The fusogenic effect of synthetic polycations on negatively charged lipid bilayers

The effect of synthetic polycations, polyallylamine, and polyethylenimine, on liposomes containing phosphatidylserine was investigated along with that of polylysine and divalent cations. The addition of polycations caused aggregation of sonicated vesicles composed of phosphatidylserine and phosphatidylcholine (molar ratio 1:4) as determined by measuring the turbidity changes. Liposomal turbidity increased 10 times compared with that of control liposomes at charge ratios of polymer/vesicle from 0.23 (polylysine) to 2.5 (linear polyethylenimine), while the turbidity was unchanged by the addition of Ca2+ or Mg2+ at charge ratios up to 500. These polycations also induced intermixing of liposomal membranes as indicated by resonance energy transfer between fluorescent lipids incorporated in lipid bilayers, without inducing drastic permeability changes as determined from the calcein release. Fifty percent intermixing of liposomes (0.05 mM as lipid concentration) was induced by these polycations at charge ratios of around 1.0. However, the highest resonance energy transfer was produced by the addition of polyallylamine, which caused multicycles of membrane intermixing between vesicles. Polycation-induced membrane intermixing and permeability changes of phosphatidylserine liposomes were also investigated. At charge ratios of around 1.0, these polymers caused resonance energy transfer of fluorescent lipids incorporated in separate vesicles; however, polyallylamine and branched polyethylenimine also caused permeability increases of liposomal membranes. Membrane intermixing and permeability changes of phosphatidylserine vesicles induced by polyallylamine were dependent on the polymer/vesicle charge ratio, and were different from those induced by Ca2+ since the latter caused half-maximal membrane intermixing or permeability change of phosphatidylserine vesicles at about 1 mM at the liposomal concentrations investigated.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

Studies on vinblastine-induced autophagocytosis in mouse liver. IV. Origin of membranes

The origin of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied by cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). The marker enzymes used were adenosine triphosphatase for the plasma membrane, glucose-6-phosphatase for the endoplasmic reticulum and thiamine pyrophosphatase for the Golgi apparatus and the endoplasmic reticulum. All the three enzymes showed a characteristic localization in both control and vinblastine-treated hepatocytes. The space between the limiting membranes of a few apparently newly formed AV's showed weak glucose-6-phosphatase activity. Neither adenosine triphosphatase nor thiamine pyrophosphatase activities were observed on or between the AV membranes. It was suggested that endoplasmic reticulum membranes may be used as a source of AV membranes in hepatocytes. The lack of glucose-6-phosphatase activity in the limiting membranes even of most of the newly formed AV's suggests a transformation process of the membranes destined to form AV, during which the enzyme activity characteristic for endoplasmic reticulum may disappear from them.     

Studies on vinblastine-induced autophagocytosis in mouse liver

Summary The origin of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied by cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). The marker enzymes used were adenosine triphosphatase for the plasma membrane, glucose-6-phosphatase for the endoplasmic reticulum and thiamine pyrophosphatase for the Golgi apparatus and the endoplasmic reticulum. All the three enzymes showed a characteristic localization in both control and vinblastine-treated hepatocytes. The space between the limiting membranes of a few apparently newly formed AV's showed weak glucose-6-phosphatase activity. Neither adenosine triphosphatase nor thiamine pyrophosphatase activities were observed on or between the AV membranes. It was suggested that endoplasmic reticulum membranes may be used as a source of AV membranes in hepatocytes. The lack of glucose-6-phosphatase activity in the limiting membranes even of most of the newly formed AV's suggests a transformation process of the membranes destined to form AV, during which the enzyme activity characteristic for endoplasmic reticulum may disappear from them.     

Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry

Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes “artificial plasma membrane mimicking” (“PMm”) vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca2+ concentration was increased to a threshold of 3--5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 micrometer diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2+-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca2+ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca2+ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca2+ concentration.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca²⁺ concentration was increased to a threshold of 3–5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 μm diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca²⁺-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca²⁺ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca²⁺ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca²⁺ concentration.     

Cholesterol transfer between lipid vesicles. Effect of phospholipids and gangliosides.

The effect of lipid composition on the rate of cholesterol movement between cellular membranes is investigated using lipid vesicles. The separation of donor and acceptor vesicles required for rate measurement is achieved by differential centrifugation so that the lipid effect can be quantified in the absence of a charged lipid generally used for ion-exchange-based separation. The rate of cholesterol transfer from small unilamellar vesicles (SUVs) containing 50 mol% cholesterol to a common large unilamellar vesicle (LUV) acceptor containing 20 mol% cholesterol decreases with increasing mol% of sphingomyelin in the SUVs, while phosphatidylethanolamine and phosphatidylserine have no appreciable effect at physiologically relevant levels. There is a large decrease in rate when phosphatidylethanolamine constitutes 50 mol% of donor phospholipids. Interestingly, gangliosides which have the same hydrocarbon moiety as sphingomyelin exert an opposite effect. The effect of spingomyelin seems to be mediated by its ability to decrease the fluidity of the lipid matrix, while that of gangliosides may arise from a weakening of phosphatidylcholine-cholesterol interactions or from a more favourable (less polar) microenvironment for the desorption of cholesterol provided by the head-group interactions involving sugar residues. If the effect of asymmetric transbilayer distribution of lipids is taken into consideration, the observed composition-dependent rate changes could partly account for the large difference in the rates of cholesterol desorption from the inner and outer layers of plasma membrane. Such rate differences may be responsible for an unequal steady-state distribution of cholesterol among various cellular membranes and lipoproteins.     

Membrane events involved in myoblast fusion

Myoblast fusion has been studied in cultures of chick embryonic muscle utilizing ultrastructural techniques. The multinucleated muscle cells (myotubes) are generated by the fusion of two plasma membranes from adjacent cells, apparently by forming a single bilayer that is particle-free in freeze-fracture replicas. This single bilayer subsequently collapses, and cytoplasmic continuity is established between the cells. The fusion between the two plasma membranes appears to take place primarily within particle-free domains (probably phospholipid enriched), and cytoplasmic unilamellar, particle-free vesicles are occasionally associated with these regions. These vesicles structurally resemble phospholipid vesicles (liposomes). They are present in normal myoblasts, but they are absent in certain fusion-arrested myoblast popluations, such as those treated with either 5-bromo-deoxyuridine (BUdR), cycloheximide (CHX), or pospholipase C (PLC). The unilamellar, particle-free vesicles are present in close proximity to the plasma membranes, and physical contact is observed frequently between the vesicle membrane and the plasma membrane. The regions of vesicle membrane-plasma membrane interaction are characteristically free of intramembrane particles. A model for myoblast fusion is presented that is based onan interpretation of these observations. This model suggests that the cytoplasmic vesicles initiate the generation of particle-depleted membrane domains, both being essential components in the fusion process.     

ATP-dependent fusion of liposomes with the Golgi apparatus of perforated cells

We examined the interactions of lipid vesicles (liposomes) labeled with various fluorescent markers with the intracellular membranes of semi-intact ("perforated") fibroblasts. When incubations were performed in the presence of an ATP-regenerating system, both vesicle lipids and entrapped water soluble markers were transferred to the Golgi apparatus of treated cells, indicative of membrane fusion. Fusion occurred using unilamellar vesicles 30-80 nm in diameter and composed of phosphatidylcholine alone, but was inhibited when equimolar amounts of either phosphatidylserine or sphingomyelin were present in the vesicles. Lipid vesicle-Golgi membrane fusion was also inhibited by pretreatment of the perforated cells with N-ethylmaleimide. These findings suggest that lipid vesicles may be useful for delivery of labeled lipids, macromolecules, and dyes to the Golgi apparatus, and for modeling the interactions of transport vesicles with the Golgi apparatus.     

Pardaxin induces aggregation but not fusion of phosphatidylserine vesicles

The effects on membranes of pardaxin, an amphipathic polypeptide, purified from the gland secretion of the Red Sea Moses sole flatfish Pardachirus marmoratus are dose-dependent and range from formation of voltage-gated, cation-selective pores to lysis. We have now investigated the interactions of pardaxin with small unilamellar liposomes. Light scattering showed that pardaxin (10⁻⁷–10⁻⁹M) mediated the aggregation of liposomes composed of phosphatidylserine but not of phosphatidylcholine. Aggregation of phosphatidylserine vesicles was impaired by vesicle depolarization. Furthermore, pardaxin-mediated aggregation between fluorescent-labeled PS vesicles was accompanied by leakage of the vesicle contents, and not by fusogenic process within the aggregates. We suggest that pardaxin is a unique polypeptide, that induces vesicle aggregation and membrane destabilization, but not membrane fusion; the mechanism of the aggregation activity of pardaxin is related to its amphipathic properties.     

Effects of proteins on phospholipid vesicle aggregation and lipid vesicle-monolayer interactions

The effects of proteins on divalent cation-induced phospholipid vesicle aggregation and phospholipid vesicle-monolayer membrane interactions (fusion) were examined. Glycophorin (from human erythrocytes) suppressed the membrane interactions more than N-2 protein (from human brain myelin) when these proteins were incorporated into acidic phospholipid vesicle membranes. The threshold concentrations of divalent cations which induced vesicle aggregation were increased by protein incorporation, and the rate of vesicle aggregation was reduced. A similar inhibitory effect by the proteins, incorporated into lipid vesicle membranes, was observed for Ca²⁺-induced lipid vesicle-monolayer interactions. However, when these proteins were incorporated only in the acidic phospholipid monolayers, the interaction (fusion) of the lipid vesicle-monolayer membranes, induced by divalent cations, was not appreciably altered by the presence of the proteins.In contrast to these two proteins, the presence of synexin in the solution did enhance the Ca²⁺-induced aggregation of phosphatidylserine vesicles, but did not seem to affect the degree of Ca²⁺-induced fusion between phosphatidylserine/phosphatidylcholine (1:1) and phosphatidylserine vesicles and monolayer membranes.     

Reversible Liposome Association Induced by LAH4: A Peptide with Potent Antimicrobial and Nucleic Acid Transfection Activities

We report on the reversible association of anionic liposomes induced by an antimicrobial peptide (LAH4). The process has been characterized for mixed membranes of POPC and POPS at molar ratios of 1:1, 3:1, and 9:1. Although the vesicles remain in suspension in the presence of excess amounts of peptide, the addition of more lipids results in surface charge neutralization, aggregation of the liposomes, and formation of micrometer-sized structures that coexist in equilibrium with vesicles in suspension. At low ratios of anionic lipids, vesicle aggregation is a reversible process, and vesicle disassembly is observed upon inversion of the surface charge by further supplementation with anionic vesicles. In contrast, a different process, membrane fusion, occurs in the presence of high phosphatidylserine concentrations. Upon binding to membranes containing low POPS concentrations, the peptide adopts an in-plane α-helical structure, a secondary structure that is conserved during vesicle association and dissociation. Our finding that peptides are essential for vesicle aggregation contributes to a better understanding of the activity of antimicrobial peptides, and suggests an additional layer of complexity in membrane-protein lipid interactions.     

Low pH induced membrane fusion of lipid vesicles containing proton-sensitive polymer

For the purpose of cytoplasmic delivery of aqueous content in liposomes through endosomes, we synthesized a pH-sensitive polymer, cetylacetyl(imidazol-4-ylmethyl)polyethylenimine (CAIPEI), which generates polycations at acidic pH. CAIPEI in its aqueous phase caused aggregation of sonicated vesicles composed of phosphatidylserine (PS) and phosphatidylcholine (PC) (molar ratio 1:4) when the pH of the solution was lowered. The polymer also induced membrane intermixing as measured by resonance energy transfer between vesicles containing N-(7-nitro-2,1,3-benz[d]oxadiazol-4-yl)phosphatidylethanolamine and those containing N-Rhodamine phosphatidylethanolamine at pH 4-5, while the addition of CAIPEI caused neither aggregation of PC vesicles nor the intermixing of liposomal membranes between PC and PC/PS vesicles at any pH. The CAIPEI-induced membrane intermixing was dependent on the polymer/vesicle ratio rather than on the polymer concentration. Then the polymer was incorporated into the bilayers of PC vesicles. These CAIPEI vesicles also caused membrane intermixing with liposomes containing PS under acidic conditions. The reconstituted CAIPEI did not reduce the trapping efficiency of vesicles or increase their permeability to glucose even at low pH. The vesicles caused the low pH induced aggregation and membrane intermixing with other negatively charged liposomes containing phosphatidic acid or phosphatidylglycerol. These results suggest that the protonation of the polymer at acidic pH endows the CAIPEI vesicles with the activity to fuse with negatively charged liposomes.     

Enhanced binding of phosphatidylserine-containing lipid vesicle targets to RAW264 macrophages

Summary Phosphatidylserine was found to significantly enchance the binding of phospholipid vesicles to RAW264 macrophages. We have measured the kinetics of non-specific uptake of unilamellar vesicles as a function of phosphatidylserine concentration in these model target membranes. Dimyristoylphosphatidylcholine was the principle component of these phospholipid vesicles. In most experiments, radiolabeled phospholipid and 1 mol % each of both a fluorescent phospholipid and a hapten-containing lipid headgroup were utilized. In the presence of specific anti-hapten antibody phosphatidylserine-containing vesicles are rapidly taken up via phagocytosis. The antibody-independent non-specific uptake of phosphatidylserine-free vesicles was low, as previously reported. However, the presence of 5 mol % phosphatidylserine dramatically enhanced the uptake of phospholipid vesicles by macrophages. This uptake was shown to be principally due to binding to the macrophage surface. Incubation of macrophages in the presence of sodium azide or at 4°C, conditions which are known to inhibit phagocytosis, do not influence the uptake of the lipid vesicles. Fluorescence video-intensification microscopy was used to observe the interaction of carboxyfluorescein-loaded vesicles with macrophages. Fluorescence could not be observed when using phosphatidylserine-free vesicles. However, phosphatidylserine-containing vesicles can be observed bound to the cell periphery. Intracellular fluorescence could not be observed. The binding of phosphatidylserine-containing vesicles was enhanced roughly four-fold over phosphatidylserine because the effect could not be observed with membranes containing 1 mol % or 2.5 mol% phosphatidylserine. In addition, the binding enhancement required the presence of divalent cations in the incubation medium.Abbreviations DMPC dimyristoylphosphatidylcholine - PS phosphatidylserine - DNP-PE dinitrophenyl---minocaproyl-phosphatidylethanolamime - NBDPE N-4-nitrobenzo-2-oxa-1, 3-diazole phosphatidylethanolamine - EDTA ethylenediaminetetraacetic acid     

Characterization of P4 ATPase Phospholipid Translocases (Flippases) in Human and Rat Pancreatic Beta Cells: THEIR GENE SILENCING INHIBITS INSULIN SECRETION*

The negative charge of phosphatidylserine in lipid bilayers of secretory vesicles and plasma membranes couples the domains of positively charged amino acids of secretory vesicle SNARE proteins with similar domains of plasma membrane SNARE proteins enhancing fusion of the two membranes to promote exocytosis of the vesicle contents of secretory cells. Our recent study of insulin secretory granules (ISG) (MacDonald, M. J., Ade, L., Ntambi, J. M., Ansari, I. H., and Stoker, S. W. (2015) Characterization of phospholipids in insulin secretory granules in pancreatic beta cells and their changes with glucose stimulation. J. Biol. Chem. 290, 11075–11092) suggested that phosphatidylserine and other phospholipids, such as phosphatidylethanolamine, in ISG could play important roles in docking and fusion of ISG to the plasma membrane in the pancreatic beta cell during insulin exocytosis. P4 ATPase flippases translocate primarily phosphatidylserine and, to a lesser extent, phosphatidylethanolamine across the lipid bilayers of intracellular vesicles and plasma membranes to the cytosolic leaflets of these membranes. CDC50A is a protein that forms a heterodimer with P4 ATPases to enhance their translocase catalytic activity. We found that the predominant P4 ATPases in pure pancreatic beta cells and human and rat pancreatic islets were ATP8B1, ATP8B2, and ATP9A. ATP8B1 and CDC50A were highly concentrated in ISG. ATP9A was concentrated in plasma membrane. Gene silencing of individual P4 ATPases and CDC50A inhibited glucose-stimulated insulin release in pure beta cells and in human pancreatic islets. This is the first characterization of P4 ATPases in beta cells. The results support roles for P4 ATPases in translocating phosphatidylserine to the cytosolic leaflets of ISG and the plasma membrane to facilitate the docking and fusion of ISG to the plasma membrane during insulin exocytosis.     

Docking and fast fusion of synaptobrevin vesicles depends on the lipid compositions of the vesicle and the acceptor SNARE complex-containing target membrane

The influence of the lipid environment on docking and fusion of synaptobrevin 2 (Syb2) vesicles with target SNARE complex membranes was examined in a planar supported membrane fusion assay with high time-resolution. Previously, we showed that approximately eight SNARE complexes are required to fuse phosphatidylcholine (PC) and cholesterol model membranes in ∼20 ms. Here we present experiments, in which phosphatidylserine (PS) and phosphatidylethanolamine (PE) were added to mixtures of PC/cholesterol in different proportions in the Syb2 vesicle membranes only or in both the supported bilayers and the Syb2 vesicles. We found that PS and PE both reduce the probability of fusion and that this reduction is fully accounted for by the lipid composition in the vesicle membrane. However, the docking efficiency increases when the PE content in the vesicle (and target membrane) is increased from 0 to 30%. The fraction of fast-activating SNARE complexes decreases with increasing PE content. As few as three SNARE complexes are sufficient to support membrane fusion when at least 5% PS and 10% PE are present in both membranes or 5% and 30% PE are present in the vesicle membrane only. Despite the smaller number of required SNAREs, the SNARE activation and fusion rates are almost as fast as previously reported in reconstituted PC/cholesterol bilayers, i.e., of 10 and ∼20 ms, respectively.     

Disappearance of macrophage surface folds after antibody-dependent phagocytosis

We have employed the method of Burwen and Satir (J. Cell Biol., 1977, 74:690) to measure the disappearance of surface folds from resident guinea pig peritoneal macrophages after antibody-dependent phagocytosis. Unilamellar phospholipid vesicles containing dimyristoylphosphatidylcholine and 1 mol % dinitrophenyl-epsilon- aminocaproyl-phosphatidylethanolamine, a lipid that possesses a hapten headgroup, were prepared by an ether injection technique. These vesicles were taken up by macrophages in a time- and temperature- dependent fashion. Vesicles that contained ferritin trapped in the internal aqueous volume were identified within macrophages by transmission electron microscopy. Scanning electron microscopy has shown that macrophage surface folds decrease dramatically after phagocytosis. The surface fold length (micrometer) per unit smooth sphere surface area (micrometer2) decreases from 1.3 +/- 0.3 micrometer- 1 to 0.53 +/- 0.25 micrometer-1 when cells are incubated in the presence of specific anti-DNP antibody and vesicles at 37 degrees C. No significant effect was observed in the presence of antibody only or vesicles only. Our studies shown that phagocytosis is associated with a loss of cell surface folds and a loss of cell surface area, which is consonant with current views of the endocytic process. On the basis of our uptake data, we estimate that approximately 400 micrometer2 of vesicle surface membrane is internalized. The guinea pig macrophage plasma membrane has a total area of approximately 400 micrometer2 in control studies, whereas the cells have roughly 300 micrometer2 after phagocytosis. These estimates of surface areas include membrane ruffles and changes directly related to changes in cell volume. We suggest that during antibody-dependent phagocytosis a membrane reservoir is made available to the cell surface.     

Role of Phosphatidylserine in Phospholipid Flippase-Mediated Vesicle Transport in Saccharomyces cerevisiae

Phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. The genome of budding yeast encodes four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), which associate with the Cdc50 family noncatalytic subunit, and one monomeric flippase Neo1p. Flippases have been implicated in the formation of transport vesicles, but the underlying mechanisms are largely unknown. We show here that overexpression of the phosphatidylserine synthase gene CHO1 suppresses defects in the endocytic recycling pathway in flippase mutants. This suppression seems to be mediated by increased cellular phosphatidylserine. Two models can be envisioned for the suppression mechanism: (i) phosphatidylserine in the cytoplasmic leaflet recruits proteins for vesicle formation with its negative charge, and (ii) phosphatidylserine flipping to the cytoplasmic leaflet induces membrane curvature that supports vesicle formation. In a mutant depleted for flippases, a phosphatidylserine probe GFP-Lact-C2 was still localized to endosomal membranes, suggesting that the mere presence of phosphatidylserine in the cytoplasmic leaflet is not enough for vesicle formation. The CHO1 overexpression did not suppress the growth defect in a mutant depleted or mutated for all flippases, suggesting that the suppression was dependent on flippase-mediated phospholipid flipping. Endocytic recycling was not blocked in a mutant lacking phosphatidylserine or depleted in phosphatidylethanolamine, suggesting that a specific phospholipid is not required for vesicle formation. These results suggest that flippase-dependent vesicle formation is mediated by phospholipid flipping, not by flipped phospholipids.     

Evidence that the platelet plasma membrane does not contain a (Ca2+ + Mg2+)-dependent ATPase

The present study was designed to determine the subcellular distribution of the platelet (Ca2+ + Mg2+)-ATPase. Human platelets were surface labeled by the periodate-boro[3H]hydride method. Plasma membrane vesicles were then isolated to a purity of approx. 90% by a procedure utilizing wheat germ agglutinin affinity chromatography. These membranes were found to be 2.6-fold enriched in surface glycoproteins compared to an unfractionated vesicle fraction and almost 7-fold enriched compared to intact platelets. In contrast, the isolated plasma membranes showed a decreased specific activity of the (Ca2+ + Mg2+)-ATPase compared to the unfractionated vesicle fraction. This decrease in specific activity was found to be similar to that of an endoplasmic reticulum marker, glucose-6-phosphatase, and to that of a platelet inner membrane marker, phospholipase A2. We conclude, therefore, that the (Ca2+ + Mg2+)-ATPase is not located in the platelet plasma membrane but is restricted to membranes of intracellular origin.