Phospholipid Translocation Research Articles

The role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes.

To elucidate the role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes, we examined the interaction of erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion of human erythrocytes was monitored by light microscopy as well as spectrophotometrically by the octadecylrhodamine dequenching assay. Phospholipid translocation and distribution between the inner and the outer leaflet of intact red blood cells were determined with spin-labeled phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Significant fusion of lipid-asymmetric red blood cells where PS and PE are predominantly oriented to the inner leaflet was only observed at Ca2+ concentrations greater than or equal to 10 mM (in the presence of 10 mM phosphate buffer) while fusion of lipid-symmetric erythrocyte membranes was established at greater than or equal to 1.5 mM Ca2+. The Ca2+ threshold of fusion of lipid-asymmetric red blood cells was significantly reduced (i) after exposure of PS to the outer layer but not after redistribution of PE alone, and (ii) upon incorporation of spin-labeled PS into the outer leaflet of red blood cells. Spin-labeled PE or PC did not affect fusion, suggesting that the serine headgroup is an important factor in calcium-phosphate-induced fusion.

Relationships between the surface exposure of acidic phospholipids and cell fusion in erythrocytes subjected to electrical breakdown

The procoagulant activity of human erythrocytes, which provides a measure of the translocation of acidic phospholipids from the inner to the outer monolayer of the plasma membrane, has been compared with the percentage cell fusion in experiments on the effects of electrical breakdown pulses under differing experimental conditions. After treatment with breakdown pulses of 20 μs or longer (5 kV cm−1), the plasma membranes of erythrocytes in 250 mM sucrose exhibited an almost complete loss of asymmetry with respect to acidic phospholipids. As the breakdwon voltage was increased from 2 to 5 kV cm−1 (with breakdown pulses of 99 μs), the surface exposure of acidic phospholipids and cell fusion increased approximately in parallel. Furthermore, with 99 μs pulses and a voltage of 3 kV cm−1, a decrease in the osmolarity from 250 to 150 mM of the source medium was accompanied by an increase in both the surface exposure of acidic phospholipids and the extent of cell fusion. Breakdwon pulses of 2–5 μs were sufficient to cause a marked loss of asymmetry, but no cell fusion was observed unless the pulse length was at least 20 μs. Kinetic experiments indicated that exposure of the acidic phospholipids at the cell surface was more likely to be due to a direct effect of the electric field pulses on plasma membrane structure than to secondary effects, such as the action of endogenous proteinases on the membrane skeleton. It seems possible that a localised, surface exposure of acidic phospholipids may contribute to the ‘long-lived fusogenic state’ (Sowers, A.E. (1986) J. Cell Biol. 102, 1358–1362) and the ‘transient permeant structures’ (Teissié, J. and Rols, M.P. (1986) Biochem. Biophys. Res. Commun. 140, 258–266) than enable cell fusion to occur when contact between cells is established after they have been subjected to field pulses. Our observations alos provide circumstantial support for the concept that changes in the phospholipid asymmetry of membrane may be important in physiologically-occurring instances of biomembrane fusion.

Mitochondrial membrane contact sites of yeast. Characterization of lipid components and possible involvement in intramitochondrial translocation of phospholipids

Submitochondrial membrane fractions from yeast that are enriched in inner and outer membrane contact sites were analyzed with respect to their lipid composition. Characteristic features were the significantly reduced content of phosphatidylinositol, the decreased amount of phosphatidylcholine, and the enrichment in phosphatidylethanolamine and cardiolipin. Coisolation of phosphatidylserine synthase with the outer membrane portion and enrichment of phosphatidylserine decarboxylase in the inner membrane portion of isolated contact sites provided the basis for a metabolic assay to study phosphatidylserine transfer from the outer to the inner mitochondrial membrane via contact sites. The efficient conversion to [3H]phosphatidylethanolamine of [3H]phosphatidylserine synthesized from [3H]serine in situ supports the notion that mitochondrial membrane contact sites are zones of intramitochondrial translocation of phosphatidylserine.

Permeability Characteristics of Deoxygenated Sickle Cells

Abstract This study investigated the effect of acute deoxygenation on membrane permeability characteristics of sickle cells. Measured fluxes of Na+ and K+ in ouabain-inhibited cells, of chloride and sulfate exchange in 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS)-inhibited and untreated cells, and of erythritol, mannitol, and arabinose in cytochalasin B-inhibited cells indicated that a deoxygenation-induced permeability change occurred in sickle cells only for cations and chloride. Monovalent cation permeabilities increased five-fold, and chloride influx into DIDS treated cells was enhanced nearly threefold on sickle cell deoxygenation. In contrast, no detectable increase in permeability to the other solutes was found. To gain perspective on these findings, similar measurements were performed in normal cells treated with diamide, an agent shown by others to induce a coupled increase in membrane permeability and phospholipid translocation, reminiscent of deoxygenation-induced changes in sickle cells. Although the increase in cation permeability was no greater than that in sickled cells, treatment with 2 mmol/L diamide also produced a twofold increase in the first order rate constants for sulfate exchange and mannitol efflux, indicating a relatively nonselective permeability increase that permitted flux of larger solutes than in the case of deoxygenated sickle cells. These results suggest that the deoxygenation of sickle cells induces a permeability increase that is relatively insensitive to charge, but is restrictive with respect to solute size.

Permeability characteristics of deoxygenated sickle cells

This study investigated the effect of acute deoxygenation on membrane permeability characteristics of sickle cells. Measured fluxes of Na+ and K+ in ouabain-inhibited cells, of chloride and sulfate exchange in 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS)-inhibited and untreated cells, and of erythritol, mannitol, and arabinose in cytochalasin B-inhibited cells indicated that a deoxygenation-induced permeability change occurred in sickle cells only for cations and chloride. Monovalent cation permeabilities increased five-fold, and chloride influx into DIDS treated cells was enhanced nearly threefold on sickle cell deoxygenation. In contrast, no detectable increase in permeability to the other solutes was found. To gain perspective on these findings, similar measurements were performed in normal cells treated with diamide, an agent shown by others to induce a coupled increase in membrane permeability and phospholipid translocation, reminiscent of deoxygenation-induced changes in sickle cells. Although the increase in cation permeability was no greater than that in sickled cells, treatment with 2 mmol/L diamide also produced a twofold increase in the first order rate constants for sulfate exchange and mannitol efflux, indicating a relatively nonselective permeability increase that permitted flux of larger solutes than in the case of deoxygenated sickle cells. These results suggest that the deoxygenation of sickle cells induces a permeability increase that is relatively insensitive to charge, but is restrictive with respect to solute size.

Halothane and Isoflurane Alter Phospholipid Transmethylation in Rat Brain Synaptosomes

The mechanism of action of inhalational anesthetics is unknown, but neuronal membrane alteration is a favored hypothesis. Since phospholipid methylation and translocation play a key role in the transmission of biologic signals across cell membranes, we examined the effect of two commonly used halogenated anesthetics, halothane and isoflurane, on phospholipid methylation in rat brain synaptosomes. Using S-adenosyl-L-[3H-methyl]methionine as a donor, we found a two-fold increase in 3H-methyl incorporation into phospholipids in synaptosomes taken from rats exposed to concentrations that just abolish pain response, but not in rats exposed to higher or lower concentrations. Methylation was not increased in rats newly recovered from anesthesia. Halothane added to synaptosomes taken from rats not previously exposed to anesthetics stimulated 3H-methyl incorporation over a wide range of concentrations. Enhancement of phospholipid methylation by halothane and isoflurane may effect an alteration of neural signal transduction that results in the anesthetic state.

Babesia bovis—effects of phospholipid translocation and adenosine tri-phosphate consumption

The adenosine tri-phosphate concentration of Babesia bovis-infected erythrocytes was significantly ( P < 0.001) less than that of pre-infection erythrocytes. In addition, phosphatidyl serine was detected on the plasmatic surface of the infected erythrocyte. These two related findings could play important roles in the microvascular stasis characteristic of acute B. bovis infection.

Protein-mediated phospholipid translocation in the endoplasmic reticulum with a low lipid specificity

The outside-inside translocation rate of various amphiphilic spin-labeled phospholipids has been measured in rat liver endoplasmic reticulum vesicles. The eight spin-labels tested experienced a fast flip-flop rate with the same half-time of approximately 20 min at 37 degrees C. The stationary distribution of these phospholipid analogues was ca. 45% on the inner vesicular leaflet and 55% on the external one, showing that there is no net enrichment of some lipid in one layer under the experimental conditions used. The initial rate of translocation was reduced 4-fold if membranes were preincubated with N-ethylmaleimide (2 mM) and was about an order of magnitude lower in liposomes made from the extracted lipids. An apparent saturability of the transbilayer diffusion can be deduced from the variation of the initial velocity of the relocation kinetics vs the amount of analogue incorporated in the membrane. Moreover, translocation rates of two different spin-labeled phospholipids introduced simultaneously in the membrane were almost equally reduced by the presence of the other lipid. On the other hand, no competition between the water-soluble dibutyroylphosphatidylcholine and the amphiphilic spin-labeled phospholipids could be detected. Overall, these results suggest that phospholipid translocation in the endoplasmic reticulum is a protein-mediated process with a low specificity, which tends, in the absence of any other metabolic event, to equilibrate the phospholipid composition of the two membrane halves.

Intramitochondrial transfer of phospholipids in the yeast, Saccharomyces cerevisiae.

Translocation of phosphatidylinositol, which is synthesized on the outer aspect of the outer membrane of isolated yeast mitochondria, to the inner membrane is linked to phosphatidylinositol synthesis and is therefore a vectorial process. Phosphatidylinositol once integrated into the inner mitochondrial membrane is not transferred back to the mitochondrial surface. Phosphatidylserine is also translocated from the outer to the inner mitochondrial membrane, where it is decarboxylated to phosphatidylethanolamine. We made use of this metabolic modification to characterize the intramitochondrial transfer of phosphatidylserine and phosphatidylethanolamine. Intramitochondrial phosphatidylserine transfer is insensitive to the uncoupler carbonyl cyanide m-chlorophenylhydrazone and to valinomycin and is thus independent of an electrochemical gradient across the inner membrane. Transfer of phosphatidylserine from the outer to the inner mitochondrial membrane occurs not only in intact mitochondria but also in mitoplasts which are devoid of intermembrane space proteins but have the outer membrane still adherent to the inner membrane. This result suggests that specific contact sites are involved in the intramitochondrial translocation of phospholipids. 3H-Labeled phosphatidylethanolamine synthesized from [3H]serine in isolated mitochondria is readily exported from the inner to the outer mitochondrial membrane without prior mixing with the pool of phosphatidylethanolamine of the inner membrane.

Aminophospholipid translocase in the plasma membrane of Friend erythroleukemic cells can induce an asymmetric topology for phosphatidylserine but not for phosphatidylethanolamine

The ATP-dependent translocation of phospholipids in the plasma membrane of intact Friend erythroleukemic cells (FELCs) was studied in comparison with that in the membrane of mature murine erythrocytes. This was done by following the fate of radiolabeled phospholipid molecules, previously inserted into the outer monolayer of the plasma membranes by using a non-specific lipid transfer protein. The transbilayer equilibration of these probe molecules was monitored by treating the cells — under essentially non-lytic conditions — with phospholipases A 2 of different origin. Rapid reorientations of the newly introduced aminophospholipids in favour of the inner membrane leaflet were observed in fresh mouse erythrocytes; the inward translocation of phosphatidylcholine (PC) in this membrane proceeded relatively slow. In FELCs, on the other hand, all three glycerophospholipids equilibrated over both halves of the plasma membrane very rapidly, i.e. within 1 h; nevertheless, an asymmetric distribution in favour of the inner monolayer was only observed for phosphatidylserine (PS). Lowering the ATP-level in the FELCs caused a reduction in the rate of inward translocation of both aminophospholipids, but not of that of PC, indicating that this translocation of PS and phosphatidylethanolamine (PE) is clearly ATP-dependent. Hence, the situation in the plasma membrane of the FELC is rather unique in a sense that, though an ATP-dependent translocase is present and active both for PS and PE, its activity results in an asymmetric distribution of PS, but not of PE. This remarkable situation might be the consequence of the fact that, in contrast to the mature red cell, this precursor cell still lacks a complete membrane skeletal network.

Measurement of outward translocation of phospholipids across human erythrocyte membrane.

Spin-labeled phospholipids have been used to study the outside----inside and inside----outside transport of phospholipids across the human erythrocyte membrane at 37 degrees C. As already shown, inward transport is much faster for aminophospholipids than for phosphatidylcholine. In addition, we show here that outward transport of the phosphatidylserine and phosphatidylethanolamine analogues is three to four times faster than that of phosphatidylcholine. Magnesium depletion of the erythrocytes considerably decreases the outward rate of both aminophospholipids to values close to that of phosphatidylcholine. These results suggest that the outward aminophospholipid translocation is, at least partly, protein mediated. The protein involved could be identical to the inward Mg-ATP-dependent aminophospholipid carrier.

ChemInform Abstract: Effect of Pressure on Phospholipid Translocation in Lipid Bilayers

ChemInform Abstract: Effect of Pressure on Phospholipid Translocation in Lipid Bilayers

Effect of pressure on phospholipid translocation in lipid bilayers

Effect of pressure on phospholipid translocation in lipid bilayers

Influence of beta-lactam antibiotics and ciprofloxacin on cell envelope of Escherichia coli.

The effects of subinhibitory concentrations of different beta-lactam antibiotics and one quinolone on the quantitative composition of the outer membrane (OM) of two strains of Escherichia coli, on lipid translocation into the OM, and on the production of capsular K1 polysaccharide were studied. The phospholipid/amino acid ratio was reduced in almost all OM preparations from antibiotic-treated bacteria. In one strain, antibiotic treatment increased the lipopolysaccharide/amino acid ratio. The amount of peptidoglycan fragments bound to the OM was increased by all the antibiotics. In pulse-chase experiments with a radioactive lipid precursor, ciprofloxacin, imipenem, and aztreonam inhibited phospholipid translocation into the OM. Furthermore, imipenem, cephaloridine, and ciprofloxacin induced a pronounced reduction of the production of capsular K1 polysaccharide. Thus, antibiotics seem to induce marked changes of the quantitative composition of the cell envelope of E. coli. Possible connections of these data with findings on the influence of antibiotics on functional parameters of the host-parasite relationship such as OM immunogenicity and serum resistance are discussed.

Membrane lipid biogenesis and transport

Membrane biogenesis is an essential feature of cellular development and growth. The initial assembly of membrane lipids and proteins occurs primarily in the endoplasmic reticulum (ER). It has been demonstrated that the enzymes involved in the de novo biosynthesis of phospholipids are exclusively located on the cytoplasmic surface of the ER. A rapid transbilayer movement of phospholipids has also been reported in isolated liver microsomes, which is compatible with the movement of newly synthesized lipids to the lumenal surface of the ER. Comparison with the transbilayer movement of phospholipids across protein-free lipid bilayers, has lead to the proposal that a protein which would catalyze the translocation of phospholipids across the ER membrane (“flipase”), might be involved in the assembly of the lipid bilayer of the ER. Since the various membranes in a eukaryotic cell differ markedly in their lipid composition, it is clear that specific sorting and transport of these membrane components must occur.

Phospholipid transfer activity in synchronous populations of Rhodobacter sphaeroides

Studies of intracytoplasmic membrane biogenesis employing steady-state synchronously dividing populations of Rhodobacter sphaeroides reveal that the translocation of pre-existing phospholipid into the growing membrane is concurrent with cell division (Cain, B.D., Deal, C.D., Fraley, R.T. and Kaplan, S. (1981) J. Bacteriol. 145, 1154–1166), yet the mechanism of phospholipid movement is unknown. However, the discovery of phospholipid transfer protein activity in R. sphaeroides (Cohen, L.K., Lueking, D.R. and Kaplan, S. (1979) J. Biol. Chem. 254, 721–728) provides one possible mechanism for phospholipid movement. Therefore the level of phospholipid transfer activity in cell lysates of synchronized cultures was measured and was shown to increase stepwise coinciding precisely with the increase in cell number of the culture. Although the amount of transfer activity per cell remained constant throughout the cell cycle, the specific activity of the phospholipid transfer activity showed a cyclical oscillation with its highest value coincident with the completion of cell division. Purified intracytoplasmic membrane can be used as phospholipid acceptor in the developed phospholipid transfer assay by employing either cytoplasmic membrane or liposomes as the phospholipid donor. Intracytoplasmic membrane isolated from the cells prior to division (high protein to phospholipid ratio) served as a better phospholipid acceptor in the phospholipid transfer system when compared with membranes derived from the cells following cell division (low protein to phospholipid ratio).

The translocation of phospholipids and wax esters from the cytoplasmic to outer membrane of the cell envelope in Micrococcus cryophilus

Conference Article| April 01 1986 The translocation of phospholipids and wax esters from the cytoplasmic to outer membrane of the cell envelope in Micrococcus cryophilus G. M. LLOYD; G. M. LLOYD * Search for other works by this author on: This Site PubMed Google Scholar N. J. RUSSELL N. J. RUSSELL 1Department of Biochemistry, University College, P.O. Box 78, Cardiff CF1 1XL, Wales, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1986) 14 (2): 302–303. https://doi.org/10.1042/bst0140302 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation G. M. LLOYD, N. J. RUSSELL; The translocation of phospholipids and wax esters from the cytoplasmic to outer membrane of the cell envelope in Micrococcus cryophilus. Biochem Soc Trans 1 April 1986; 14 (2): 302–303. doi: https://doi.org/10.1042/bst0140302 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: CM, cytoplasmic membrane, OM, outer membrane This content is only available as a PDF. © 1986 Biochemical Society1986 Article PDF first page preview Close Modal You do not currently have access to this content.

Translocation énergie dépendante des amino-phospholipides dans la membrane des globules rouges

In this review, we show how the stability of the asymmetric transverse distribution of phospholipids and the physiological role of the asymmetric distribution can be explained. Experiments with paramagnetic or fluorescent lipids enabled us to show that in fresh red blood cells, i.e. containing ATP, and in resealed ghosts containing ATP (1 mM) the amino derivatives (phosphatidylserine and phosphatidylethanolamine) are selectively transported from the outer monolayer to the inner monolayer of the membranes. On the other hand, phosphatidylcholine and sphingomyelin are not carried and diffuse spontaneously with a very long characteristic time. The ATP-dependent carrier mechanism can be inhibited by protein reacting groups (N-ethyl maleimide and ortho-vanadate), which very probably implies a transmembrane protein specific for amino phospholipids. The affinity for phosphatidylserine seems slightly higher than that for phosphatidylethanolamine. In addition we show the close parallel between the transverse distribution of phospholipids and cell shape. This leads us to suggest that the phospholipid translocation would be used to maintain the natural discoid shape of red blood cells. A possible generalisation of this mechanism to other cells and its implications for endocytosis are discussed.

Maintenance of epithelial surface membrane lipid polarity: a role for differing phospholipid translocation rates.

Large differences in lipid composition of apical and basolateral membranes from epithelial cells exist. To determine the responsible mechanism(s), rat renal cortical brush border and basolateral membrane phospholipids were labeled using 32P and either [3H]-glycerol or [2-3H] acetate for incorporation and degradation studies, respectively. Brush border and basolateral membrane fractions were isolated simultaneously from the same cortical homogenate. Different phospholipid classes were degraded at variable rates with phosphatidylcholine having the fastest decay rate. Decay rates for individual phospholipid classes were, however, similar in both brush border and basolateral membrane fractions. In phospholipid incorporation studies, again, large variations existed between individual phospholipid classes with phosphatidylcholine and phosphatidylinositol showing the most rapid rates of incorporation. Sphingomyelin and phosphatidylserine showed extremely slow incorporation rates and did not enter into the isotopic decay phase for 48 hr. In contrast to degradation studies, however, the same phospholipid class labeled the two surface membrane domains at highly variable rates. The difference in these rates, with the exception of phosphatidylinositol, were identical to the differences in phospholipid compositions between the two membranes. For example, phosphatidylcholine was incorporated into the basolateral membrane 2.5 X faster than into the brush border membrane and its relative composition was 2.5 X greater in the basolateral membrane. The opposite was true for sphingomyelin. These results indicate incorporation and not degradation rates of individual phospholipids play a major role in regulating the differing phospholipid composition of brush border and basolateral membranes.

ATP-dependent translocation of amino phospholipids across the human erythrocyte membrane

Trace amounts of radiolabeled phospholipids were inserted into the outer membrane leaflet of intact human erythrocytes, using a non-specific lipid transfer protein. Phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine were transferred from the donor lipid vesicles to the membrane of the intact red cell with equal ease, whilst sphingomyelin was transferred 6-times less efficiently. The transbilayer mobility and equilibrium distribution of the labeled phospholipids were assessed by treatment of the intact cells with phospholipases. In fresh erythrocytes, the labeled amino phospholipids appeared to move rapidly towards the inner leaflet. The choline phospholipids, on the other hand, approached an equilibrium distribution which strongly favoured the outer leaflet. In ATP-depleted erythrocytes, the relocation of the amino phospholipids was markedly retarded.