Polar Localization Analysis of Anionic Phospholipids and Their Binding Proteins in Arabidopsis Pollen Tubes.

Pollen tubes are typical polarized growth cells whose elongation occurs only in tip regions and is highly dependent on precise and ordered exocytosis/endocytosis in the top regions of the tubes. Although anionic phospholipids have been proven to be involved in regulating vesicle trafficking and the proper localization and functions of proteins in pollen tubes, the underlying cellular and molecular mechanisms remain poorly understood. To further understand how anionic phospholipids are involved in vesicle trafficking and in the control of protein localization and functions, assay methods to analyze the polar localization of anionic phospholipids and their binding proteins, and identifying phospholipid-protein interactions, should be developed. Here, we describe detailed protocols for analyzing anionic phospholipid polar localization and colocalization with their binding proteins in Arabidopsis pollen tubes and examining phospholipid-protein interactions in vitro.

The impact of volatile anesthetics and propofol on phosphatidylinositol 4,5-bisphosphate signaling.

Phosphatidylinositol 4,5-bisphosphate (PIP2), as well as other anionic phospholipids, play a pivotal role in various cellular processes, including ion channel regulation, receptor trafficking, and intracellular signaling pathways. The binding of volatile anesthetics and propofol to PIP2 leads to alterations in PIP2-mediated signaling causing modulation of ion channels such as ɣ-aminobutyric acid type A (GABAA) receptors, voltage-gated calcium channels, and potassium channels through various mechanisms. Additionally, the interaction between anionic phospholipids and G protein-coupled receptors plays a critical role in various anesthetic pathways, with these anesthetic-induced changes impacting PIP2 levels which cause cascading effects on receptor trafficking, including GABAA receptor internalization. This comprehensive review of various mechanisms of interaction provides insights into the intricate interplay between PIP2 signaling and anesthetic-induced changes, shedding light on the molecular mechanisms underlying anesthesia.

A global LC-MS2 -based methodology to identify and quantify anionic phospholipids in plant samples.

Anionic phospholipids (PS, PA, PI, PIPs) are low-abundant phospholipids with impactful functions in cell signaling, membrane trafficking and cell differentiation processes. They can be quickly metabolized and can transiently accumulate at defined spots within the cell or an organ to respond to physiological or environmental stimuli. As even a small change in their composition profile will produce a significant effect on biological processes, it is crucial to develop a sensitive and optimized analytical method to accurately detect and quantify them. While thin-layer chromatography (TLC) separation coupled with gas chromatography (GC) detection methods already exist, they do not allow for precise, sensitive, and accurate quantification of all anionic phospholipid species. Here we developed a method based on high-performance liquid chromatography (HPLC) combined with two-dimensional mass spectrometry (MS2 ) by MRM mode to detect and quantify all molecular species and classes of anionic phospholipids in one shot. This method is based on a derivatization step by methylation that greatly enhances the ionization, the separation of each peak, the peak resolution as well as the limit of detection and quantification for each individual molecular species, and more particularly for PA and PS. Our method universally works in various plant samples. Remarkably, we identified that PS is enriched with very long chain fatty acids in the roots but not in aerial organs of Arabidopsis thaliana. Our work thus paves the way for new studies on how the composition of anionic lipids is finely tuned during plant development and environmental responses.

Interactions of REF1 and SRPP1 rubber particle proteins from Hevea brasiliensis with synthetic phospholipids: Effect of charge and size of lipid headgroup.

According to the fatty acid and headgroup compositions of the phospholipids (PL) from Hevea brasiliensis latex, three synthetic PL were selected (i.e. POPA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) to investigate the effect of PL headgroup on the interactions with two major proteins of Hevea latex, i.e. Rubber Elongation Factor (REF1) and Small Rubber Particle Protein (SRPP1). Protein/lipid interactions were screened using two models (lipid vesicles in solution or lipid monolayers at air/liquid interface). Calcein leakage, surface pressure, ellipsometry, microscopy and spectroscopy revealed that both REF1 and SRPP1 displayed stronger interactions with anionic POPA and POPG, as compared to zwitterionic POPC. A particular behavior of REF1 was observed when interacting with POPA monolayers (i.e. aggregation + modification of secondary structure from α-helices to ß-sheets, characteristic of its amyloid aggregated form), which might be involved in the irreversible coagulation mechanism of Hevea rubber particles.

The pollen-specific class VIII-myosin ATM2 from Arabidopsis thaliana associates with the plasma membrane through a polybasic region binding anionic phospholipids.

Pollen tubes display polarized tip-growth and are a model to study the coordination of vesicular trafficking and cytoskeletal control. The molecular details of how dynamic actin filaments associate with the plasma membrane are currently unclear. In Arabidopsis thaliana, plasma membrane attachment of actin filaments may be mediated by four myosins representing the plant-specific myosin-subclass VIII, which localize to the plasma membrane and display only minor motor-activity. Here we explore the mode of membrane attachment of the pollen-expressed class VIII-myosins ATM2 and VIII-B through interaction with anionic membrane phospholipids. A fluorescent mCherry-ATM2-fusion decorated plasma membrane-peripheral actin filaments when expressed in tobacco pollen tubes, consistent with a role of class VIII-myosins at the membrane-cytoskeleton interface. As recombinant proteins, class VIII-myosins are prone to aggregation and to proteolysis, creating a challenge for their biochemical characterization. We describe a purification scheme for guanidinium chloride (GdmCl)-denatured recombinant proteins, followed by a renaturation protocol to obtain pure, soluble protein fragments of ATM2 and VIII-B. The fragments represent the C-terminal tail and coiled-coil-regions and lack the N-terminal actin-binding regions, IQ or motor domains. Based on lipid-overlays and liposome-sedimentation assays, the fragments of ATM2 and VIII-B bind anionic phospholipids. Small polybasic regions at the extreme C-termini were sufficient for lipid-binding of the respective protein fragments. When expressed in tobacco pollen tubes, a fluorescence-tagged variant of ATM2 lacking its lipid-binding region displayed substantially reduced plasma membrane association. The data indicate that class VIII-myosins may facilitate actin-plasma membrane attachment through interaction with anionic phospholipids, mediated by polybasic C-terminal lipid-binding domains.

Characterization of phospholipid-modified lung surfactant in vitro and in a neonatal ARDS model reveals anti-inflammatory potential and surfactant lipidome signatures.

A strong inflammatory immune response drives the lung pathology in neonatal acute respiratory distress syndrome (nARDS). Anti-inflammatory therapy is therefore a promising strategy for improved treatment of nARDS. We demonstrate a new function of the anionic phospholipids POPG, DOPG, and PIP2 as inhibitors of IL-1ß release by LPS and ATP-induced inflammasome activation in human monocyte-derived and lung macrophages. Curosurf® surfactant was enriched with POPG, DOPG, PIP2 and the head-group derivative IP3, biophysically characterized and applicability was evaluated in a piglet model of nARDS. The composition of pulmonary surfactant from piglets was determined by shotgun lipidomics screens. After 72 h of nARDS, levels of POPG, DOPG, and PIP2 were enhanced in the respective treatment groups. Otherwise, we did not observe changes of individual lipid species in any of the groups. Surfactant proteins were not affected, with the exception of the IP3 treated group. Our data show that POPG, DOPG, and PIP2 are potent inhibitors of inflammasome activation; their enrichment in a surfactant preparation did not induce any negative effects on lipid profile and reduced biophysical function in vitro was mainly observed for PIP2. These results encourage to rethink the current strategies of improving surfactant preparations by inclusion of anionic lipids as potent anti-inflammatory immune regulators.

Implications of a Diabetic Foot Xerosis Treatment With an Emulsion Containing the Plant-Based Anionic Phospholipids.

PURPOSE: This study compares and contrasts a skin cream containing plant-based anionic polar phospholipid (APP) technology with a mineral oil hydrocarbon (petrolatum)-based (MHB) skin cream technology in the treatment of skin xerosis (dryness) in diabetic feet. Skin cream with APP technology promotes phospholipid absorption, reparation of intercellular lamellae, and organization of water promoting hydration; whereas skin cream with mineral hydrocarbon-based (MHB) technology principally covers skin, preventing dehydration. METHODS: Subjects (n = 54) with diagnoses of diabetes mellitus and foot skin dryness were studied using a multicenter, double-blind, masked-study design. An emulsion cream containing 0.05% APP in triglycerides (APP preparation) was compared to MHB skin cream, Eucerin® (MHB preparation) applied topically to skin of the feet. Graded measurements were recorded on 4 efficacy variables including dryness, erythema, fissures, and itching and neurovascular assessments. Implications of the plant-based and mineral-based skin creams in the context of skin xerosis are contrasted. RESULTS: APP and MHB preparations were similar in effectiveness and safety. There was no significant difference among any of the 4 efficacy variables (P < .5) including neurovascular measurements. The APP preparation is absorbed into the skin, whereas the MHB skin cream leaves detectable residues after each application. CONCLUSION: Although the APP and MHB preparations were not significantly different in effectiveness and safety, distinctively, application of the APP skin cream preparation absorbed into the skin leaving no discernible residue in contrast to the MHB preparation leaving residues potentiating textile damage. Both of these technologies function in the hydration of skin; however, they differ in their modes of action. The plant-based APP preparation functions actively by phospholipid and triglyceride absorption, reparation of skin lamellae, and in the consequent delivery and organization of waters of hydration in skin. The MHB preparation functions passively, hydrating the skin it covers by sealing the skin against dehydration.

Periodic bilayer organization in the complexes of Beta-2 Glycoprotein I with anionic lipid membranes.

ß2 glycoprotein I (ß2GPI) is a soluble protein that participates in blood coagulation, clearance of apoptotic bodies and generation of antigens in antiphospholipid syndrome among many other functions. We studied the aggregates formed by ß2GPI with the anionic phospholipids palmitoyloleoylphosphatidyl glycerol, dimyristoylphosphatidyl glycerol, dipalmitoylphosphatidyl glycerol and cardiolipin using small angle X-ray scattering. The complexes obtained in a medium containing 0.01 M NaCl showed Bragg peaks up to the sixth order in a well-defined integer sequence indicating the presence of a lamellar stacking with a periodicity of 17.8 nm and with largely reduced membrane fluctuations. Modeling the complex signal allowed us to conclude that the coherence length was only two bilayers and that about 15% of the total surface was actually stacked. The space between bilayers allows accommodating an extended ß2GPI molecule making a bridge between the interacting bilayers. The interactions between membranes mediated by ß2GPI was favored when the membranes were in the liquid crystalline state.

Alteration of Membrane Fluidity or Phospholipid Composition Perturbs Rotation of MreB Complexes in Escherichia coli.

Gram-negative bacteria such as Escherichia coli are surrounded by inner and outer membranes and peptidoglycan in between, protecting the cells from turgor pressure and maintaining cell shape. The Rod complex, which synthesizes peptidoglycan, is composed of various proteins such as a cytoplasmic protein MreB, a transmembrane protein RodZ, and a transpeptidase PBP2. The Rod complex is a highly motile complex that rotates around the long axis of a cell. Previously, we had reported that anionic phospholipids (aPLs; phosphatidylglycerol and cardiolipin) play a role in the localization of MreB. In this study, we identified that cells lacking aPLs slow down Rod complex movement. We also found that at higher temperatures, the speed of movement increased in cells lacking aPLs, suggesting that membrane fluidity is important for movement. Consistent with this idea, Rod complex motion was reduced, and complex formation was disturbed in the cells depleted of FabA or FabB, which are essential for unsaturated fatty acid synthesis. These cells also showed abnormal morphology. Therefore, membrane fluidity is important for maintaining cell shape through the regulation of Rod complex formation and motility.

Two distinct anionic phospholipid-dependent events involved in SecA-mediated protein translocation.

Protein translocation across the bacterial cytoplasmic membrane is an essential process catalyzed by the Sec translocase, which in its minimal form consists of the protein-conducting channel SecYEG, and the motor ATPase SecA. SecA binds via its positively charged N-terminus to membranes containing anionic phospholipids, leading to a lipid-bound intermediate. This interaction induces a conformational change in SecA, resulting in a high-affinity association with SecYEG, which initiates protein translocation. Here, we examined the effect of anionic lipids on the SecA-SecYEG interaction in more detail, and discovered a second, yet unknown, anionic lipid-dependent event that stimulates protein translocation. Based on molecular dynamics simulations we identified an anionic lipid-enriched region in vicinity of the lateral gate of SecY. Here, the anionic lipid headgroup accesses the lateral gate, thereby stabilizing the pre-open state of the channel. The simulations suggest flip-flop movement of phospholipid along the lateral gate. Electrostatic contribution of the anionic phospholipids at the lateral gate may directly stabilize positively charged residues of the signal sequence of an incoming preprotein. Such a mechanism allows for the correct positioning of the entrant peptide, thereby providing a long-sought explanation for the role of anionic lipids in signal sequence folding during protein translocation.

Maspin binds to cardiolipin in mitochondria and triggers apoptosis.

A central question in cell biology is how cells respond to stress signals and biochemically regulate apoptosis. One critical pathway involves the change of mitochondrial function and release of cytochrome c to initiate apoptosis. In response to apoptotic stimuli, we found that maspin-a noninhibitory member of the serine protease inhibitor superfamily-translocates from the cytosol to mitochondria and binds to cardiolipin in the inner mitochondrial membrane. Biolayer interferometry assay revealed that recombinant maspin binds cardiolipin with an apparent Kd,of ∼15.8 µM and competes with cytochrome c (apparent Kd of ∼1.31 µM) for binding to cardiolipin-enriched membranes. A hydrophobic, lysine-rich domain in maspin consists of 27 aa, is located at position 268-294, and is responsible for the interaction of this protein with cardiolipin. Depletion of cardiolipin in cells significantly prevents maspin binding to the inner mitochondrial membrane and decreases cytochrome c release and apoptosis. Alteration to maspin's cardiolipin binding domain changes its ability to bind cardiolipin, and tumor cells expressing this mutant have a low frequency of apoptosis. We propose a model of apoptosis in which maspin binds to cardiolipin, displaces cytochrome c from the membrane, and facilitates its release to the cytoplasm.-Mahajan, N., Hoover, B., Rajendram, M., Shi, H. Y., Kawasaki, K., Weibel, D. B., Zhang, M. Maspin binds to cardiolipin in mitochondria and triggers apoptosis.

Rcs Phosphorelay Activation in Cardiolipin-Deficient Escherichia coli Reduces Biofilm Formation.

Biofilm formation is a complex process that requires a number of transcriptional, proteomic, and physiological changes to enable bacterial survival. The lipid membrane presents a barrier to communication between the machinery within bacteria and the physical and chemical features of their extracellular environment, and yet little is known about how the membrane influences biofilm development. We found that depleting the anionic phospholipid cardiolipin reduces biofilm formation in Escherichia coli cells by as much as 50%. The absence of cardiolipin activates the regulation of colanic acid synthesis (Rcs) envelope stress response, which represses the production of flagella, disrupts initial biofilm attachment, and reduces biofilm growth. We demonstrate that a reduction in the concentration of cardiolipin impairs translocation of proteins across the inner membrane, which we hypothesize activates the Rcs pathway through the outer membrane lipoprotein RcsF. Our study demonstrates a molecular connection between the composition of membrane phospholipids and biofilm formation in E. coli and suggests that altering lipid biosynthesis may be a viable approach for altering biofilm formation and possibly other multicellular phenotypes related to bacterial adaptation and survival.IMPORTANCE There is a growing interest in the role of lipid membrane composition in the physiology and adaptation of bacteria. We demonstrate that a reduction in the anionic phospholipid cardiolipin impairs biofilm formation in Escherichia coli cells. Depleting cardiolipin reduced protein translocation across the inner membrane and activated the Rcs envelope stress response. Consequently, cardiolipin depletion produced cells lacking assembled flagella, which impacted their ability to attach to surfaces and seed the earliest stage in biofilm formation. This study provides empirical evidence for the role of anionic phospholipid homeostasis in protein translocation and its effect on biofilm development and highlights modulation of the membrane composition as a potential method of altering bacterial phenotypes related to adaptation and survival.

Specific binding of human C-reactive protein towards supported monolayers of binary and engineered phospholipids.

Circulating C-reactive protein (CRP) recognizes altered plasma membranes and activates complements systems in the acute phase of inflammation and infection in human. We have shown previously the calcium-independent adsorption of CRP toward 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and lysophosphatidylcholine (LPC) on supported phospholipid monolayers. Here, we extended our study to other phospholipids and additives to elucidate the pattern recognition of CRP using a surface plasmon resonance biosensor. Surface density and lateral fluidity depended on the type of phospholipids in the monolayers as characterized by SPR and fluorescence recovery after photobleaching measurements. CRP recognized 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) in the supported POPC monolayers without calcium at pH 7.4 and 5.5. As opposed to LPC, CRP did not recognize 3-sn-lysophosphatidylethanolamine in the POPC monolayers in calcium-free conditions. While, the addition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or sphingomyelin to supported POPC monolayers blocked CRP adsorption. Calcium-dependent CRP binding was observed only at pH 5.5 on supported monolayers of engineered phospholipids with inverted headgroups relative to POPC. The complement 1q (C1q) protein recognized the active form of CRP on the supported phospholipid monolayers. The discovery of CRP recognition with these phospholipids aids our understanding of the activation dynamics of CRP with phospholipid-based biomaterials when used during the acute phase.

Mechanisms of thrombosis in systemic lupus erythematosus and antiphospholipid syndrome.

The presence of antiphospholipid antibodies is one of the most common acquired risk factors for thrombosis. Antiphospholipid antibodies is a collective term for a set of autoantibodies with closely related but different specificity. Experiments in which isolated patient antibodies were injected into mice have shown that a specific subset of autoantibodies, those directed against the first domain of plasma protein ß2-glycoprotein I, can explain the increased risk of thrombosis. Experiments performed with these mice have shown that autoantibodies against ß2-glycoprotein I bind to and activate cells such as endothelial cells, monocytes, and platelets. Activation of these cells, all involved in the regulation of hemostasis, results in a shift towards a prothrombotic state. How this process is regulated, whether this is the only mechanism involved, and whether this is the only subpopulation responsible for the increased thrombotic risk is unknown. In this review, we will critically discuss what is known and what is debatable on the pathophysiology of antiphospholipid syndrome.

Polar localization of MreB actin is inhibited by anionic phospholipids in the rod-shaped bacterium Escherichia coli.

Bacterial actin MreB is required for the maintenance of cell polarity. MreB is located underneath the cell membrane and mainly localizes at a central cylindrical part of the cell. In addition, it has recently been found that anionic phospholipids (aPLs: phosphatidylglycerol and cardiolipin) play a crucial role in excluding MreB from the cell poles. Subcellular localization of MreB is positively and negatively regulated by membrane curvature and aPLs, respectively.

Lipids regulate the hydrolysis of membrane bound glucosylceramide by lysosomal ß-glucocerebrosidase.

Glucosylceramide (GlcCer) is the primary storage lipid in the lysosomes of Gaucher patients and a secondary one in Niemann-Pick disease types A, B, and C. The regulatory roles of lipids on the hydrolysis of membrane bound GlcCer by lysosomal ß-glucocerebrosidase (GBA1) was probed using a detergent-free liposomal assay. The degradation rarely occurs at uncharged liposomal surfaces in the absence of saposin (Sap) C. However, anionic lipids stimulate GlcCer hydrolysis at low pH by up to 1,000-fold depending on the nature and position of the negative charges in their head groups while cationic lipids inhibit the degradation, thus showing the importance of electrostatic interactions between the polycationic GBA1 and the negatively charged vesicle surfaces at low pH. Ceramide, fatty acids, monoacylglycerol, and diacylglycerol also stimulate GlcCer hydrolysis while SM, sphingosine, and sphinganine play strong inhibitory roles, thereby explaining the secondary storage of GlcCer in Niemann-Pick diseases. Surprisingly, cholesterol stimulates GlcCer degradation in the presence of bis(monoacylglycero)phosphate (BMP). Sap C strongly stimulates GlcCer hydrolysis even in the absence of BMP and the regulatory roles of the intraendolysosomal lipids on its activity is discussed. Our data suggest that these strong modifiers of GlcCer hydrolysis affect the genotype-phenotype correlation in several cases of Gaucher patients independent of the types.

Organization and function of anionic phospholipids in bacteria.

In addition to playing a central role as a permeability barrier for controlling the diffusion of molecules and ions in and out of bacterial cells, phospholipid (PL) membranes regulate the spatial and temporal position and function of membrane proteins that play an essential role in a variety of cellular functions. Based on the very large number of membrane-associated proteins encoded in genomes, an understanding of the role of PLs may be central to understanding bacterial cell biology. This area of microbiology has received considerable attention over the past two decades, and the local enrichment of anionic PLs has emerged as a candidate mechanism for biomolecular organization in bacterial cells. In this review, we summarize the current understanding of anionic PLs in bacteria, including their biosynthesis, subcellular localization, and physiological relevance, discuss evidence and mechanisms for enriching anionic PLs in membranes, and conclude with an assessment of future directions for this area of bacterial biochemistry, biophysics, and cell biology.

Studies on the interactions of bisphenols with anionic phospholipids of decomposer membranes in model systems.

Bisphenol A (BPA) and other bisphenols constitute a class of organic pollutants, which because of their estrogenic properties, low dose activity and bioaccumulation pose considerable risk for public health as well as for the environment. Accumulated in the sediment bisphenols can endanger the decomposers' populations being incorporated into their cellular membranes; however, the mechanism of their membrane activity is unknown. Therefore, to study these phenomena we applied anionic phospholipid Langmuir monolayers as simple but versatile models of decomposers biomembranes. Phosphatidylglycerols and cardiolipins are not only the main components of bacterial membranes but also of crucial importance in mitochondrial and thylakoid membranes in eukaryotic cells. In our investigations we applied five compounds of the bisphenol class most commonly detected in the environment. To characterize the bisphenols-model membrane interactions we applied multiple mutually independent methods of physical chemistry; namely: the Langmuir monolayer technique, surface potential measurements, Brewster angle microscopy for the visualization of the monolayers' texture and grazing incidence X-ray diffraction for the discussion of the phospholipids packing within the monolayers. Our studies indicated that all the investigated bisphenols interact with the model membrane, but the strength of the interactions is dependent on the bisphenol structure and hydrophobicity and the fluidity of the model membranes. We proved that bisphenol S often treated as the least toxic BPA analog can also be incorporated to the model membranes changing their structure and fluidity.

The synaptotagmin juxtamembrane domain is involved in neuroexocytosis.

Synaptotagmin is a synaptic vesicle membrane protein which changes conformation upon Ca(2+) binding and triggers the fast neuroexocytosis that takes place at synapses. We have synthesized a series of peptides corresponding to the sequence of the cytosolic juxtamembrane domain of synaptotagmin, which is highly conserved among different isoforms and animal species, with or without either a hexyl hydrophobic chain or the hexyl group plus a fluorescein moiety. We show that these peptides inhibit neurotransmitter release, that they localize on the presynaptic membrane of the motor axon terminal at the neuromuscular junction and that they bind monophosphoinositides in a Ca(2+)-independent manner. Based on these findings, we propose that the juxtamembrane cytosolic domain of synaptotagmin binds the cytosolic layer of the presynaptic membrane at rest. This binding brings synaptic vesicles and plasma membrane in a very close apposition, favouring the formation of hemifusion intermediates that enable rapid vesicle fusion.

Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2.

During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747-1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion.