Effects of pH on structural and functional properties of porin from the outer membrane of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>. I. Functionally important conformational transitions of yersinin

The changes in the structural and functional properties of yersinin, a porin from the outer membrane of Yersinia pseudotuberculosis, were studied in the pH range 8.0–2.0 using SDs-PAGE, scanning microcalorimetry, optical spectroscopy and bilayer lipid membrane technique. It was found that in the pH range under study the changes in the spatial structure of yersinin were biphasic. In the first steps of pH titration (pH 8.0–4.5), porin underwent a series of conformational transitions, which did not affect the trimeric structure of its molecule. In the second step (pH 4.0–2.0), structural rearrangements led to dissociation of the protein trimers into monomers. It is noteworthy that complete unfolding of the polypeptide chain of the protein was not observed even at low values of pH. Thus, at pH 2.0 the conformational intermediate of the protein retained up to 50% of its regular secondary structure. Studies of current fluctuations in the bilayer lipid membrane revealed that in weakly acidic media the conductivity of yersinin pores was decreased by one order of magnitude. The most drastic changes in the conductivity of the model membrane were observed at pH 5.8, whereas a further decrease of pH to 5.0 resulted in the closure of porin channels. It was concluded that the observed changes in the pore-forming properties of yersinin in a narrow range of pH represent an early step in the adaptation of bacteria to the changing conditions of the environment and entail control over the biosynthesis of nonspecific porins. The pH-dependent changes in the structure and pore-forming properties of yersinin provide additional evidence in favor of conformational and functional plasticity of porins.     

Adaptation of Escherichia coli O157:H7 to pH Alters Membrane Lipid Composition, Verotoxin Secretion, and Resistance to Simulated Gastric Fluid Acid

The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37°C) was determined for Escherichia coli O157:H7 (HEC, ATCC 43895), an rpoS-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic E. coli (NPEC, ATCC 25922). Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min. Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased cis-vaccenic acid (C18:1ω7c) in the membrane lipids of all strains. The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity. HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 10⁸ CFU/ml. In addition, the ratio of extracellular to intracellular verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity. These results suggest that while the rpoS gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease verotoxin secretion.     

Probing the interaction between vesicular stomatitis virus and phosphatidylserine

The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion between the viral envelope and the endosomal membrane at the acidic environment of this compartment. In this work, we evaluated VSV interactions with membranes of different phospholipid compositions, at neutral and acidic pH, using atomic force microscopy (AFM) operating in the force spectroscopy mode, isothermal calorimetry (ITC) and molecular dynamics simulation. We found that the binding forces differed dramatically depending on the membrane phospholipid composition, revealing a high specificity of G protein binding to membranes containing phosphatidylserine (PS). In a previous work, we showed that the sequence corresponding amino acid 164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Here, we used this sequence to explore VSV–PS interaction using ITC. We found that peptide binding to membranes was exothermic, suggesting the participation of electrostatic interactions. Peptide–membrane interaction at pH 7.5 was shown to be specific to PS and dependent on the presence of His residues in the fusion peptide. The application of the simplified continuum Gouy–Chapman theory to our system predicted a pH of 5.0 at membrane surface, suggesting that the His residues should be protonated when located close to the membrane. Molecular dynamics simulations suggested that the peptide interacts with the lipid bilayer through its N-terminal residues, especially Val¹⁴⁵ and His¹⁴⁸. Fabiana A.Carneiro and Pedro A. Lapido-Loureiro contributed equally to this work An erratum to this article can be found at     

Characterization of the channel properties of tetanus toxin in planar lipid bilayers.

A detailed characterization of the properties of the channel formed by tetanus toxin in planar lipid bilayers is presented. Channel formation proceeds at neutral pH. However, an acidic pH is required to detect the presence of channels in the membrane rapidly and effectively. Acid pH markedly lowers the single-channel conductance, for phosphatidylserine at 0.5 M KCl gamma = 89 pS at pH 7.0 while at pH 4.8, gamma = 30 pS. The toxin channel is cation selective without significant selectivity between potassium and sodium (gamma [K+]/gamma [Na+] greater than or equal to 1.35). In all the lipids studied gamma is larger at positive than at negative voltages. The toxin channel is voltage dependent both at neutral and acidic pH: for phosphatidylserine membranes, the probability of the channel being open is much greater at positive than at negative voltage. In different phospholipids the channel exhibits different voltage dependence. In phosphatidylserine membranes the channel is inactivated at negative voltages, whereas in diphytanoylphosphatidylcholine membranes channels are more active at negative voltages than at positive. The presence of acidic phospholipids in the bilayers increases both the single-channel conductance as well as the probability of the channel being open at positive voltage. A subconductance state is readily identifiable in the single-channel recordings. Accordingly, single-channel conductance histograms are best fitted with a sum of 3 Gaussian distributions corresponding to the closed state, the open subconductance state and the full open state. Channel activity occurs in bursts of openings separated by long closings. Probability density analysis of the open dwell times of the toxin channel indicate the existence of a single open state with a lifetime greater than or equal to 1 ms in all lipids studied. Analysis of intra-bursts closing lifetimes reveals the existence of two components; the slow component is of the order of 1 ms, the fast one is less than or equal to 0.5 ms. The channel activity induced by tetanus toxin in lipid bilayers suggests a mechanism for its neurotoxicity: a voltage dependent, cation selective channel inserted in the postsynaptic membrane would lead to continuous depolarization and, therefore, persistent activation of the postsynaptic cell.     

Microbiological Survey of Adirondack Lakes with Various pH Values

Nine high-altitude oligotrophic Adirondack lakes in upstate New York having water of pH 4.3 to 7.0 were surveyed for total bacterial numbers and possible adaptation of the microbial communities to environmental pH. The number of heterotrophic bacteria from water samples recoverable on standard plate count agar were low (10¹ to 10³ per ml) for most of the lakes. Acridine orange direct counts were approximately two orders of magnitude higher than plate counts for each lake. Sediment aerobic heterotrophs recovered on standard plate count agar ranged from 1.4 × 10⁴ to 1.3 × 10⁶ per g of sediment. Direct epifluorescence counts of bacteria in sediment samples ranged from 3.0 × 10⁶ to 1.4 × 10⁷ per g. Low density values were consistent with the oligotrophic nature of all the lakes surveyed. There were no apparent differences in numbers of bacteria originally isolated at pH 5.0 and pH 7.0 between circumneutral lakes (pH > 6.0) and acidic lakes (pH < 5.0). Approximately 1,200 isolates were recultured over a range of pH from 3.0 to 7.0. Regardless of the original isolation pH (pH 5.0 or pH 7.0), less than 10% of the isolates grew at pH < 5.0. Those originally isolated at pH 5.0 also grew at pH 6.0 and 7.0. Those originally isolated at pH 7.0 preferred pH 7.0, with 98% able to grow at pH 6.0 and 44% able to grow at pH 5.0. A chi-square contingency test clearly showed (P < 0.005) that two distinct heterotrophic populations had been originally isolated at pH 5.0 and pH 7.0, although there is undoubtedly some overlap between the two populations.     

Erratum

Escherichia coli cells (unsaturated fatty acid auxotroph) have been adapted to grow on branched-chain fatty acids. Membrane vesicles were isolated from cells grown on a mixture of branched-chain fatty acids isolated from the lipids of Bacillus subtilis (E. coli (B. subtilis) membranes) and on a pure synthetic anti-isononadecanoic acid (E. coli (aC19) membranes).We have shown, using wide-angle X-ray diffraction and differential scanning calorimetry, that the ordered state of the lipids is perturbed in the case of E. coli (aC19) membranes. The perturbation leads to the presence of a large wide-angle X-ray diffraction at 4.25–4.3 Å, as opposed to the presence of a sharp 4.2 Å reflection in unperturbed systems.We have shown, using freeze-fracture electron microscopy, that a protein segregation exists in the case of E. coli (aC19) membranes (at low temperature the integral membrane proteins aggregate in the membrane domains containing the disordered lipids); we do not observe such segregation in the case of E. coli (B. subtilis) membranes. We conclude that in cases where the branching of the fatty acids introduces a perturbation of the lipid order, the integral membrane proteins can still be accommodated in membrane domains containing the ‘perturbed’ ordered lipids.Finally, we have determined the rate of β-galactoside transport in E. coli (aC19) and E. coli (B. subtilis) membranes as a function of temperature. We have shown that, in both cases, the Arrhenius representations display an increased slope in the region of the disorder-to-order transition. We conclude that such an increased slope may have different origins. In the case of E. coli (aC19) membranes, it is the result of the aggregation of the β-galactoside carriers together with other integral membrane proteins which may lead to the inactivation of the carriers; in the case of E. coli (B. subtilis) membranes, it is the result of the partial immobilisation of the carriers embedded in a lipid environment, of which the fluidity, despite the perturbation of its lipid order, is still much less than that associated with lipids in a totally disordered state.     

Membrane simulations mimicking acidic pH reveal increased thickness and negative curvature in a bilayer consisting of lysophosphatidylcholines and free fatty acids

Phospholipids are key components of biological membranes and their lipolysis with phospholipase A₂ (PLA₂) enzymes occurs in different cellular pH environments. Since no studies are available on the effect of pH on PLA₂-modified phospholipid membranes, we performed 50-ns atomistic molecular dynamics simulations at three different pH conditions (pH 9.0, 7.5, and 5.5) using a fully PLA₂-hydrolyzed phosphatidylcholine (PC) bilayer which consists solely of lysophosphatidylcholine and free fatty acid molecules. We found that a decrease in pH results in lateral squeezing of the membrane, i.e. in decreased surface area per headgroup. Thus, at the decreased pH, the lipid hydrocarbon chains had larger S_CD order parameter values, and also enhanced membrane thickness, as seen in the electron density profiles across the membrane. From the lateral pressure profiles, we found that the values of spontaneous curvature of the two opposing monolayers became negative when the pH was decreased. At low pH, protonation of the free fatty acid headgroups reduces their mutual repulsion and accounts for the pH dependence of all the above-mentioned properties. The altered structural characteristics may significantly affect the overall surface properties of biomembranes in cellular vesicles, lipid droplets, and plasma lipoproteins, play an important role in membrane fission and fusion, and modify interactions between membrane lipids and the proteins embedded within them.     

Adaptation of Escherichia coli O157:H7 to pH alters membrane lipid composition, verotoxin secretion, and resistance to simulated gastric fluid acid

The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37 degrees C) was determined for Escherichia coli O157:H7 (HEC, ATCC 43895), an rpoS-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic E. coli (NPEC, ATCC 25922). Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min. Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased cis-vaccenic acid (C18:1 omega 7c) in the membrane lipids of all strains. The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity. HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 10(8) CFU/ml. In addition, the ratio of extracellular to intracellular verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity. These results suggest that while the rpoS gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease verotoxin secretion.     

Lipid packing variations induced by pH in cardiolipin-containing bilayers: the driving force for the cristae-like shape instability

Cardiolipin is a four-tailed acidic lipid found predominantly within the inner membrane of mitochondria, and is thought to be a key component in determining inner membrane properties and potential. Thus, cardiolipin may be involved in the dynamics of the inner membrane characteristic invaginations (named cristae) that protrude into the matrix space. In previous studies, we showed the possibility to induce, by localized proton flow, a macroscopic cristae-like shape remodeling of an only-lipid model membrane mimicking the inner mitochondrial membrane. In addition, we reported a theoretical model describing the dynamics of a chemically driven membrane shape instability caused by a modification of the plane-shape equilibrium density of the lipids in the membrane. In the present work, we focus on the lipid-packing modifications observed in a model cardiolipin-containing lipid membrane submitted to pH decrease because this is the driving force of the instability. Laurdan fluorescence and ζ-potential measurements show that under pH decrease, membrane surface charge decreases, but that significant modification of the lipid packing is observed only for CL-containing membranes. Our giant unilamellar vesicle experiments also indicate that cristae-like morphologies are only observed for CL-containing lipid membranes. Taken together, these results highlight the fact that only a strong modulation of the lipid packing of the exposed monolayer leads to membrane shape instability and suggest that mitochondrial lipids, in particular the cardiolipin, play a specific role under pH modulation in inner mitochondrial membrane morphology and dynamics.     

Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane‐adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel‐fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large‐scale lipid phase separation and protein segregation in intact, protein‐crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.     

Membrane lipids and soluble sugars dynamics of the alkaliphilic fungus Sodiomyces tronii in response to ambient pH

Alkaliphily, the ability of an organism to thrive optimally at high ambient pH, has been well-documented in several lineages: archaea, bacteria and fungi. The molecular mechanics of such adaptation has been extensively addressed in alkaliphilic bacteria and alkalitolerant fungi. In this study, we consider an additional property that may have enabled fungi to prosper at alkaline pH: altered contents of membrane lipids and cytoprotectant molecules. In the alkaliphilic Sodiomyces tronii, we showed that at its optimal growth pH 9.2, the fungus accumulates abundant cytosolic trehalose (4–10% dry weight) and phosphatidic acids in the membrane lipids, properties not normally observed in neutrophilic species. At a very high pH 10.2, the major carbohydrate, glucose, was rapidly substituted by mannitol and arabitol. Conversely, lowering the pH to 5.4–7.0 had major implications both on the content of carbohydrates and membrane lipids. It was shown that trehalose dominated at pH 5.4. Fractions of sphingolipids and sterols of plasma membranes rapidly elevated possibly indicating the formation of membrane structures called rafts. Overall, our results reveals complex dynamics of the contents of membrane lipids and cytoplasmic sugars in alkaliphilic S. tronii, suggesting their adaptive functionality against pH stress.

     

pH-Induced Conformational Change of the β-Barrel-Forming Protein OmpG Reconstituted into Native E. coli Lipids

A gating mechanism of the β-barrel-forming outer membrane protein G (OmpG) from Escherichia coli was recently presented. The mechanism was based on X-ray structures revealed from crystals grown from solubilized OmpG at both neutral pH and acidic pH. To investigate whether these conformations represent the naturally occurring gating mechanism, we reconstituted OmpG in native E. coli lipids and applied high-resolution atomic force microscopy. The reconstituted OmpG molecules assembled into both monomers and dimers. Single monomeric and dimeric OmpG molecules showed open channel entrances at pH 7.5 and at room temperature. The extracellular loops connecting the β-strands that form the transmembrane β-barrel pore exhibited elevated structural flexibility. Upon lowering the pH to 5.0, the conformation of OmpG molecules changed to close the extracellular entrance of their channel. It appears that one or more of the extracellular loops collapsed onto the channel entrance. This conformational change was fully reversible. Our data confirm that the previously reported gating mechanism of OmpG occurs at physiological conditions in E. coli lipid membranes.     

Melatonin Alters Fluid Phase Coexistence in POPC/DPPC/Cholesterol Membranes

The structure and biophysical properties of lipid membranes are important for cellular functions in health and disease. In Alzheimer’s disease, the neuronal membrane is a target for toxic amyloid-β (Aβ). Melatonin is an important pineal gland hormone that has been shown to protect against Aβ toxicity in cellular and animal studies, but the molecular mechanism of this protection is not fully understood. Melatonin is a small membrane-active molecule that has been shown to interact with model lipid membranes and alter the membrane biophysical properties, such as membrane molecular order and dynamics. This effect of melatonin has been previously studied in simple model bilayers with one or two lipid components. To make it more relevant to neuronal membranes, we used a more complex ternary lipid mixture as our membrane model. In this study, we used ²H-NMR to investigate the effect of melatonin on the phase behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and cholesterol lipid membranes. We used deuterium-labeled POPC-d₃₁ and DPPC-d₆₂,separately to probe the changes in hydrocarbon chain order as a function of temperature and melatonin concentration. We find that POPC/DPPC/cholesterol at molar proportions of 3:3:2 is close to liquid-disordered/liquid-ordered phase separation and that melatonin can induce phase separation in these ternary mixtures by preferentially incorporating into the disordered phase and increasing its level of disorder. At 5 mol% melatonin, we observed phase separation in samples with POPC-d₃₁, but not with DPPC-d₆₂, whereas at 10 mol% melatonin, phase separation was observed in both samples with either POPC-d₃₁ or DPPC-d₆₂. These results indicate that melatonin can have a strong effect on membrane structure and physical properties, which may provide some clues to understanding how melatonin protects against Aβ, and that choice of chain perdeuteration is an important consideration from a technical point of view.     

FisB relies on homo-oligomerization and lipid binding to catalyze membrane fission in bacteria

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, fission protein B (FisB), is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here, we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of approximately 12 molecules that give way to an immobile cluster at the engulfment pole containing approximately 40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest that FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests that homo-oligomerization and trans-interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid binding, and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature sensing.

Little is known about how membrane fission occurs in bacteria; this study suggests that the membrane fission protein FisB exploits the unique cellular geometry encountered during sporulation to enable its localization to the fission site through a novel mechanism, where it catalyzes membrane scission.     

Permeability of Lipid Bilayer Membranes to Organic Solutes

A sensitive fluorescence technique was used to measure transport of organic solutes through lipid bilayer membranes and to relate permeability to the functional groups of the solute, lipid composition of the membrane, and pH of the medium. Indole derivatives having ethanol, acetate, or ethylamine in the 3-position, representing neutral, acidic, and basic solutes, respectively, were the primary models. The results show: (a) Neutral solute permeability is not greatly affected by changes in lipid composition but presence or absence of cholesterol in the membranes could greatly alter permeability of the dissociable substrates. (b) Indole acetate permeability was reduced by introduction of phosphatidylserine into membranes to produce a net negative charge on the membranes. (c) Permeability response of dissociable solutes to variation in pH was in the direction predicted but not always of the magnitude expected from changes in the calculated concentrations of the undissociated solute in the bulk aqueous phase. Concentration gradients of amines across the membranes caused substantial diffusion potentials, suggesting that some transport of the cationic form of the amine may occur. It is suggested that factors such as interfacial charge and hydration structure, interfacial polar forces, and lipid organization and viscosity, in addition to the expected solubility-diffusion relations, may influence solute flux.     

Binding of adenovirus and its external proteins to Triton X-114. Dependence on pH

35S-Labeled adenovirus type 2 (Ad2) (10 ng/ml) was incubated with 1% Triton X-114 at various pH values varying from 3.0 to 8.0. The detergent phase was separated from the aqueous phase by centrifugation, and the amounts of Ad2 were determined in the two phases. At pH 7.0-8.0, less than 5% of Ad2 was associated with the detergent phase; at pH 5.0 or below, about 60% of Ad2 was associated with the detergent phase. When a mixture of 35S-labeled capsid proteins was used at pH 7.0, 60-70% of the total proteins were associated with the detergent at pH 5.0, but less than 5% of the proteins interacted with detergent at pH 7.0. Among the three major external proteins (hexon, penton base, and fiber), penton base had the highest association with Triton X-114 at pH 5.0. Both intact virus and the capsid proteins that were associated with Triton X-114 at pH 5.0 were released into the aqueous phase on subsequent incubation at pH 7.0. On the basis of these results, it is suggested that mildly acidic pH induces amphiphilic properties in adenovirus capsid proteins and may help Ad2 escape from acidic endocytic vesicles.     

Partly folded states of bovine carbonic anhydrase interact with zwitterionic and anionic lipid membranes

The acidic, partly folded states of bovine carbonic anhydrase II (BCAII) were used as an experimental system to study the interactions of partly denatured proteins with lipid membranes. The pH dependence of their interactions with palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidylglycerol (POPG) membranes was studied. A filtration binding assay shows that acidic partly folded states of BCAII bind to POPC membranes. Fluorescence emission spectra from Trp residues of the bound protein are slightly shifted to shorter wavelength and can be quenched by a water-soluble quencher of fluorescence, indicating that the binding occurs without deep penetration of Trp residues into the membrane. The content of beta-structures of the protein in solution, as revealed by FT-IR spectroscopy, decreases in the partly folded states and the binding to POPC membrane occurs without further changes of secondary structure. In the presence of 0.1 M NaCl, a partly folded state self-aggregates and does not bind to POPC membrane. At acidic pH, BCAII binds to POPG membranes both at high and low ionic strength. The binding to the anionic lipid occurs with protein self-aggregation within the lipid-protein complexes and with changes in the secondary structure; large blue shifts in the fluorescence emission spectra and the decrease in the exposure to water-soluble acrylamide quencher of Trp fluorescence strongly suggest that BCAII penetrates the hydrocarbon domain in the POPG-protein complexes.     

pH-dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen

Protective antigen (PA) of anthrax toxin forms ion-conductive channels in planar lipid bilayers and liposomes under acidic pH conditions. We show here that PA has a similar permeabilizing action on the plasma membranes of CHO-K1 and three other mammalian cell lines (J774A.1, RAW264.7 and Vero). Changes in membrane permeability were evaluated by measuring the efflux of the K⁺ analogue, ⁸⁶Rb⁺, from prelabelled cells, and the influx of ²²Na⁺. The permeabilizing activity of PA was limited to a proteolytically activated form (PA_N) and was dependent on acidic pH for membrane insertion (optimal at pH 5.0), but not for sustained ion flux. The flux was reduced in the presence of several known channel blockers: tetrabutyl-, tetrapentyl-, and tetrahexylammonium bromides. PA_N facilitated the membrane translocation of anthrax edema factor under the same conditions that induced changes in membrane permeability to ions. These results indicate that PA_N permeabilizes cellular membranes under conditions that are believed to prevail in the endosomal compartment of toxin-sensitive cells; and they provide a basis for more detailed studies of the relationship between channel formation and translocation of toxin effector moieties in vivo.     

Novel organization and properties of annexin 2-membrane complexes

Annexin 2 belongs to the annexin family of proteins that bind to phospholipid membranes in a Ca(2+)-dependent manner. Here we show that, under mild acidic conditions, annexin 2 binds to and aggregates membranes containing anionic phospholipids, a fact that questions the mechanism of its interaction with membranes via Ca(2+) bridges only. The H(+) sensitivity of annexin 2-mediated aggregation is modulated by lipid composition (i.e. cholesterol content). Cryo-electron microscopy of aggregated liposomes revealed that both the monomeric and the tetrameric forms of the protein form bridges between the liposomes at acidic pH. Monomeric annexin 2 induced two different organizations of the membrane junctions. The first resembled that obtained at pH 7 in the presence of Ca(2+). For the tetramer, the arrangement was different. These bridges seemed more flexible than the Ca(2+)-mediated junctions allowing the invagination of membranes. Time-resolved fluorescence analysis at mild acidic pH and the measurement of Stokes radius revealed that the protein undergoes conformational changes similar to those induced by Ca(2+). Labeling with the lipophilic probe 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine indicated that the protein has access to the hydrophobic part of the membrane at both acidic pH in the absence of Ca(2+) and at neutral pH in the presence of Ca(2+). Models for the membrane interactions of annexin 2 at neutral pH in the presence of Ca(2+) and at acidic pH are discussed.     

Lipid Metabolism of Caddisfly Larvae at Low pH

The influence of low pH (5.0 and 4.0) on lipid metabolism of caddisfly larvae Hydropsyche contubernalis L. (Trichoptera) was studied in 48 h toxicity experiments. The results were correlated with lipid composition of caddisfly larvae directly isolated from natural water. Phospholipids, cholesterol, mono-, di-, triacylglycerols, and fatty acids were detected by thin-layer and liquid chromatography. Minimal environmental changes were shown to initiate the biochemical adaptation mechanisms strengthening the cellular membranes through their condensation due to additional phospholipid and cholesterol synthesis. In the natural medium the adaptation processes are more active than in the artificial medium. More serious changes, such as pH decrease to 4.0, suppress the adaptation processes in the first medium and terminate them in the second one.