Pierre Fellmann

Ion regulation of phosphatidylserine and phosphatidylethanolamine outside-inside translocation in human erythrocytes

In previous publications, we have shown, by using spin-labeled derivatives, that the translocation of phosphatidylserine and phosphatidylethanolamine from the outer to the inner monolayer of human erythrocyte membrane is a protein-mediated phenomenon, which requires hydrolisable Mg2+-ATP. The inhibition by intracellular Ca2+ (0.2 microM) or by extracellularly added vanadate (50 microM) was reported (Seigneuret, M. and Devaux, P.F. (1984) Proc. Natl. Acad. Sci. USA 81, 3751-3755; Zachowski, A., Favre, E., Cribier, S., Hervé, P. and Devaux, P.F. (1986) Biochemistry 25, 2585-2590). The present article gives further insight into the effects of intracellular and extracellular ions on the aminophospholipid translocation in human erythrocytes. By measuring the cell ATP concentration, we now show that the inhibitory effect of intracellular calcium on spin-labeled aminophospholipid translocation is partly due to the ATP depletion, which follows the increased consumption by the calcium pump. However, a direct inhibitory effect of cytosolic Ca2+ on the aminophospholipid translocase can be demonstrated by measuring the initial rate of aminophospholipid translocation in the presence of variable amounts of intracellular calcium, at fixed ATP concentrations. Moreover, the transmembrane equilibrium distribution of phosphatidylserine and phosphatidylethanolamine are affected differently by Ca2+: when cytosolic Ca2+ concentration is increased, alteration of phosphatidylethanolamine distribution begins as soon as the inward translocation is affected by Ca2+ (approx. 50 nM), whereas phosphatidylserine distribution remains unchanged within a large inhibitory range of cytosolic Ca2+ concentrations and decreases above 0.2 microM of free Ca2+ within the cytosol. Decrease of the intracellular Mg2+ concentration below its physiological value (approx. 2 mM) results in the inhibition of aminophospholipid inward transport, whereas increase of Mg2+ concentration does not modify this transport. If Mn2+ is substituted for Mg2+, part of the aminophospholipid translocation is maintained, whereas if Co2+ is substituted for Mg2+, the rapid translocation is completely abolished. Concentrations as high as a millimolar of extracellular Ca2+, Mg2+ or Mn2+ have no effect on the aminophospholipid translocation. The less usual cations Cr3+, Fe2+, Cu2+, Sn2+ and Eu3+ are also uneffective. With extracellular Ni2+ or Co2+, some inhibition can be observed, half inhibition by Ni2+ corresponding to 500 microM. Vanadyl (VO2+), on the other hand, is a potent inhibitor of the aminophospholipid translocation when applied on the extracellular surface, half-inhibition being reached around 30 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Transmembrane diffusion of fluorescent phospholipids in human erythrocytes

The outside-inside passage and transmembrane equilibrium distribution of several amphiphilic fluorescent phospholipids were examined in human erythrocytes. The results were compared with previous kinetic data obtained with spin-labeled phospholipids and with the equilibrium distribution of endogenous lipids in erythrocytes. When a nitro benzoxadiazole (NBD) was at the terminal position of a 6 carbon beta-chain, the outside-inside diffusion of the fluorescent phosphatidylserine (PS) analogue was slower, and the plateau lower than with long chain radioactive PS or spin-labeled PS. The corresponding phosphatidylethanolamine (PE) did not flip nor did the phosphatidylcholine (PC) analogue. With a NBD at the 12th carbon of a 18C alpha-chain, the amino-derivatives behaved more like endogenous PS and PE, i.e. they accumulated rapidly on the inner monolayer; however, the phosphatidylcholine analogue reached a plateau corresponding to 50% inside within 2 h at 37 degrees C, indicative of an abnormal rapid diffusion. In the latter case, changing the beta-chain from four to eight carbons had no influence on this rapid diffusion. We conclude that when the NBD is close to the glycerol moiety, it diminishes the affinity of the aminophospholipids for the aminophospholipid translocase. When it is close to the methyl terminal of an acyl chain, there is an acceleration of the spontaneous flip-flop. Presumably the polarity of the NBD is responsible for an unconventional orientation of the flexible acyl chain, thereby causing the transmembrane destabilization of the phospholipid. Overall these results illustrate the respective roles of spontaneous diffusion and translocase activity on transmembrane equilibrium distribution of phospholipids. They also show that NBD derivatives should be used cautiously as indicators of endogenous phospholipids.

Boundary lipids and protein mobility in rhodopsin-phosphatidylcholine vesicles. Effect of lipid phase transitions.

Purified rhodopsin from bovine retina has been incorporated into phospholipid bilayers. Dimiristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleylphosphatidylcholine and egg phosphatidylcholine were used as host lipids, with ratio of lipid to protein of 120 : 1 (mol to mol). In order to probe the lipid-protein interface specifically, a spin-labeled fatty acid was covalently bound to rhodopsin via an isocyanate reacting group. A spin-labeled phospholipid was used to probe the bulk lipidic phase while a tightly bound maleimide spin label was used to obtain the protein rotational correlation time by the saturation transfer technique. The following results were obtained: (1) The kinetics of reduction by ascorbate of the spin-labeled fatty acid covalently bound to rhodopsin demonstrate that the alkyl chain attached to the protein is positioned in the membrane in the same way as the alkyl chains of a phospholipid. (2) The EPR spectra of the latter shows two components: a strongly immobilized component and a weakly immobilized component. The ratio of the two depends upon the temperature and on the nature of the phospholipids. (3) The signal of the weakly immobilized component is compared to that obtained in the corresponding pure lipids. The latter signal, assumed to represent non-bounded lipids, indicates a sharp transition at the phospholipid phase transition with dimytristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The former signal (corresponding to the lipid-protein interface) indicates only a broad transition extending over 7 degrees C with dipalmitoylphosphatidylcholine and almost no transition with dimyristoylphosphatidylcholine. (4) In a similar way, the rotational correlation time of the protein only changes progressively when the phase transition occurs. Our interpretation of the data can be summarized as follows: The immobilized component seen by the EPR technique in the hydrophobic environment of this intrinsic protein very probably reflects protein-protein contacts and thus corresponds to hindrance of the labeled chains, when they are trapped between neighbouring proteins. Below the phase transition lipid segregation whould increase the probability of protein contact. However, over a certain range of temperature, the contact with the protein interface probably at the same time prevents the non-segregated phospholipids from feezing. The differences in the results obtained with the various phosphatidylcholines above their transition temperature suggest that the solubility of rhodopsin in bilayers depends not only on the fluidity of the lipids, but also, to some extent, on the phospholipid chain length.

Investigation on lipid asymmetry using lipid probes Comparison between spin-labeled lipids and fluorescent lipids

Synthetic lipids with a nitroxide or a fluorescent probe have been extensively used during the last 30 years to determine the transmembrane diffusion of phospholipids in artificial or biological membranes. However, the relevance of data obtained with these modified lipids has sometimes been questioned. Beside possible artefacts introduced by the reporter probe, synthetic lipids used in cells often contain a short fatty acid chain in the sn-2 position, which gives them higher water solubility than naturally occurring lipids. In the present review, we have attempted to give a critical appraisal. Main strategies are recalled and important discoveries obtained with lipid probes on transmembrane lipid traffic in eukaryotic cells are briefly summarized. Examples of artefacts caused by lipid probes are given. Comparisons between data obtained by different techniques such as ESR and fluorescence allow us to emphasize the complementary character of the two approaches and more generally show the necessity to use several probes before drawing conclusions concerning endogenous lipids. In spite of these pitfalls, overall, lipid probes have provided a wealth of useful information that, to date, cannot be obtained with unlabeled lipids.

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.

Transmembrane distribution and translocation of spin-labeled plasmalogens in human red blood cells

We have synthesized two new spin-labeled alkenylacyl phospholipids (plasmalogens) in order to investigate the transmembrane distribution and transport of this subclass of glycerophospholipids in human red blood cells. The plasmenylethanolamine analogue diffuses rapidly from the outer to the inner leaflet with a half time at 37 degrees C of 30 min comparable to that of the corresponding diacyl-phosphatidylethanolamine spin-label in an ATP-requiring and N-ethyl maleimide sensitive manner. The plateau corresponds to 79% of the aminophospholipids on the inner leaflet. By contrast, after 4 h incubation less than 20% of the plasmenylcholine spin-labels reach the interior. Thus plasmalogens behave as the corresponding diacyl-lipids. We infer that plasmenylethanolamine is transported from the outer to the inner leaflet of the red cell membrane by the aminophospholipid translocase.

High-resolution 31P-1H two-dimensional Nuclear Magnetic Resonance spectra of unsonicated lipid mixtures spinning at the magic-angle

Abstract For multilamellar suspensions of phospholipids, the 1H and 31P Nuclear Magnetic Resonance (NMR) spectra obtained with magic-angle spinning (MAS) exhibit resolution comparable to that of sonicated vesicles. However, specific lipid head groups cannot be recognized in a lipid mixture using one-dimensional NMR spectroscopy. We show here that the combination of MAS and two-dimensional Heteronuclear Overhauser Effect SpectroscopY (HOESY) reveals magnetic interactions between the phosphate and its neighbouring protons and thus allows the distinction in situ of several lipids in a mixture. The 31P-1H HOESY spectra of suspensions of phosphatidylcholine and phosphatidylglycerol or phosphatidylcholine, phosphatidylethanolamine and sphingomyelin are shown as examples. In the course of these experiments, intramolecular spin-diffusion as well as intermolecular interactions between lipids and water were observed. The technique should enable the investigation of lipid-lipid and lipid-protein interactions, lipid hydration as well as lipid asymmetry in membranes without the use of isotopically labeled lipids.

Labeling of human erythrocyte membrane proteins by photoactivatable radioiodinated phosphatidylcholine and phosphatidylserine A search for the aminophospholipid translocase

We have synthesized radioiodinated photoactivatable phosphatidylcholine (125‐N3‐PC) and phosphatidylserine (125I‐N3‐PS). After incubation with red blood cells in the dark, the labeled PC could be extracted but not the corresponding PS molecule, indicating that the latter was transported by the aminophospholipid translocase, but not the former. When irradiated immediately after incorporation, N3‐PS, but not N3‐PC, partially blocked subsequent translocation of spin‐labeled aminophospholipids. Analysis of probe distribution by SDSpolyacrylamide gel electrophoresis revealed that 125I‐N3‐PS labeled seven membrane bound components with molecular masses between 140 and 27 kDa: one (or several) of these components should correspond to the aminophospholipid translocase.

Photoaffinity spin-labeling of the Ca2+ ATPase in sarcoplasmic reticulum : Evidence for oligomeric structure

Abstract A spin labeled fatty acid (16-doxylstearic acid) was linked to a photochemical reacting group (azido derivative). When the molecule is introduced, at a low concentration, into rabbit sarcoplasmic reticulum membranes, the spectrum before illumination is identical to the spectrum obtained with the corresponding spin labeled fatty acid. After illumination, a large immobilized components is seen. It corresponds to about 70% of the ESR signal of the effectively bound label, at room temperature. The fraction of immobilized component varies with temperature, from 100% at 0°C to 50% at 35°C. Addition of a small amount of detergent (dodecyl octaethylene glycol monoether), under non solubilizing conditions, decreases the fraction of signal due to a strongly immobilized probe. A possible interpretation is that the immobilized signal reflects protein bound spin labels trapped in Ca2+ ATPase oligomers, which are partially dissociated by detergent addition or temperature increase.