pH dependence of listeriolysin O aggregation and pore-forming ability

Listeriolysin O (LLO) is the major factor implicated in the escape of Listeria monocytogenes from the phagolysosome. It is the only representative of cholesterol-dependent cytolysins that exhibits pH-dependent activity. Despite intense studies of LLO pH-dependence, this feature of the toxin still remains incompletely explained. Here we used fluorescence and CD spectroscopy to show that the structure of LLO is not detectably affected by pH at room temperature. We observed slightly altered haemolytic and permeabilizing activities at different pH values, which we relate to reduced binding of LLO to the lipid membranes. However, alkaline pH and elevated temperatures caused rapid denaturation of LLO. Aggregates of the toxin were able to bind Congo red and Thioflavin T dyes and were visible under transmission electron microscopy as large, amorphous, micrometer-sized assemblies. The aggregates had the biophysical properties of amyloid. Analytical ultracentrifugation indicated dimerization of the protein in acidic conditions, which protects the protein against premature denaturation in the phagolysosome, where toxin activity takes place. We therefore suggest that LLO spontaneously aggregates at the neutral pH found in the host cell cytosol and that this is a major mechanism of LLO inactivation.     

Sec16p potentiates the action of COPII proteins to bud transport vesicles

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is approximately 10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.     

Listeriolysin O: cholesterol inhibits cytolysis but not binding to cellular membranes

Listeriolysin O (LLO) binds to cholesterol-containing membranes in which it oligomerizes to form pores. Preincubation of the toxin with cholesterol is known to inhibit haemolysis, whereas the oxidized form of cholesterol has no inhibitory effect. Using immunoblot analyses and flow cytometry we demonstrate that preincubation with cholesterol does not influence binding of the listeriolysin–cholesterol complex to red blood cells, eukaryotic cells or artificial membranes. Lytic activity of membrane-bound LLO inactivated by cholesterol can be restored by enzymatic treatment with cholesterol oxidase. To determine the step at which cholesterol inhibits lytic activity, we looked for pore formation using electron microscopy. Pores formed by purified listeriolysin could be directly visualized using erythrocyte ghosts. This property was lost upon incubation of the toxin with cholesterol. Quantitative analysis strongly suggest that inhibition of lysis by cholesterol is not due to decreased binding of listeriolysin to target membranes, but rather to an interference with a subsequent step leading to polymerization of the toxin.     

Drosophila S2 cells: an alternative infection model for Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that infects humans and animals. Its pathogenic strategy involves the expression of virulence proteins that mediate intracytosolic growth and cell-to-cell spread. A key virulence protein is the cholesterol-dependent cytolysin, listeriolysin O (LLO), which is largely responsible for mediating escape from the phagosome into the host cytosol. To study further the host processes exploited during L. monocytogenes infection, we sought to develop Drosophila S2 cells as a model for infection. Here, we show that S2 cells share a number of properties with mammalian cell culture models of infection. As with mouse macrophages, LLO was required for phagosomal escape from S2 cells. Furthermore, vacuolar escape was dependent on their acidification via the ATPase proton pumps, as bafilomycin A1 treatment sharply decreased escape. However, unlike in mouse macrophages, LLO mutants replicated in the phagosome of S2 cells. Drosophila cells are cholesterol auxotrophs, and exogenous cholesterol increased the infection rate of L. monocytogenes (LLO independent) and also augmented the efficiency of vacuolar escape (LLO dependent). With available genetic tools such as RNA interference, S2 cells could become an important model in the study of host-pathogen interactions.     

The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.     

Bacterial protein listeriolysin O induces nonmonotonic dynamics because of lipid ejection and crowding

Membrane-bound protein complexes involving pore forming toxins (PFTs) released by virulent bacteria are known to form transmembrane pores leading to host cell lysis. Developing alternative strategies against PFT mediated bacterial virulence factors requires an understanding of the cellular membrane response. However, membrane disruption and related lipid reorganization events during attack by PFTs remain largely unexplored. We report counterintuitive and nonmonotonic variations in lipid diffusion, measured using confocal fluorescence correlation spectroscopy, due to interplay of lipid ejection and crowding by membrane-bound oligomers of a prototypical cholesterol-dependent cytolysin, listeriolysin O (LLO). The observed dynamical crossover is correlated with concentration dependent transitions of LLO oligomeric state populations from rings to arc-like pore complexes, predicted using a proposed two-state free area-based diffusion model. At low PFT concentrations, a hitherto unexplored regime of increased lipid diffusivity is attributed to lipid ejection events because of a preponderance of ring-like pore states. At higher protein concentrations in which membrane-inserted arc-like pores dominate, lipid ejection is less efficient and the ensuing crowding results in a lowering of lipid diffusion. These variations in lipid dynamics are corroborated by macroscopic rheological response measurements of PFT bound vesicles. Our study correlates PFT oligomeric state transitions, membrane remodeling, and mechanical property variations, providing unique insights into the pore forming mechanisms of cholesterol-dependent cytolysins.     

Listeriolysin O: a phagosome-specific lysin

Listeriolysin O (LLO) is a pore-forming toxin of the cholesterol-dependent cytolysin family and a primary virulence factor of the gram-positive, facultative intracellular pathogen Listeria monocytogenes. During the intracellular life cycle of L. monocytogenes, LLO is largely responsible for mediating rupture of the phagosomal membrane, thereby allowing the bacterium access to the host cytosol, its replicative niche. In the host cytosol, LLO activity is controlled at numerous levels to prevent perforation of the plasma membrane and loss of the intracellular environment. In this review, we focus primarily on the role of LLO in phagosomal escape and the multiple regulatory mechanisms that control LLO activity in the host cytosol.     

Listeriolysin O: A phagosome‐specific cytolysin revisited

Listeriolysin O (LLO) is an essential determinant of Listeria monocytogenes pathogenesis that mediates the escape of L. monocytogenes from host cell vacuoles, thereby allowing replication in the cytosol without causing appreciable cell death. As a member of the cholesterol‐dependent cytolysin (CDC) family of pore‐forming toxins, LLO is unique in that it is secreted by a facultative intracellular pathogen, whereas all other CDCs are produced by pathogens that are largely extracellular. Replacement of LLO with other CDCs results in strains that are extremely cytotoxic and 10,000‐fold less virulent in mice. LLO has structural and regulatory features that allow it to function intracellularly without causing cell death, most of which map to a unique N‐terminal region of LLO referred to as the proline, glutamic acid, serine, threonine (PEST)‐like sequence. Yet, while LLO has unique properties required for its intracellular site of action, extracellular LLO, like other CDCs, affects cells in a myriad of ways. Because all CDCs form pores in cholesterol‐containing membranes that lead to rapid Ca²⁺ influx and K⁺ efflux, they consequently trigger a wide range of host cell responses, including mitogen‐activated protein kinase activation, histone modification, and caspase‐1 activation. There is no debate that extracellular LLO, like all other CDCs, can stimulate multiple cellular activities, but the primary question we wish to address in this perspective is whether these activities contribute to L. monocytogenes pathogenesis.     

Membrane cholesterol and sphingomyelin,and ostreolysin A are obligatory for pore-formation by a MACPF/CDC-like pore-forming protein,pleurotolysin B

The mushroom Pleurotus ostreatus has been reported to produce the hemolytic proteins ostreolysin (OlyA), pleurotolysin A (PlyA) and pleurotolysin B (PlyB). The present study of the native and recombinant proteins dissects out their lipid-binding characteristics and their roles in lipid binding and membrane permeabilization. Using lipid-binding studies, permeabilization of erythrocytes, large unilamellar vesicles of various lipid compositions, and electron microscopy, we show that OlyA, a PlyA homolog, preferentially binds to membranes rich in sterol and sphingomyelin, but it does not permeabilize them. The N-terminally truncated Δ48PlyB corresponds to the mature and active form of native PlyB, and it has a membrane attack complex-perforin (MACPF) domain. Δ48PlyB spontaneously oligomerizes in solution, and binds weakly to various lipid membranes but is not able to perforate them. However, binding of Δ48PlyB to the cholesterol and sphingomyelin membranes, and consequently, their permeabilization is dramatically promoted in the presence of OlyA. On these membranes, Δ48PlyB and OlyA form predominantly 13-meric oligomers. These are rosette-like structures with a thickness of ∼9 nm from the membrane surface, with 19.7 nm and 4.9 nm outer and inner diameters, respectively. When present on opposing vesicle membranes, these oligomers can dimerize and thus promote aggregation of vesicles. Based on the structural and functional characteristics of Δ48PlyB, we suggest that it shares some features with MACPF/cholesterol-dependent cytolysin (CDC) proteins. OlyA is obligatory for the Δ48PlyB permeabilization of membranes rich in cholesterol and sphingomyelin.     

Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers

Ostreolysin, a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), recognizes and binds specifically to membrane domains enriched in cholesterol and sphingomyelin (or saturated phosphatidylcholine). These events, leading to permeabilization of the membrane, suggest that a cholesterol-rich liquid-ordered membrane phase, which is characteristic of lipid rafts, could be its possible binding site. In this work, we present effects of ostreolysin on membranes containing various steroids. Binding and membrane permeabilizing activity of ostreolysin was studied using lipid mono- and bilayers composed of sphingomyelin combined, in a 1/1 molar ratio, with natural and synthetic steroids (cholesterol, ergosterol, β-sitosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, cholesteryl acetate, and 5-cholesten-3-one). Binding to membranes and lytic activity of the protein are both shown to be dependent on the intact sterol 3β-OH group, and are decreased by introducing additional double bonds and methylation of the steroid skeleton or C17-isooctyl chain. The activity of ostreolysin mainly correlates with the ability of the steroids to promote formation of liquid-ordered membrane domains, and is the highest with cholesterol-containing membranes. Furthermore, increasing the cholesterol concentration enhanced ostreolysin binding in a highly cooperative manner, suggesting that the membrane lateral distribution and accessibility of the sterols are crucial for the activity of this new member of cholesterol-dependent cytolysins.     

A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway

The signalling pathway for the hepatocyte growth factor receptor, Met/HGF-R, is hijacked by the bacterial surface protein InlB to induce Listeria monocytogenes entry into non-phagocytic cells. We previously showed that Listeria invades host cells by interacting with specialized microdomains of the host plasma membrane called lipid rafts. In this study, we analysed in living cells signalling events that are crucial for Listeria entry using a fluorescence resonance energy transfer-based microscopic method. Phosphoinositide (PI) 3-kinase activity and Rac1 signalling induced by Listeria interacting with epithelial cells were monitored as well as signalling induced by soluble InlB and the Met natural ligand HGF. We found that InlB and HGF induced similar kinetics of PI 3-kinase and Rac1 activation. PI 3-kinase activation was upstream and independent of Rac1 activation. Cholesterol-depletion experiments were performed to address the role of lipid rafts in Met signalling. The amount of 3'-phosphoinositides produced by PI 3-kinase was not affected by cholesterol depletion, while their membrane dynamic was cholesterol-dependent. Rac1 activation, downstream from PI 3-kinase, was cholesterol-dependent suggesting that the spatial distribution of 3'-phosphoinositides within membrane microdomains is critical for Rac1 activation and consequently for F-actin assembly at bacterial entry site.     

Listeria monocytogenes listeriolysin O and phosphatidylinositol-specific phospholipase C affect adherence to epithelial cells

Listeria monocytogenes, a foodborn intracellular animal and human pathogen, produces several exotoxins contributing to virulence. Among these are listeriolysin O (LLO), a pore-forming cholesterol-dependent hemolysin, and a phosphatidylinositol-specific phospholipase C (PI-PLC). LLO is known to play an important role in the escape of bacteria from the primary phagocytic vacuole of macrophages, and PI-PLC supports this process. Evidence is accumulating that LLO and PI-PLC are multifunctional virulence factors with many important roles in the host-parasite interaction other than phagosomal membrane disruption. LLO and PI-PLC may induce a number of host cell responses by modulating signal transduction of infected cells via intracellular Ca2+ levels and the metabolism of phospholipids. This would result in the activation of host phospholipase C and protein kinase C. In the present study, using Bacillus sub tilis strains expressing LLO, PI-PLC, and simultaneously LLO and PI-PLC, we show that LLO and PI-PLC enhance bacterial binding to epithelial cells Int407, with LLO being necessary and PI-PLC playing an accessory role. The results of this work suggest that these two listerial proteins act on epithelial cells prior to internalization.     

The innate immunity role of cathepsin-D is linked to Trp-491 and Trp-492 residues of listeriolysin O

Listeriolysin O (LLO) is a thiol-activated cytolysin secreted by Listeria monocytogenes . LLO and phosphatidylinositol phospholipase C are two essential virulence factors, which this bacterium needs to escape from the phagosomal compartment to the cytoplasm. Cathepsin-D specifically cleaves LLO, between the Trp-491 (tryptophan amino acid in three letter nomenclature) and Trp-492 residues of the conserved undecapeptide sequence, ECTGLAWEWWR, in the domain 4 of LLO (D4). Moreover, these residues also correspond to the phagosomal-binding epitope. Cathepsin-D had no effect on phosphatidylinositol phospholipase C. We have observed that cathepsin-D cleaved the related cholesterol-dependent cytolysin pneumolysin at the same undecapeptide sequence between Trp-435 and Trp-436 residues. These studies also revealed an additional cathepsin-D cleavage site in the pneumolysin D4 domain localized in the 361-GDLLLD-366 sequence. These differences might confer a pathogenic advantage to listeriolysin O, increasing its resistance to phagosomal cathepsin-D action by reducing the number of cleavages sites in the D4 domain. Using ΔLLO/W491A and ΔLLO/W492A bacterial mutants, we reveal that the Trp-491 residue has an important role linked to cathepsin-D in Listeria innate immunity.     

Attenuated mutants of the intracellular bacterium Listeria monocytogenes obtained by single amino acid substitutions in listeriolysin O

Listeriolysin O (LLO), a major virulence factor of the intracellular bacterium Listeria monocytogenes, shares with other known 'thiol-activated toxins' a conserved undecapeptide, ECTGLAWEWWR, located in the C-terminal region of the protein and containing the unique cysteine of the molecule. Single amino acid substitutions were created in this region to study the role of cysteine and tryptophan residues in the lytic activity of LLO as well as in the virulence of the bacterium. Transformation of a transposon-induced non-haemolytic mutant with plasmids carrying the mutated genes allowed allele exchange and transfer of mutations on to the chromosome by in vivo recombination. The mutant strains secreted a full-length 59 kilodalton LLO. A decrease of 25% in the haemolytic activity in culture supernatants was observed in the case of mutation Cys-484 to Ala and of 80% for mutation Cys-484 to Ser. Mutations Trp-491 and Trp-492 to Ala decreased activity by, respectively, 95% and 99.9%. LLOs produced by the mutants, as the wild type, were active at low pH, inhibited by cholesterol, and able to bind to cell membranes. A close relationship was found between virulence of mutants in the mouse model and haemolytic activity in their culture supernatants. These results demonstrate that the thiol group of Cys-484 is not essential for either haemolytic activity in vitro or virulence in vivo. In contrast, Trp-492 appears to be required for both haemolytic activity and virulence. The finding that the nearly non-haemolytic mutant Trp-492-Ala persisted in the spleen for several days after inoculation indicates that mutagenesis of a virulence determinant can attenuate virulence and provides a novel approach to the development of live vaccine strains.     

Conformational changes that effect oligomerization and initiate pore formation are triggered throughout perfringolysin O upon binding to cholesterol

Pore formation by the cholesterol-dependent cytolysins (CDCs) requires the presence of cholesterol in the target membrane. Cholesterol was long thought to be the cellular receptor for these toxins, but not all CDCs require cholesterol for binding. Intermedilysin, secreted by Streptococcus intermedius, only binds to membranes containing the human protein CD59 but forms pores only if the membrane contains sufficient cholesterol. In contrast, perfringolysin O (PFO), secreted by Clostridium perfringens, only binds to membranes containing substantial amounts of cholesterol. Given that different steps in the assembly of various CDC pores require cholesterol, here we have analyzed to what extent cholesterol molecules, by themselves, can modulate the conformational changes associated with PFO oligomerization and pore formation. PFO binds to cholesterol when dispersed in aqueous solution, and this binding triggers the distant rearrangement of a beta-strand that exposes an oligomerization interface. Moreover, upon binding to cholesterol, PFO forms a prepore complex, unfolds two amphipathic transmembrane beta-hairpins, and positions their nonpolar surfaces so they associate with the hydrophobic cholesterol surface. The interaction of PFO with cholesterol is therefore sufficient to initiate an irreversible sequence of coupled conformational changes that extend throughout the toxin molecule.     

Liposome-doped nanocomposites as artificial-cell-based biosensors: detection of listeriolysin O

Listeriolysin O (LLO) is a pore-forming hemolysin secreted by the foodborne pathogen Listeria monocytogenes and is required for bacterial virulence. Current detection methods for L. monocytogenes are time-consuming, labor-intensive, and expensive, which is impractical considering the limitations of food storage. To overcome these problems, we developed a liposome-doped silica nanocomposite as a simple, inexpensive, and highly stable biosensor material that mimics existing whole-cell assays for LLO. Small unilamellar liposomes containing fluorescent dyes were immobilized within porous silica using alcohol-free sol-gel synthesis methods. The immobilized liposomes served as cellular surrogates for membrane insertion and pore formation by LLO. The integrity of liposomes in the solid-state sol-gel glass was investigated by fluorescence quenching and leaching assays. The materials were stable for at least 5 months in ambient conditions. Both free and immobilized liposomes responded to LLO at pH 6.0 with concentration dependent kinetics. The pore formation of LLO in liposome-doped silica composites displayed similar kinetic curves as free liposomes but with slower rates. LLO insertion into the immobilized liposomes was pH dependent. No increase in membrane permeability was observed at pH 7.4 for the liposome-doped composites in the presence of LLO. Immobilized liposomes can detect LLO in approximately 1.5 h using a steady state calibration and within 30 min using a kinetic calibration. These liposome silica composites potentially could be used for the detection of hemolysin producing L. monocytogenes as well as the many other bacteria that produce pore-forming toxins.     

Switch-like Responses of Two Cholesterol Sensors Do Not Require Protein Oligomerization in Membranes

Many cellular processes are sensitive to levels of cholesterol in specific membranes and show a strongly sigmoidal dependence on membrane composition. The sigmoidal responses of the cholesterol sensors involved in these processes could arise from several mechanisms, including positive cooperativity (protein effects) and limited cholesterol accessibility (membrane effects). Here, we describe a sigmoidal response that arises primarily from membrane effects due to sharp changes in the chemical activity of cholesterol. Our models for eukaryotic membrane-bound cholesterol sensors are soluble bacterial toxins that show an identical switch-like specificity for endoplasmic reticulum membrane cholesterol. We show that truncated versions of these toxins fail to form oligomers but still show sigmoidal binding to cholesterol-containing membranes. The nonlinear response emerges because interactions between bilayer lipids control cholesterol accessibility to toxins in a threshold-like fashion. Around these thresholds, the affinity of toxins for membrane cholesterol varies by >100-fold, generating highly cooperative lipid-dependent responses independently of protein-protein interactions. Such lipid-driven cooperativity may control the sensitivity of many cholesterol-dependent processes.     

Listeriolysin O: a genuine cytolysin optimized for an intracellular parasite

Cholesterol-dependent cytolysins (CDCs)* are produced by a large number of pathogenic gram-positive bacteria. A member of this family, listeriolysin O (LLO), is produced by the intracellular pathogen Listeria monocytogenes. A unique feature of LLO is its low optimal pH activity (approximately 6) which permits escape of the bacterium from the phagosome into the host cell cytosol without damaging the plasma membrane of the infected cell. In a recent study (Glomski et al., 2002, this issue), Portnoy's group has addressed the molecular mechanism underlying the pH sensitivity of LLO. Unexpectedly, a single amino acid substitution in LLO L461T results in a molecule more active at neutral pH and promoting premature permeabilization of the infected cells, leading to attenuated virulence. This finding highlights how subtle changes in proteins can be exploited by bacterial pathogens to establish and maintain the integrity of their specific niches.     

Chondrocytes utilize a cholesterol-dependent lipid translocator to externalize phosphatidylserine

During endochondral ossification, growth plate chondrocytes release plasma membrane (PM) derived matrix vesicles (MV), which are the site of initial hydroxyapatite crystal formation. MV constituents which facilitate the mineralization process include the integral membrane ectoenzymes alkaline phosphatase (ALPase) and nucleotide pyrophosphatase phosphodiesterase (NPP1/PC-1), along with a phosphatidylserine- (PS-) rich membrane surface that binds annexins and calcium, resulting in enhanced calcium entry into MV. In this study, we determined that chick growth plate MV were highly enriched in membrane raft microdomains containing high levels of cholesterol, glycophosphatidylinositol- (GPI-) anchored ALPase, and phosphatidylserine (PS) localized to the external leaflet of the bilayer. To determine how such membrane microdomains arise during chondrocyte maturation, we explored the role of PM cholesterol-dependent lipid assemblies in regulating the activities of lipid translocators involved in the externalization of PS. We first isolated and determined the composition of detergent-resistant membranes (DRMs) from chondrocyte PM. DRMs isolated from chondrocyte PM were enhanced in ganglioside 1 (GM1) and cholesterol as well as GPI-anchored ALPase. Furthermore, these membrane domains were enriched in PS (localized to the external leaflet of the bilayer) and had significantly higher ALPase activity than non-cholesterol-enriched domains. To understand the role of cholesterol-dependent lipid assemblies in the externalization of PS, we measured the activities of two lipid transporters involved in PS externalization, aminophospholipid translocase (APLT) and phospholipid scramblase (PLSCR1), during maturation of a murine chondrocytic cell line, N1511. In this report, we provide the first evidence that maturing chondrocytes express PLSCR1 and have scramblase activity. We propose that redistribution of PS is dependent on an increase in phospholipid scramblase activity and a decrease in APLT activity. Lastly, we show that translocator activity is most likely to be modulated by membrane cholesterol levels through a membrane raft microdomain.