). Due to its localization, VDAC is involved in a wide range of processes, such as passage of ATP out of mitochondria, and particularly plays a central role in apoptosis. Importantly, the assembly of VDAC provides interaction with a wide range of proteins, some implying oligomerization. However, many questions remain as to the VDAC structure, its supramolecular assembly, packing density, and oligomerization in the MOM is unknown. Here we report the so far highest resolution view of VDAC and its native supramolecular assembly. We have studied yeast MOM by high-resolution atomic force microscopy (AFM) in physiological buffer and found VDAC in two distinct types of membrane domains. We found regions where VDAC was packed at high density (approximately 80%), rendering the membrane a voltage-dependent molecular sieve. In other domains, VDAC has a low surface density (approximately 20%) and the pore assembly ranges from single molecules to groups of up to 20. We assume that these groups are mobile in the lipid bilayer and allow association and dissociation with the large assemblies. VDAC has no preferred oligomeric state and no long-range order was observed in densely packed domains. High-resolution topographs show an eye-shaped VDAC with 3.8 nm x 2.7 nm pore dimensions. Based on the observed VDAC structure and the pair correlation function (PCF) analysis of the domain architectures, we propose a simple model that could explain the phase behavior of VDAC, and illustrates the sensitivity of the molecular organization to conditions in the cell, and the possibility for modulation of its assembly. The implication of VDAC in cytochrome c release from the mitochondria during cell apoptosis has made it a target in cancer research.     

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.     

Functional Characterization of the Conserved “GLK” Motif in Mitochondrial Porin from Neurospora crassa

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 [-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 -barrel that is not directly involved in ATP binding.     

Regulation of the mitochondrial outer membrane channel,VDAC

The channel-forming protein, VDAC, located in the mitochondrial outer membrane, is probably responsible for the high permeability of the outer membrane to small molecules. The ability to regulate this channelin vitro raises the possibility that VDAC may perform a regulatory rolein vivo. VDAC exists in multiple, quasi-degenerate conformations with different permeability properties. Therefore a modest input of energy can change VDAC's conformation. The ability to use a membrane potential to convert VDAC from a high (open) to a low (closed) conducting form indicates the presence of a sensor in the protein that allows it to respond to the electric field. Titration and modification experiments point to a polyvalent, positively charged sensor. Soluble, polyvalent anions such as dextran sulfate and Konig's polyanion seem to be able to interact with the sensor to induce channel closure. Thus there are multiple ways of applying a force on the sensor so as to induce a conformational change in VDAC. Perhaps cells use one or more of these methods.     

Voltage gating in VDAC is markedly inhibited by micromolar quantities of aluminum

Summary The mitochondrial outer membrane contains voltagegated channels called VDAC that are responsible for the flux of metabolic substrates and metal ions across this membrane. The addition of micromolar quantities of aluminum chloride to phospholipid membranes containing VDAC channels greatly inhibits the voltage dependence of the channels' permeability. The channels remain in their high conducting (open) state even at high membrane potentials. An analysis of the change in the voltage-dependence parameters revealed that the steepness of the voltage dependence decreased while the voltage needed to close half the channels increased. The energy difference between the open and closed states in the absence of an applied potential did not change. Therefore, the results are consistent with aluminum neutralizing the voltage sensor of the channel. pH shift experiments showed that positively charged aluminum species in solution were not involved. The active form was identified as being either (or both) the aluminum hydroxide or the tetrahydroxoaluminate form. Both of these could reasonably be expected to neutralize a positively charged voltage sensor. Aluminum had no detectable effect of either single-channel conductance or selectivity, indicating that the sensor is probably not located in the channel proper and is distinct from the selectivity filter.     

High-conductance pathways in mitochondrial membranes

The outer and inner membranes of mitochondria have recently been studied with the patch clamp technique. What has emerged is still an ill-defined picture for either membrane, primarily for the wide range of conductances found. Interestingly, however, a few conductances (in the range of 10–80 pS) seem to be ubiquitously distributed. Parallel studiesin situ and in reconstituted systems have allowed the assignment to distinct membrane locations of some conductances, whose physiological role is, however, not yet elucidated.     

A novel stimulator of protein phosphorylation in Neurospora crassa

Summary An endogenous thermostable activator of Protein kinase III (PKIII) was purified from 100000 × g supernatants of Neurospora crassa mycelial extracts. This 38 000 dalton polypeptide, clearly separable from calmodulin on P-60 gel filtration, specifically stimulated N. crassa PKIII activity on casein or phosvitin in vitro phosphorylation.The factor was only present in the initial growth phase of the fungus. The mechanism of PKIII activation and its possible regulatory role are discussed.Abbreviations PK protein kinase - MES 2-N-Morpholino ethane-sulfonic acid - PMSF phenylmethylsulfonyl fluoride - S₁₀₀ 100000 × g Supernatant     

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.     

Behaviour of DNA-induced inositol-independent transformants of Neurospora crassa in sexual crosses

Summary Treatment of inositolless (inl) strains of Neurospora crassa with DNA from the wild type (allo-DNA) gives rise to inositol-independent (inl ⁺) colonies. Some of these DNA-induced inl ⁺ strains (transformants) are sterile in sexual crosses on minimal medium that selects for the maintaining of the inl ⁺ character. The same inl ⁺ transformants, when crossed with an inl standard strain, are fertile on complete (inositol-containing) medium. There are, however, an increased number of unusual non-Mendelian tetrads (24%) among the progeny. The inl ⁺ and inl progeny from these complete non-Mendelian tetrads were further examined for the inheritance of the inl ⁺ trait. Several inl ⁺ progeny of these tetrads segregate inl conidia if growing on inositol-containing medium. The number of inl ⁺ conidia in certain inl ⁺ cultures decreases quickly under non-selective conditions. In transformants carrying mutant markers in linkage groups III, IV and VI non-Mendelian segregation of these traits can also be detected.The mechanism of the development of sterility and of the aberrant segregation is discussed.     

Cobalt transport in a cobalt-resistant strain ofNeurospora crassa

Uptake of Co²⁺ by cobalt-resistant strain is dependent on Co²⁺ concentration in the medium and is linear with time. The uptake is unaffected by metabolic inhibitors and decreased at low pH values. The uptake is independent of temperature in the range 0–40‡ C. The transport system is a passive diffusion process, unlike in the parent wild type strain where it is energy-dependent. It is possible that Mg²⁺ transport system is not involved in Co²⁺ transport in this strain, since the Co²⁺ uptake is not suppressed by Mg²⁺ as in the parent strain.     

Carbon starvation mediated changes in carbohydrate metabolism inNeurospora crassa

Carbon starvation conditions were found to increase the activities of gluconeogenic enzymes such as malic enzyme, cytosolic malate dehydrogenase and isocitrate lyase along with proteases and inhibition in glucose catabolic enzymes such as G6P dehydrogenase and FDP aldolase inNeurospora crassa     

Heat-induced changes in respiratory pathways and mitochondrial structure during microcycle conidiation of Neurospora crassa

Changes in both respiratory pathways and mitochondrial structure of Neurospora crassa occurred under conditions of microcycle conidiation. Upon heat-treatment at 46°C, conidia developed a highly cyanide-insensitive, hydroxamate-sensitive respiration associated with morphological alterations in mitochondrial membranes; such changes were time-dependent. When heat-treated conidia were shifted down to 25°C, the alternate, hydroxamate-sensitive respiration decreased significantly, paralleling the recovery of well-cristated mitochondria with an electron-dense matrix in the germ tubes. The decrease in hydroxamate-sensitivity was associated with two periods of increase in cyanide sensitivity corresponding to the events of germination and precocious proconidial budding.     

Stress factor-induced changes in the activity of antioxidant protective mechanisms in the wild type strain of <Emphasis Type="Italic">Neurospora crassa</Emphasis> and in its photoreceptor complex mutants

The effect of stress factors (changes in oxygen content, temperature, and illumination) on superoxide dismutase (SOD) and catalase activity, as well as on the content of thiol and disulfide groups, in low-molecular-weight compounds and proteins of Neurospora crassa mycelium was studied in the wild type strain and white collar-(wc-1) and white collar-2 (wc-2) mutants. Environmental stress factors induced the activation of both SOD and catalase, as well as an increase in the thiol level in the wild type strain of Neurospora crassa. In the wc-1 and wc-2 mutants, an increase in catalase activity and in the total thiol level was revealed; however, activation of superoxide dismutase was not observed. A decrease in the formation of disulfide bonds in the proteins of wc-1 and wc-2 mutants (as compared with the wild type strain) was recorded. These results indicate disrupted transduction of stress factor signals that promotes reactive oxygen species (ROS) formation in the WCC mutants.     

The circadian conidiation rhythm inNeurospora crassa

The filamentous fungusNeurospora crassais one of the best organisms for analysing the molecular basis of the circadian rhythm observed in asexual spore formation, conidiation. Many clock mutants in which the circadian conidiation rhythm has different characteristics compared to those in the wild-type strain have been isolated since the early 1970s. With the cloning of one of these clock genes,frq, the molecular basis of the circadian clock inNeurosporahas become gradually clearer. Physiological and pharmacological studies have also contributed to our understanding of the physiological basis of the circadian clock inNeurospora. These studies strongly indicate that the circadian clock is based on or is closely related to a network of metabolic processes for cellular activities. Based on these studies, it may be possible to isolate new types of clock mutants which should contribute to a better understanding of the molecular basis of the circadian clock inNeurospora.     

Excretion of pyruvate in nickel toxicity in wild type and Ni2+ resistant mutants ofNeurospora crassa

The parent wild strainNeurospora crassa Em 5297a and three Ni²⁺ resistantNeurospora crassa mutants have been shown to excrete pyruvate into the culture medium in Ni²⁺ and Co²⁺ toxicities. Ni²⁺ has a more pronounced effect in this regard. The excretion is progressive with growth inhibition and is abolished by Mg²⁺ in all strains and by Fe³⁺ partially in the Em strain but not inNeurospora crassa Ni^R1. Pyruvate, citrate and malate supplementation reverse growth inhibition caused by excess Ni²⁺, but with concomitant suppression of Ni²⁺ accumulation. It is suggested that one of the features of Ni²⁺ toxicity inNeurospora crassa is a derangement in carbohydrate metabolism at step(s) beyond pyruvate and that this is possibly due to decreased invivo activity of Mg²⁺ dependent processes     

Probing the orientation of yeast VDAC1 in vivo

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.