Consequences of omega -6-oleate desaturase deficiency on lipid dynamics and functional properties of mitochondrial membranes of Arabidopsis thaliana.

We probed the role of the polyunsaturated fatty acids on the dynamic and functional properties of mitochondrial membranes using the fad2 mutant of Arabidopsis thaliana, deficient in omega-6-oleate desaturase. In mitochondria of this mutant, the oleic acid content exceeded 70% of the total fatty acids, and the lipid/protein ratio was greatly enhanced. As a consequence, local microviscosity, probed by anthroyloxy fatty acid derivatives, was increased by 30%, whereas the lipid lateral diffusion, assayed using 1-pyrenedodecanoic acid, was approximately 4 times increased. Functional parameters such as oxygen consumption rate under phosphorylating and nonphosphorylating conditions and proton permeability of the inner mitochondrial membrane were significantly reduced in fad2 mitochondrial membranes, while the thermal dependence of the respiration was enhanced. Moreover, metabolic control analysis of the respiration clearly showed an enhancement of the control exerted by the membrane proton leaks. Our data suggest that the loss of omega-6-oleate desaturase activity in Arabidopsis cells induced an enhancement of both microviscosity and lipid/protein ratio of mitochondrial membranes, which in turn were responsible for the change in lateral mobility of lipids and for bioenergetic parameter modifications.

Comparison of lipid binding and transfer properties of two lipid transfer proteins from plants.

Plant lipid transfer proteins (LTPs) are soluble proteins which are characterized by their in vitro ability to transfer phospholipids between two membranes. We have compared the functional properties of two LTPs purified from maize and wheat seeds knowing that, despite a high degree of sequence identity, the two proteins exhibit structural differences. It was found that wheat LTP had a lower transfer activity than the maize LTP, consistent with a lower kinetics of fatty acid binding. The lower affinity for the fatty acids of the wheat LTP could be explained by a narrowing occurring in the middle part of the binding site, as revealed by comparing the fluorescence spectra of various anthroyloxy-labeled fatty acids associated with the two LTPs. The affinity for some natural fatty acids was studied by competition with fluorescent fatty acids toward binding to the protein. Again, wheat LTP had a lower affinity for those molecules. All together, these observations reveal the complexity of the LTP family in plants, probably reflecting the multiple roles played by these proteins.

Lipid-transfer proteins from plants: structure and binding properties.

Plant cells contain lipid-transfer proteins (LTPs) able to transfer phospholipids between membranes in vitro. Plant LTPs share in common structural and functional features. Recent structural studies carried out by NMR and X-ray crystallography on an LTP isolated from maize seeds have showed that this protein involves four helices packed against a C-terminal region and stabilized by four disulfide bridges. A most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule and able to accommodate acyl chains. It was thus of interest to study the ability of maize LTP to bind hydrophobic ligands such as acyl chains or lysophosphatidylcholine and to determine the effect of this binding on phospholipid transfer. The binding abilities of maize LTP, presented in this paper, are discussed and compared to those of lipid-binding proteins from animal tissues.

Transbilayer movement and distribution of spin-labelled phospholipids in the inner mitochondrial membrane.

The transmembrane diffusion and equilibrium distribution of spin-labelled phosphatidylethanolamine (PE*), phosphatidylcholine (PC*) and cardiolipin (CL*) were investigated in purified mitochondrial inner membranes using electron spin resonance spectroscopy. Using the back exchange technique, we found that the outside-inside movement of PE* and PC* in beef-heart inner mitochondrial membranes was rapid (t1/2 in the range 10-15 min at 30 degrees C). The steady-state distributions in non-energised mitoplasts were approximately 30% in the inner leaflet for PC* and 39% for PE*. Within the limits of probe concentration that can possibly be used in these experiments, the initial velocity of the inward movement was not saturable with respect to the amount of analogue added to the membranes, suggesting that the spin-labelled phospholipids diffused passively between the two leaflets of the inner mitochondrial membrane. In energised mitoplasts, PC* behaviour was not affected, PE* diffused approximately two times faster toward the inner monolayer but reached the same plateau. Treatment of energised mitochondria with N-ethylmaleimide did not affect PC* diffusion, while the kinetics of PE* internalisation became identical to that of PC*. Similar results were found when PC* and PE* movements were studied in mitoplasts from beef heart, rat liver or yeast. The spin-labelled cardiolipin, which possesses four long chains, had to be introduced in the mitoplast with some ethanol. After equilibration (t1/2 of the order of 13 min at 30 degrees C), the transmembrane distribution suggested that approximately half of the cardiolipin analogue remained in the outer leaflet. These results do not allow us to determine if a specific protein (or flippase) is involved in the phospholipid transmembrane traffic within inner mitochondrial membranes, but they show that lipids can rapidly flip through the mitochondrial membrane.

Cloning, expression, and chromosomal mapping of a human ATPase II gene, member of the third subfamily of P-type ATPases and orthologous to the presumed bovine and murine aminophospholipid translocase.

Recently, a P-type ATPase was cloned from bovine chromaffin granules (b-ATPase II) and a mouse teratocarcinoma cell line (m-ATPase II) and was shown to be homologous to the Saccharomyces cerevisiae DRS2 gene, the inactivation of which resulted in defective transport of phosphatidylserine. Here, we report the cloning from a human skeletal muscle cDNA library of a human ATPase II (h-ATPase II), orthologous to the presumed bovine and mouse aminophospholipid translocase (95.3 and 95.9% amino acid identity, respectively). Compared with the bovine and mouse counterparts, the cloned h-ATPase II polypeptide exhibits a similar membrane topology, but contains 15 additional amino acids (1163 vs 1148) located in the second intracytoplasmic loop, near the DKTGTLT-phosphorylation site. However, RT-PCR analysis performed with RNA from different human tissues and cell lines revealed that the coding sequence for these 15 residues is sometimes present and sometimes absent, most likely as a result of a tissue-specific alternative splicing event. The h-ATPase II gene, which was mapped to chromosome 4p14-p12, is expressed as a 9.5-kb RNA species in a large variety of tissues, but was not detected in liver, testis, and placenta, nor in the erythroleukemic cell line K562.

Characterisation of acyl binding by a plant lipid-transfer protein.

Maize lipid-transfer protein (LTP) is a small soluble protein which is able to transfer in vitro phospholipids between membranes and to bind fatty acids or lysoderivatives. In the studies reported here, fluorescent-labelled fatty acids were used to characterise the nature of the binding site on LTP. A fluorescent analogue of 12 carbons with a pyrene moiety attached at the end, alone or in conjunction with an anthroyloxy analogue, indicated that LTP could bind two fatty acids although with a marked difference in affinity. The binding capacity was strongly affected after reduction of the protein by dithiothreitol, showing that the four S-S bonds of LTP are essential for its lipid binding property. Other analogues used were 16-carbon or 18-carbon fatty acids with an anthracene moiety attached at different points of the hydrocarbon chain. Emission maxima of these molecules varied with the analogue and suggested a motional constraint for the bound fatty acid which is more important around the middle of the chain than at its extremities. Binding displacement studies were carried out with a wide range of fatty acids or fatty acyl derivatives. Fatty acids of 16 to 19 carbons were found to be the preferred ligands. The presence of one double bond did not change appreciably the affinity of LTP, although the presence of two or three double bonds or of a hydroxyl moiety significantly reduced the affinity. Fatty acyl-CoA or lysoderivatives bound as well as the corresponding fatty acid.

Calcium induces phospholipid redistribution and microvesicle release in human erythrocyte membranes by independent pathways.

The increase in intracellular Ca2+ concentration in erythrocytes and platelets results in simultaneous phospholipid scrambling and microvesicle shedding. Microvesicle formation involves membrane fusion events which were proposed either to be tightly linked to phospholipid transversal redistribution or to occur by a separate mechanism. We report here that in erythrocytes incubated in high K+ medium, or in resealed ghosts, phospholipid scrambling can be fully induced by intracellular Ca2+ without microvesicle formation. Furthermore, in ghosts resealed in the presence of spermine, intracellular Ca2+, at low concentration, was able to induce microvesicles, whereas scrambling was drastically inhibited. Surprisingly, in spermine-containing ghosts prepared from erythrocytes of a patient with a bleeding disorder, due to a lack of Ca2+-induced phospholipid scrambling and vesicle shedding (characterized as a Scott syndrome), Ca2+ also promoted microvesicle release. Data show that phospholipid scrambling and microvesicle production, although closely regulated, proceed by independent pathways.

Fodrin inhibits phospholipases A2, C, and D by decreasing polyphosphoinositide cell content.

Brain fodrin inhibited in a dose dependent manner the GTPgammaS-stimulated cytosolic PLA2 (cPLA2), PLC, and PLD activities in differentiated HL-60 cells permeabilized with streptolysin O. cPLA2 and PLD were inhibited by the same concentrations of fodrin (IC50=1.5-2 nM) but PLC was inhibited by lower concentrations (IC50=0.3 nM). Moreover, the rates of inhibition were different between the phospholipases. Spectrin, which shares 50% homology with fodrin, had similar effects on the three phospholipases. However, using cytosol-depleted cells or recombinant PLD1, we showed that fodrin was not a direct inhibitor. Studying the potential mechanisms of these inhibitions, we demonstrated that a major decrease in membrane phosphatidylinositol 4-monophosphate (PtdIns(4)P) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) amounts was induced by fodrin. Exogenous PtdIns(4,5)P2 partly reversed fodrin inhibition of GTPgammaS-stimulated phospholipase C activity. Hence, inhibition of PLC, cPLA2, and PLD activities observed with fodrin could be related to the decrease of PtdIns(4,5)P2, substrate of PLC, a cofactor of PLD and an enhancer of cPLA2 activity.

Asymmetrical distribution of cardiolipin in yeast inner mitochondrial membrane triggered by carbon catabolite repression.

Transmembrane asymmetry of cardiolipin in yeast was monitored during the switch from fermentative to gluconeogenic growth and the reverse. As soon as cells used ethanol as an electron donor to produce ATP by oxidative phosphorylation, rapid and abundant cardiolipin synthesis was observed on the matrix side of the inner mitochondrial membrane followed by a transverse rearrangement between the two leaflets. The cardiolipin distribution changed from about 20:80 (in/out) to 70:30 (in/out), and after translocation towards the outer leaflet it finally became 37:63 (in/out). At the same time, cytochrome c oxidase activity remained stable, then increased as a possible result of the topographical rearrangement. During the reverse process from gluconeogenic to fermentative growth, the amount of cardiolipin rapidly decreased by half, its bilayer distribution apparently changing to a monolayer organization before the 20:80 (in/out) asymmetry of repressed cells was re-established. Experimental impairment of cardiolipin topography by antibiotic inhibition of gene expression or in situ dissipation of mitochondrial membrane potential produced data that prove that the amount and transmembrane distribution of the phospholipid are two specific parameters of the mitochondrial inner membrane organization in both fermentative (2.2 fmol/cell and 20:80, in/out) and gluconeogenic (4.2 fmol/cell and 37:63, in/out) growing yeast cells. Finally, the inner mitochondrial membrane topography of cardiolipin appeared to be closely associated with the transmembrane redox potential.

Drug-induced transmembrane lipid scrambling in erythrocytes and in liposomes requires the presence of polyanionic phospholipids.

The asymmetric transmembrane distribution of phospholipids between the two bilayer halves of erythrocyte can be modified upon addition of cationic amphiphilic drugs, such as chlorpromazine or verapamil. We studied this phenomenon in erythrocytes and in lipid vesicles using spin-labelled analogues of the endogenous phospholipids. The extent of the rapid disappearance of the analogues from the erythrocyte outer leaflet depended on the concentration of the drug. Up to 40% of spin-labelled sphingomyelin moved to the inner erythrocyte leaflet in 10 min in the presence of 1.5 mm chlorpromazine. Verapamil or vinblastine gave similar results. On the other hand, the inside-outside movement of the aminophospholipid analogues was less evident, and did not exceed 10%. This apparent discrepancy between inward and outward movements could result from the formation of an endovesicle which is known to occur upon drug addition at high concentration. A fraction of lipids would be trapped in the intravesicular leaflet, corresponding to the cell outer leaflet, and be inaccessible both from the cytoplasm and the extracellular medium. In cells submitted to a metabolic depletion of cellular ATP the intensity of the scrambling induced by the amphipaths was drastically lowered. We attribute this effect to the important reduction of the membrane content in phosphatidylinositol-4,5-bisphosphate (PIP2). The involvement of the latter lipid in triggering scrambling was partly confirmed by experiments carried out with artificial membranes. Indeed, in large unilamellar vesicles PIP2 is required in order to obtain a rapid redistribution of phospholipids between the two leaflets upon addition of drugs. However, the extent of phospholipid redistribution was limited to 15-20%. This redistribution was also induced when the vesicle membrane contained di-anionic phospholipids (phosphatidylinositol-4-monophosphate or diphosphatidylglycerol), but did not occur when it contained mono-anionic phospholipids (phosphatidylserine or phosphatidylinositol). Some drugs such as methochlorpromazine, active in artificial membranes, were ineffective in erythrocyte membranes, probably because they could not cross the membrane and reach PIP2 molecules at the cytoplasmic leaflet.

Characterization of the correlation between ATP-dependent aminophospholipid translocation and Mg2+-ATPase activity in red blood cell membranes.

Pseudosubstrates and inhibitors of ATPases were studied with respect to their capability to modulate the kinetic behavior of Mg2+-ATPase and aminophospholipid translocation in red blood cell ghosts. ATP was substituted by the pseudosubstrates of P-type ATPases acetyl phosphate and p-nitrophenyl phosphate. With both pseudosubstrates, aminophospholipid translocation from the outer to the inner leaflets of resealed erythrocyte ghosts could be observed, although with a significantly decreased velocity compared to that in presence of ATP, both with respect to phosphate hydrolysis and translocation. Similarly, the apparent affinities for the pseudosubstrates were much lower than for ATP. Among the inhibitors studied, suramin acted as a competitive inhibitor of ATP towards both Mg2+-ATPase activity and aminophospholipid translocation. However, the inhibition of translocation occurred at a higher inhibitor concentration than the inhibition of Mg2+-ATPase activity. With elaiophylin, only a partial inhibition of Mg2+-ATPase activity could be detected, but translocation of labeled phosphatidylserine was almost completely abolished. With eosin Y, an almost complete inhibition of both Mg2+-ATPase activity and translocation could be achieved. The observed responses of aminophospholipid translocation to ATPase inhibitors strongly suggest that a P-type ATPase, part of which displays a Mg2+-ATPase activity, is involved in aminophospholipid translocation.

Aminophospholipid translocase and proteins involved in transmembrane phospholipid traffic.

The transmembrane distribution of phospholipids in the membranes of eukaryotic cells depends on specific proteins (called flippases). The aminophospholipid translocase is responsible for the sequestration of phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet of plasma membranes. Several laboratories are presently working on the identification, purification and cloning of this Mg-ATPase, first recognized in the human red cell membrane. In accordance with the 1992 hypothesis of Higgins and Gottesman, proteins of the MDR1 family appear to be able to translocate certain phospholipids from the inner to the outer monolayer of the plasma membrane. It has been reported in particular that expression of the human MDR3 and mouse mdr2 genes promote translocation of long chain phosphatidylcholine, while expression of the MDR1 gene stimulates the outward motion of phospholipids possessing at least one short chain. ATP-independent flippases activities were recognized not only in microsomes but also in Golgi membranes.

Antagonist effects of Ca2+ and spermine on phosphatidylinositol 4,5-bisphosphate-mediated transmembrane redistribution of phospholipids in large unilamellar vesicles and in erythrocytes.

We have previously suggested the involvement of a Ca(2+)-phosphatidylinositol 4,5-bisphosphate (PIP2) complex in the phospholipid transmembrane redistribution triggered by cytosolic Ca2+ in erythrocytes. Indeed, the lipid scrambling was induced by extracellular Ca2+ in erythrocytes loaded with PIP2 and was abolished in inside-out vesicles prepared from PIP2-depleted erythrocytes (Sulpice, J.C., Zachowski, A., Devaux, P.F., & Giraud, F. (1994) J. Biol. Chem. 269, 6347-6354). Here, we show that Ca2+ triggers a partial redistribution of spin-labeled phospholipids in protein-free large unilamellar vesicles (LUVs), only when they contain PIP2. Spermine, a polyamine known to interact with PIP2 and reported to inhibit lipid scrambling in resealed ghosts, was found to inhibit also the Ca(2+)-induced scrambling in LUVs and in PIP2-loaded erythrocytes, presumably by interacting with PIP2 and preventing the formation of Ca(2+)-PIP2 complexes. A similar mechanism can account for spermine inhibition in natural membranes, confirming the role of PIP2 in the scrambling process without excluding the participation of proteins. In erythrocytes, activation of the phosphoinositide phospholipase C (PLC) or a 20 h ATP depletion, which both led to a reduction in the PIP2 content by 40-60%, did not affect Ca(2+)-induced phospholipid scrambling. In contrast, longer ATP depletion, resulting in a 80% reduction in the PIP2 content, did induce a significant decrease in lipid scrambling, suggesting that only the PIP2 pool resistant to the PLC was involved. Spermine was able to inhibit hydrolysis of this pool by an exogenous PLA2. It is thus likely that spermine antagonized the Ca(2+)-induced scrambling in resealed ghosts by interacting with the PLC-resistant pool of PIP2.

An improved assay for measuring the transverse redistribution of fluorescent phospholipids in plasma membranes.

The internalization of fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-phospholipids from the plasma membrane can be assessed by the irreversible quenching of analogues in the outer leaflet by dithionite. Here we have utilized this assay to follow the redistribution of short-chain C6-NBD-sphingomyelin and C6-NBD-phosphatidylserine from the cell membrane of human gingiva fibroblasts. The significant uptake of dithionite across the plasma membrane and the subsequent reduction of NBD-analogues exposed to the cytoplasmic lumen does not allow an accurate measurement of the amount of internalized lipid probes even at low temperature. We could show that a precise determination can be achieved by extraction of analogues remaining in the exoplasmic half by a short pretreatment with bovine serum albumin prior to addition of dithionite. The fluorescence of those analogues localized to the cytoplasmic lumen was slowly destroyed by permeating dithionite. The fluorescence of those NBD-probes which are localized in the inner layer of intracellular vesicles remained almost unaffected in the time course of the assay. Thus, this approach allows to distinguish between different routes of internalization of NBD-phospholipids from the plasma membrane.

Thermal acclimation and dietary lipids alter the composition, but not fluidity, of trout sperm plasma membrane.

The effect of a long-term adaptation of rainbow trout to 8 and 18 degrees C combined with a corn oil- or a fish oil-supplemented diet on the characteristics of the spermatozoan plasma membrane was investigated. The experiment lasted up to 22 mon during which spermatozoa were collected from the mature males. Spermatozoan plasma membranes were isolated by nitrogen cavitation, and the cholesterol content, phospholipid composition and fatty acid pattern were investigated. Membrane viscosity was assessed on whole cells by electron spin resonance using spin-labeled phospholipids. Neither diet nor rearing temperature influenced the cholesterol content of the plasma membrane nor the phospholipid class distribution. The rearing temperature of the broodstock only slightly affected the phospholipid fatty acids. A minor decrease in 18:0 and increase in monounsaturated fatty acids was observed for the cold-adapted fish. These modifications were not sufficient to affect membrane fluidity, and we conclude that trout spermatozoa do not display any homeoviscous adaptations in these conditions. On the contrary, the dietary fatty acid intake greatly modified the fatty acid profile of plasma membrane phospholipids. The fish oil-fed trout displayed a much higher n-3/n-6 fatty acid ratio than did the corn oil-fed ones, but the 22:6n-3 levels remained unchanged. Modifications in plasma membrane composition by the diet were obtained although neither of the two diets was deficient in essential fatty acids. The enrichment in n-3 fatty acids, however, did not affect plasma membrane fluidity which was unchanged by the diets.

Reconstitution of ATP-dependent aminophospholipid translocation in proteoliposomes.

In addition to ion-pumping ATPases, most plasma membranes of animal cells contain a Mg2+ ATPase activity, the function of which is unknown. This enzyme, of apparent molecular mass 110 kDa, was purified from human erythrocyte membranes by a series of column chromatographic procedures after solubilization in Triton X-100. When reincorporated into artificial bilayers formed from phosphatidylcholine, it was able to transport a spin-labeled phosphatidylserine analogue from the inner to the outer membrane leaflet provided Mg2+ ATP was present in the incubation mixture. The ATP-dependent transport of the phosphatidylethanolamine analogue required the presence of an anionic phospholipid (e.g., phosphatidylinositol) in the outer membrane leaflet. In contrast the transmembrane distribution of spin-labeled phosphatidylcholine was unaffected in the same experimental conditions. This transmembrane movement of aminophospholipid analogues was inhibited by treatment of the proteoliposomes with a sulfhydryl reagent. We conclude that the Mg2+ ATPase is sufficient for the biochemical expression of the aminophospholipid translocase activity, which is responsible for the inward transport of phosphatidylserine and phosphatidylethanolamine within the erythrocyte membrane. The presence of this transport activity in many animal cell plasma membranes provides a function for the Mg2+ ATPase borne by these membranes.

Protein-dependent translocation of aminophospholipids and asymmetric transbilayer distribution of phospholipids in the plasma membrane of ram sperm cells.

We have investigated the transbilayer movement of phospholipids in the plasma membrane of ram sperm cells using spin- and fluorescence-labeled lipid analogues. After incorporation into the outer leaflet, phosphatidylcholine (PC) and sphingomyelin (SM) moved slowly to the inner cytoplasmic leaflet, whereas phosphatidylserine (PS) and phosphatidylethanolamine (PE) rapidly disappeared from the exoplasmic monolayer. Variation of the initial velocity of the relocation kinetics vs the amount of analogue incorporated into the membrane suggests a saturability of the transbilayer movement of aminophospholipids. ATP depletion or pretreatment with N-ethylmaleimide of ram sperm cells reduced the fast inward motion of PS and PE, indicating a protein-mediated aminophospholipid translocation. The results suggest for the plasma membrane of ram sperm cells the presence of an aminophospholipid translocase and an asymmetric transversal lipid distribution with aminophospholipids preferentially located in the inner leaflet and choline-containing phospholipids in the outer leaflet. The relevance of the transversal segregation of phospholipids for membrane fusion processes occurring during fertilization is discussed.

Phospholipid transverse asymmetry in trout spermatozoa plasma membrane.

The phospholipid transmembrane distribution and movement in the plasma membrane of rainbow trout spermatozoa was determined with spin-labelled phospholipid analogues. After initial incorporation in the outer membrane leaflet, only the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE) redistributed towards the inner leaflet. At equilibrium, more than 90% PS and approximately 80-85% PE were located in the cytoplasmic leaflet. The inward motion was significantly faster for PS (t1/2 approximately 5 min) than for PE (t1/2 approximately 60 min). Both the velocity and the extent of this redistribution were dependent on the cellular ATP level, arguing for the involvement of the aminophospholipid translocase. Comparison of the electron paramagnetic resonance spectra recorded from analogues located in either one of the leaflet showed an important difference of microviscosity between the cytoplasmic and the extracellular bilayer leaflets. Assuming that the analogue distribution reflects the asymmetry of the endogenous phospholipids, the higher probe mobility in the inner leaflet could be correlated to a higher degree of unsaturation of the fatty acids present in this hemi-leaflet.

Requirement for phosphatidylinositol 4,5-bisphosphate in the Ca(2+)-induced phospholipid redistribution in the human erythrocyte membrane.

In order to investigate how calcium on the cytosolic side of human erythrocytes induces the transmembrane redistribution of phospholipids, we studied the effect of this cation on the transmembrane movements of spin-labeled phospholipids (phosphatidylserine (PS) and phosphatidylcholine (PC)) incorporated into inside-out vesicles derived from human erythrocytes. We found that the extent of the Ca(2+)-induced lipid scrambling was dependent upon the level of phosphatidylinositol 4,5-bisphosphate (PIP2) contained in the external leaflet of inside-out vesicles. The level of PIP2 in this leaflet, which normally accounts for 80% of the total membrane PIP2, was manipulated either by ATP depletion of the original erythrocytes or by incorporation of exogenous PIP2. Similarly, loading the outer monolayer of the membrane of intact erythrocytes with exogenous PIP2 caused, in a dose-dependent way, the scrambling of spin-labeled phosphatidylethanolamine, sphingomyelin, PC, and PS and in parallel the stomatocytic conversion of the cells. Both scrambling and stomatocytosis were strictly dependent on the presence of divalent cations in the medium. Mg2+ could replace Ca2+ but required a 10 times higher concentration. The effect was specific for PIP2, the other phosphoinositides being unable to induce the lipid redistribution. The shape change, but not the scrambling, required a normal ATP level. These results show that Ca2+ or Mg2+ trigger the lipid redistribution either from the internal or the external side of the membrane, provided that enough PIP2 is present on that side. Thus, no specific protein is required for this process. We infer that the ATP-dependent shape change of erythrocytes after incubation with PIP2 and Ca2+ results from the bilayer imbalance due to the activity of the aminophospholipid translocase which relocates PS and phosphatidylethanolamine to the inner monolayer without simultaneous outward diffusion of PC and sphingomyelin.

Rapid determination of the transbilayer distribution of NBD-phospholipids in erythrocyte membranes with dithionite.

The assessment of the transverse distribution and mobility of NBD-labelled phospholipid analogues in biological membranes by selective chemical destruction of fluorescent label in the outer monolayer with dithionite has been investigated using resealed erythrocyte ghosts as a model system. The distribution of those analogues can be determined in < 30 s directly in the cell suspension provided the permeation of dithionite across the membrane is suppressed. The results were compared with data on translocation of either NBD- or spin-labelled phospholipid analogues obtained with the technique of back exchange to BSA. It is shown that the passage of dithionite can be mediated by anion-transport systems such as band 3 which is inhibited by DIDS. Appropriate conditions for the applicability of the assay were elucidated also using resealed ghosts having fluorescent NBD-taurine in the intracellular lumen. The application of the assay to measure fast translocation processes, e.g. those mediated by the aminophospholipid translocase, is described.