Animal Cell Membranes Research Articles

Regions conferring isoform-specific function in the catalytic subunit of the Na,K-pump

The Na,K-pump (i.e., Na,K-ATPase) is critical for maintaining the ionic gradients across the plasma membranes of animal cells. Its component subunits are expressed in multiple forms, but the physiological relevance of this subunit diversity remains unknown. The primary contributor to overall catalysis, the alpha subunit, exists in four isoforms. There are observed kinetic differences among these isoforms, but their subtlety makes them an unlikely basis for physiological significance. Instead, recent work suggests that the major functional distinction among the isoforms is their interaction with regulatory proteins. Moreover, the isoform-specific effects of modulatory agents such as protein kinase C seem to originate within two regions of structural divergence: the amino terminus and eleven residues near the center of the alpha subunit, the isoform-specific region.

Structure-Activity Relationships of Diastereomeric Lysine Ring Size Analogs of the Antimicrobial Peptide Gramicidin S: MECHANISM OF ACTION AND DISCRIMINATION BETWEEN BACTERIAL AND ANIMAL CELL MEMBRANES

Structure-activity relationships were examined in seven gramicidin S analogs in which the ring-expanded analog GS14 [cyclo-(VKLKVdYPLKVKLdYP)] is modified by enantiomeric inversions of its lysine residues. The conformation, amphiphilicity, and self-association propensity of these peptides were investigated by circular dichroism spectroscopy and reversed phase high performance liquid chromatography. (31)P nuclear magnetic resonance spectroscopic and dye leakage experiments were performed to evaluate the capacity of these peptides to induce inverse nonlamellar phases in, and to permeabilize phospholipid bilayers; their growth inhibitory activity against the cell wall-less mollicute Acholeplasma laidlawii B was also examined. The amount and stability of beta-sheet structure, effective hydrophobicity, propensity for self-association in water, ability to disrupt the organization of phospholipid bilayers, and ability to inhibit A. laidlawii B growth are strongly correlated with the facial amphiphilicity of these GS14 analogs. Also, the magnitude of the parameters segregate these peptides into three groups, consisting of GS14, the four single inversion analogs, and the two multiple inversion analogs. The capacity of these peptides to differentiate between bacterial and animal cell membranes exhibits a biphasic relationship with peptide amphiphilicity, suggesting that there may only be a narrow range of peptide amphiphilicity within which it is possible to achieve the dual therapeutic requirements of high antibiotic effectiveness and low hemolytic activity. These results were rationalized by considering how the physiochemical properties of these GS14 analogs are likely to be reflected in their partitioning into lipid bilayer membranes.

Reminiscences of Leon A. Heppel

Reminiscences of Leon A. Heppel

The complex life of simple sphingolipids

The extensive diversity of membrane lipids is rarely appreciated by cell and molecular biologists. Although most researchers are familiar with the three main classes of lipids in animal cell membranes, few realize the enormous combinatorial structural diversity that exists within each lipid class, a diversity that enables functional specialization of lipids. In this brief review, we focus on one class of membrane lipids, the sphingolipids, which until not long ago were thought by many to be little more than structural components of biological membranes. Recent studies have placed sphingolipids-including ceramide, sphingosine and sphingosine-1-phosphate-at the centre of a number of important biological processes, specifically in signal transduction pathways, in which their levels change in a highly regulated temporal and spatial manner. We outline exciting progress in the biochemistry and cell biology of sphingolipids and focus on their functional diversity. This should set the conceptual and experimental framework that will eventually lead to a fully integrated and comprehensive model of the functions of specific sphingolipids in regulating defined aspects of cell physiology.

Lipid Rafts in Higher Plant Cells: PURIFICATION AND CHARACTERIZATION OF TRITON X-100-INSOLUBLE MICRODOMAINS FROM TOBACCO PLASMA MEMBRANE

A large body of evidence from the past decade supports the existence of functional microdomains in membranes of animal and yeast cells, which play important roles in protein sorting, signal transduction, or infection by pathogens. They are based on the dynamic clustering of sphingolipids and cholesterol or ergosterol and are characterized by their insolubility, at low temperature, in nonionic detergents. Here we show that similar microdomains also exist in plant plasma membrane isolated from both tobacco leaves and BY2 cells. Tobacco lipid rafts were found to be greatly enriched in a sphingolipid, identified as glycosylceramide, as well as in a mixture of stigmasterol, sitosterol, 24-methylcholesterol, and cholesterol. Phospho- and glycoglycerolipids of the plasma membrane were largely excluded from lipid rafts. Membrane proteins were separated by one- and two-dimensional gel electrophoresis and identified by tandem mass spectrometry or use of specific antibody. The data clearly indicate that tobacco microdomains are able to recruit a specific set of the plasma membrane proteins and exclude others. We demonstrate the recruitment of the NADPH oxidase after elicitation by cryptogein and the presence of the small G protein NtRac5, a negative regulator of NADPH oxidase, in lipid rafts.

The SLC36 family: proton-coupled transporters for the absorption of selected amino acids from extracellular and intracellular proteolysis.

Whilst Na(+) has replaced H(+) as a major transport driving force at the plasma membrane of animal cells, the evolutionarily older H(+)-driven systems persist on endomembranes and at the plasma membrane of specialized cells. The first member of the SLC36 family, present in both intracellular and plasma membranes, was identified independently as a lysosomal amino acid transporter (LYAAT1) responsible for the export of lysosomal proteolysis products into the cytosol and as a proton/amino acid transporter (PAT1) responsible for the absorption of amino acids in the gut. In addition to LYAAT1/PAT1, the family comprises another characterized member, PAT2, and two orphan transporters. Both PAT1 and PAT2 mediate 1:1 symport of protons and small neutral amino acids such as glycine, alanine, and proline. Their mRNAs are broadly and differentially expressed in mammalian tissues. The PAT1 protein localizes to lysosomes in brain neurons, but is also found in the apical membrane of intestinal epithelial cells with a role in the absorption of amino acids from luminal protein digestion. In both cases, protons supplied by the lysosomal H(+)-ATPase or by the acidic microclimate of the brush border membrane drive transport of the amino acids into the cytosol. The subcellular localization and physiological role of PAT2 have still to be determined. SLC36 transporters are related distantly to other proton-coupled amino acid transporters, such as the vesicular neurotransmitter transporter VIAAT/VGAT (SLC32) and system N transporters (SLC38 family).

Liquid-liquid immiscibility in membranes.

The observation of liquid-liquid immiscibility in cholesterol-phospholipid mixtures in monolayers and bilayers has opened a broad field of research into their physical chemistry. Some mixtures exhibit multiple immiscibilities. This unusual property has led to a thermodynamic model of "condensed complexes." These complexes are the consequence of an exothermic, reversible reaction between cholesterol and phospholipids. In this quantitative model the complexes are sometimes concentrated in a separate liquid phase. The phase separation into a complex-rich phase depends on membrane composition and intensive variables such as temperature. The properties of defined cholesterol-phospholipid mixtures provide a conceptual foundation for the exploration of a number of aspects of the biophysics and biochemistry of animal cell membranes.

Glutamine 132 in the NAD(H)-binding component of proton-translocating transhydrogenase tethers the nucleotides before hydride transfer.

Transhydrogenase, found in bacterial membranes and inner mitochondrial membranes of animal cells, couples the redox reaction between NAD(H) and NADP(H) to proton translocation. In this work, the invariant Gln132 in the NAD(H)-binding component (dI) of the Rhodospirillum rubrum transhydrogenase was substituted with Asn (to give dI.Q132N). Mixtures of the mutant protein and the NADP(H)-binding component (dIII) of the enzyme readily produced an asymmetric complex, (dI.Q132N)(2)dIII(1). The X-ray structure of the complex revealed specific changes in the interaction between bound nicotinamide nucleotides and the protein at the hydride transfer site. The first-order rate constant of the redox reaction between nucleotides bound to (dI.Q132N)(2)dIII(1) was <1% of that for the wild-type complex, and the deuterium isotope effect was significantly decreased. The nucleotide binding properties of the dI component in the complex were asymmetrically affected by the Gln-to-Asn mutation. In intact, membrane-bound transhydrogenase, the substitution completely abolished all catalytic activity. The results suggest that Gln132 in the wild-type enzyme behaves as a "tether" or a "tie" in the mutual positioning of the (dihydro)nicotinamide rings of NAD(H) and NADP(H) for hydride transfer during the conformational changes that are coupled to the translocation of protons across the membrane. This ensures that hydride transfer is properly gated and does not take place in the absence of proton translocation.

From a pump to a pore: how palytoxin opens the gates.

The ion channel field has benefited enormously from a plethora of natural toxins that target specific ion channels (1–13), and those toxins continue to be key molecular tools in the postgenomics era. The ion transporter field has not been so lucky, in general. Its arsenal of specific toxins is much more limited, perhaps because it's not so easy to bring one's prey, or biological enemy, quickly to its knees by inhibiting an ion transporter or an ion pump. However, the Na/K pump, which establishes the normal sodium and potassium gradients across the outer membranes of most animal cells, is an exception. The Na/K pump is the specific target of two types of natural toxins, heart glycosides (14, 15) and palytoxin (16–18). Although heart glycosides will be well known to the great majority of readers, palytoxin is likely to be obscure. Long deserving of more careful attention, a careful functional analysis of palytoxin actions presented in this issue of PNAS by Artigas and Gadsby (19) takes an important step to pick up the slack.

Cytoskeletal protein 4.1 isoforms, newly discovered in heart, are differentially overexpressed in the myocardium of patients with deteriorating heart failure

Cytoskeletal proteins are thought to play an important role in cardiomyopathy. Proteins 4.1 and spectrin are required for maintenance of the structure of animal cell membranes. We have recently shown that the three genes encoding protein 4.1(4.1R, 4.1G and 4.1N) are expressed in the human heart. They form a complex with spectrins close to the Z-disc at the sarcoplasmic reticulum, considered to maintain SR integrity during heart contraction. The role of these proteins in heart failure is unknown. In an attempt to clarify this we used Quantitative Real-Time PCR to compare mRNA expression of these proteins in three groups: deteriorating heart failure patients undergoing LVAD support (n = 10), stable patients with advanced heart failure undergoing heart transplantation (ESHF, n = 17) and normal control donor hearts (n = 26). Protein expression and localisation were examined by confocal immunofluorescence microscopy. 4.1R and 4.1N were up-regulated 3.3-fold (p<0.01) and 1.8-fold (p<0.05) in the myocardium of patients undergoing LVAD support vs. ESHF. 4.1G expression and β-spectrin expression did not differ in LVAD vs. ESHF. Immunofluorescence microscopy for each 4.1 protein isoform indicated they are localised at the Z-disc. We compared measurements within the same samples with those for the pro-inflamatory cytokines TNF-α, IL1-β, IL-6, IL-4, IL-10 & IL-1R. Proteins 4.1G, 4.1R and 4.1N correlated with TNF-α, IL1-β and IL-1R within individual patients. In conclusion, differential overexpression of the cytoskeletal proteins 4.1R and 4.1N occurs in the myocardium of patients with deteriorating heart failure requiring LVAD insertion. This was associated with expression of pro-inflammatory cytokines. The mechanisms responsible require further investigation.

Interaction between microcystins of different hydrophobicities and lipid monolayers

Microcystins (MC) are a group of amphiphatic peptide hepatotoxins and protein phosphatase inhibitors produced by certain cyanobacteria (blue-green algae). Microcystins are believed to require an active transport mechanism to penetrate the plasma membranes of animal cells. In this study the surface barostat technique showed that two more hydrophobic microcystins MC-LF, containing Leu and Phe, and MC-LW, containing Leu and Trp, had a higher surface activity on an egg phosphatidylcholine-cholesterol (7:3, molar ratio) monolayer as compared to that of a more hydrophilic variant MC-LR, containing Leu and Arg. Fluorescence anisotropy measurements of 1-[4-(trimethylamine)phenyl]-hexa-1,3,5-trien (TMA-DPH) were used to assess changes in the fluidity or lipid packing of model membranes in the presence of toxins. All three toxins caused a decrease in the steady-state anisotropy of TMA-DPH, suggesting that the toxins interacted with the membranes. The change in anisotropy was more pronounced for MC-LF and MC-LW than for MC-LR. Moreover, the fluorescence emission maximum of Trp in MC-LW was shifted slightly towards a shorter wavelength and the intensity was enhanced when allowed to interact with lipid vesicles, suggesting that the single Trp in MC-LW moved into a more unpolar environment when interacting with the vesicles. The differences between hydrophilic and hydrophobic microcystins could result in changes in organotropism, toxicokinetics and bioaccumulation.

Water pumps.

The transport of water across epithelia has remained an enigma ever since it was discovered over 100 years ago that water was transported across the isolated small intestine in the absence of osmotic and hydrostatic pressure gradients. While it is accepted that water transport is linked to solute transport, the actual mechanisms are not well understood. Current dogma holds that active ion transport sets up local osmotic gradients in the spaces between epithelial cells, the lateral intercellular spaces, and this in turn drives water transport by local osmosis. In the case of the small intestine, which in humans absorbs about 8 l of water a day, there is no direct evidence for either local osmosis or aquaporin gene expression in enterocytes. Intestinal water absorption is greatly enhanced by glucose, and this is the basis for oral rehydration therapy in patients with secretory diarrhoea. In our studies of the intestinal brush border Na+-glucose cotransporter we have obtained evidence that there is a direct link between the transport of Na+, glucose and water transport, i.e. there is cotransport of water along with Na+ and sugar, that will account for about 50 % of the total water transport across the human intestinal brush border membrane. In this short review we summarize the evidence for water cotransport and propose how this occurs during the enzymatic turnover of the transporter. This is a general property of cotransporters and so we expect that this may have wider implications in the transport of water and other small polar molecules across cell membranes in animals and plants.

Photodynamic assistance increases the efficiency of the process of microinjection in animal cells

A microinjection process is described that involves the photodynamically-assisted puncture of an animal cell membrane by means of a brief and partial degeneration resulting from cell membrane damage. The ratio of dye-injected rat pheochromocytoma cells that became detached three and six days after photodynamically-assisted microinjection was 9%; however, it was 83% when a conventional mechanical microinjection process was used.

DIFFERENCES IN OXIDATIVE STRESS BETWEEN YOUNG CANADA GEESE AND MALLARDS EXPOSED TO LEAD-CONTAMINATED SEDIMENT

Lead (Pb) exposure results in an increase in tissue lipid peroxides and variation in glutathione (GSH) concentrations, which can be related to peroxidative damage of cell membranes in Pb-poisoned animals. Species and individual variation in sensitivity to Pb poisoning among animals may be due to differential resistance to oxidative stress. The effects of oxidative stress caused by Pb exposure (1.7, 414, and 828 µg/g of diet) were compared for the first 6 wk in growing young of two species of waterfowl, Canada geese ( Branta canadensis ) and mallards ( Anas platyrhynchos ), with the first species being possibly more sensitive to Pb poisoning based on previous field and laboratory observations. Blood and liver Pb concentrations increased more in mallards than in geese. This may be explained on the basis of body weight, being 3.2 times higher in geese, and by hepatic metabolism, where GSH S -transferase (GST) activity is 2.9-fold higher in geese and presumably has a role in the binding of Pb to GSH and subsequent biliary excretion. In contrast, mallards showed higher hepatic levels of GSH and activities of GSH peroxidase (GPX) and GSH reductase (GR). Although both species showed a rise in hepatic GSH concentration with Pb exposure, the relationship between increased lipid peroxidation and Pb exposure was only significant in geese. Within treatment groups, hepatic GSH concentrations were inversely related to liver Pb concentrations in both species, which may correspond to the role of GSH in Pb excretion. Hepatic GSH was also inversely related to hepatic lipid peroxidation, but only in mallards and in agreement with the species differences observed in GPX and GR activities. The lower resistance to lipid peroxidation of Canada geese may explain why geese can die in the field from Pb poisoning after ingesting fewer shot than found in the gizzards of mallards and with lower liver Pb concentrations than in mallards.

Cholesterol Does Not Induce Segregation of Liquid-Ordered Domains in Bilayers Modeling the Inner Leaflet of the Plasma Membrane

A fluorescence-quenching method has been used to assess the potential formation of segregated liquid-ordered domains in lipid bilayers combining cholesterol with mixtures of amino and choline phospholipids like those found in the cytoplasmic leaflet of the mammalian cell plasma membrane. When present in proportions >20–30 mol %, different saturated phospholipids show a strong proclivity to form segregated domains when combined with unsaturated phospholipids and cholesterol, in a manner that is only weakly affected by the nature of the phospholipid headgroups. By contrast, mixtures containing purely unsaturated phospholipids and cholesterol do not exhibit detectable segregation of domains, even in systems whose components differ in headgroup structure, mono- versus polyunsaturation and/or acyl chain heterogeneity. These results indicate that mixtures of phospholipids resembling those found in the inner leaflet of the plasma membrane do not spontaneously form segregated liquid-ordered domains. Instead, our findings suggest that factors extrinsic to the inner-monolayer lipids themselves (e.g., transbilayer penetration of long sphingolipid acyl chains) would be essential to confer a distinctive, more highly ordered organization to the cytoplasmic leaflet of “lipid raft” structures in animal cell membranes.

Mixed-chain phospholipids: structures and chain-melting behavior.

It has long been established that diacyl phospholipids isolated from animal cell membranes are predominantly of a mixed-chain variety, meaning that the sn-1 and sn-2 acyl chains are saturated and unsaturated acyl chains, respectively. In general, monoenoic and dienoic acids are found in the sn-2 acyl chain of phosphatidylcholine (PtdCho), whereas polyenoic acids are in phosphatidylethanolamine (PtdEth). These unsaturated chains contain only cis-double bonds, which are always methylene-interrupted. In recent years, the structures and the chain-melting behavior of mixed-chain PtdCho and PtdEth have been systematically studied in this laboratory. Specifically, we have examined the effects of chain unsaturation of the sn-2 acyl chain on the phase transition temperature (Tm) of many PtdCho and PtdEth by high-resolution differential scanning calorimetry (DSC). The Tm values, for instance, obtained from all-unsaturated mixed-chain PtdEth derived from a common precursor can be grouped together according to their chemical formula to form a Tm-diagram. Hence, all the Tm values can be compared simply, systematically, and simultaneously using the Tm-diagram. In addition, the energy-minimized structures of mixed-chain phospholipids containing different numbers/positions of methylene-interrupted cis-double bonds have been simulated by molecular mechanics calculations (MM). In this review, the results of our MM and DSC studies carried out with various mixed-chain phospholipids are summarized. In addition, we emphasize that the combined approach of MM and DSC yields unique information that can correlate the various Tm-profiles seen in the Tm-diagram with the structural variation of mixed-chain lipids as caused by the introduction of different numbers/positions of methylene-interrupted cis-double bonds.

Expression of sodium pump isoforms and other sodium or calcium ion transporters in the heart of hypertensive patients

The sodium pump (Na +,K +-ATPase; EC 3.6.1.37) of animal cell membranes is the enzyme responsible for the maintenance of membrane potential, for the function of secondary active transporters, and for osmoregulation of the cell. Since inhibition of the enzyme by cardiac glycosides results in increased contractility of the heart muscle and increased blood pressure, we were interested in whether there is a correlation between hypertension and expression of the various isoforms of the sodium pump. In addition, we also examined the expression of the isoforms of the sarcoplasmic and plasma membrane Ca 2+-ATPase, the Na +/Ca 2+- and Na +/H +-exchangers, and Na + channel and Ca 2+ channel isoforms. Total mRNA was isolated from 50 mg tissue from the right atrium of hypertensive and normotensive patients who were undergoing cardiac surgery. After reverse transcription and subsequent amplification of ion transporter-specific cDNA fragments by polymerase chain reaction (PCR) in the presence of [α- 32P]dCTP, quantification of the amplified fragments was carried out by the Phosphorimager technique. The data obtained show that the αl subunit mRNA is expressed similarly in normotensive and hypertensive patients. The amount of α2 subunit mRNA, however, is increased 5-fold in hypertensive patients. In the same group, the amount of α3 isoform is also significantly increased, although not as dramatically as the α2 isoform. Besides the Na +,K +-ATPase isoforms, a significant increase in the expression of mRNA for the Na +/Ca 2+-exchanger and the plasma membrane Ca 2+-ATPase isoforms was detected. It is possible that the observed changes in mRNA expression for these ion transporters reflect compensatory mechanisms to overcome a defective Na + and Ca 2+ metabolism in the tissues of hypertensive patients or reflect defects directly involved in the cause of hypertension. The expression of mRNA for all other transporters investigated was unaltered.

Subcellular Localization and Oligomerization of the Arabidopsis thaliana Somatic Embryogenesis Receptor Kinase 1 Protein

The Arabidopsis thaliana somatic embryogenesis receptor kinase 1 ( AtSERK1) gene is expressed in developing ovules and early embryos. AtSERK1 is also transiently expressed during somatic embryogenesis. The predicted AtSERK1 protein contains an extracellular domain with a leucine zipper motif followed by five leucine-rich repeats, a proline-rich region, a single transmembrane region and an intracellular kinase domain. The AtSERK1 cDNA was fused to two different variants of green fluorescent protein (GFP), a yellow-emitting GFP (YFP) and a cyan-emitting GFP (CFP), and transiently expressed in both plant protoplasts and insect cells. Using confocal laser scanning microscopy it was determined that the AtSERK1-YFP fusion protein is targeted to plasma membranes in both plant and animal cells. The extracellular leucine-rich repeats, and in particular the N-linked oligosaccharides that are present on them appear to be essential for correct localization of the AtSERK1-YFP protein. The potential for dimerization of the AtSERK1 protein was investigated by measuring the YFP/CFP fluorescence emission ratio using fluorescence spectral imaging microscopy. This ratio will increase due to fluorescence resonance energy transfer if the AtSERK1-CFP and AtSERK1-YFP fusion proteins interact. In 15 % of the cells the YFP/CFP emission ratio for plasma membrane localized AtSERK1 proteins was enhanced. Yeast-protein interaction experiments confirmed the possibility for AtSERK1 homodimerization. Elimination of the extracellular leucine zipper domain reduced the YFP/CFP emission ratio to control levels indicating that without the leucine zipper domain AtSERK1 is monomeric.

Cystic fibrosis: a brief look at some highlights of a decade of research focused on elucidating and correcting the molecular basis of the disease.

The disease Cystic Fibrosis (CF) is caused by mutations in the protein called CFTR, cystic fibrosis transmembrane conductance regulator, an ABC-transporter-like protein found in the plasma membrane of animal cells. CFTR is believed to function primarily as a Cl- channel, but evidence is mounting that this protein has other roles as well. Structurally, CFTR consists of a single polypeptide chain (1480 amino acids) that folds into 5 distinct domains. These include 2 transmembrane domains that are involved in channel formation; 2 nucleotide-binding domains (NBF1 and NBF2), the first of which clearly binds and hydrolyzes ATP; and 1 regulatory domain (R) that is phosphorylated in a cAMP-dependent process. Currently, the 3D structure of neither CFTR nor its domains has been elucidated, although both nucleotide domains have been modeled in 3D, and solution structures in 3D have been obtained for peptide segments of NBF1. The most common mutation causing CF is the deletion (delta) of a single phenylalanine (F) in position 508 within a putative helix located in NBF1. CF patients bearing this deltaF508 mutation frequently experience chronic lung infections, particularly by Pseudomonas aeruginosa, and have a life span that rarely exceeds the age of 30. Since the CFTR gene was cloned and sequenced in 1989, there has been over a decade of research focused on understanding the molecular basis of CF caused by the deltaF508 mutation, with the ultimate objective of using the knowledge gained to carry out additional research designed to correct the underlying defect. In general, this pioneering or "ground roots" research has succeeded according to plan. This brief review summarizes some of the highlights with a focus on those studies conducted in the authors' laboratory. For us, this research has been both exciting and rewarding mainly because the results obtained, despite very limited funding, have provided considerable insight, not only into the chemical, molecular, and pathogenic basis of CF, but have made it possible for us and others to now develop novel, chemically rational, and "cost effective" strategies to identify agents that correct the structural defect in the deltaF508 CFTR protein causing most cases of CF.

Vibrio cholerae cytolysin: assembly and membrane insertion of the oligomeric pore are tightly linked and are not detectably restricted by membrane fluidity

Hemolytic strains of Vibrio cholerae secrete a cytolysin that, upon binding as a monomer, forms pentameric pores in animal cell membranes. Pore formation is inhibited at low temperature and in the absence of cholesterol. We here posed the following questions: firstly, can oligomerization be observed in the absence of pore formation? Secondly, is membrane fluidity responsible for the effect of temperature or of cholesterol upon pore formation? The first issue was approached by chemical cross-linking, by electrophoretic heteromer analysis, and by electron microscopy. None of these methods yielded any evidence of a non-lytic pre-pore oligomer. The second question was addressed by the use of two susceptible liposome models, consisting of cholesterol admixed to bovine brain lipids and to asolectin, respectively. The two liposome species clearly differed in membrane fluidity as judged by diphenylhexatriene fluorescence polarization. Nevertheless, their permeabilization by the cytolysin decreased with temperature in a closely parallel fashion, virtually vanishing at 5°C. Omission of cholesterol from the liposomes uniformly led to an increase in membrane fluidity but prevented permeabilization by the cytolysin. The effects of temperature and of cholesterol upon cytolysin activity are thus not mediated by fluidization of the target membrane. The findings of our study distinguish V. cholerae cytolysin from several previously characterized pore-forming toxins.