Phospholipid outside-inside translocation in lymphocyte plasma membranes is a protein-mediated phenomenon

Zachowski, Alain; Herrmann, Andreas; Paraf, A; Devaux, Philippe F.

doi:10.1016/0005-2736(87)90328-2

Cited by 89 publications

(28 citation statements)

References 16 publications

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“…Protein-mediated inward motion of aminophospholipids discovered first in erythrocytes has been found also in other cells (6)(7)(8). Thus, the outward protein-mediated motion of aminophospholipids probably also exists in other cell membranes, which could have important physiological consequences.…”

Section: Discussionmentioning

confidence: 98%

“…The washed erythrocytes were stored on ice and used within 6 hr. All samples contained 5 mM diisopropyl fluorophosphate (6) to minimize hydrolysis of spin-labeled phospholipids.…”

Section: Methodsmentioning

confidence: 99%

“…This selective and active transport has been demonstrated also in other cells (6)(7)(8). To explain the observed phospholipid transbilayer asymmetry (9) in terms of a dynamic equilibrium, existence of an outward motion was postulated (1).…”

Section: Introductionmentioning

confidence: 99%

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Measurement of outward translocation of phospholipids across human erythrocyte membrane.

Bitbol

Devaux

1988

Proc. Natl. Acad. Sci. U.S.A.

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Spin-labeled phospholipids have been used to study the outside -. inside and inside -* outside transport of phospholipids across the human erythrocyte membrane at 3TC. As already shown, inward transport is much faster for aminophospholipids than for phosphatidycholine. In addition, we show here that outward transport of the phosphatidylserine and phosphatidylethanolamine analogues is three to four times faster than that ofphosphatidycholine. 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-ATPdependent aminophospholipid carrier.Inward translocation of phosphatidylserine and phosphatidylethanolamine across human erythrocyte membrane is mediated by a specific ATP-dependent protein (1)(2)(3)(4)(5). This selective and active transport has been demonstrated also in other cells (6)(7)(8). To explain the observed phospholipid transbilayer asymmetry (9) in terms of a dynamic equilibrium, existence of an outward motion was postulated (1). Thus, the rate of the outward transport of a given phospholipid can be estimated from knowing both its rate of inward translocation and its asymptotic distribution. Such a calculation was made by Herrmann and Muller (1986) (10) and more recently by Williamson et al. (11).In the present paper we give direct experimental evidence for outward phospholipid motion across the human erythrocyte membrane, and we assess this diffusion rate by using spin-labeled analogues of phospholipids. This outward motion of aminophospholipids is shown to be more than passive diffusion through the bilayer. Evidence for the role of a selective protein carrier is presented.MATERIALS AND METHODS Buffers. The buffer used was 140 mM NaCl/10 mM Hepes, pH 7.4; in some experiments 1 or 2 mM EDTA was added.Erythrocytes. Fresh human blood was obtained from healthy volunteers or from a local blood bank (Hopital Cochin). Blood collected on EDTA was washed three times in 3 vol of buffer by centrifugation (1000 g at 4°C for 5 min). The washed erythrocytes were stored on ice and used within 6 hr. All samples contained 5 mM diisopropyl fluorophosphate (6) to minimize hydrolysis of spin-labeled phospholip- . Spin labels were synthesized as described (1,12,13). Suitable amounts of spin labels in chloroform or chloroform/methanol 2:1 (vol/vol) were dried under vacuum and resuspended in buffer. Two volumes of this aqueous dispersion of labels (ice-cold) were then added to one volume of ice-cold washed erythrocyte pellet. We verified, as in ref.1, that the spin-labeled phospholipids incorporate in erythrocyte membranes within 3 min under such conditions. The spin-label concentration, unless specified, was 2 mol % ofthe endogenous phospholipids. Spontaneous reduction of the spin-labeled concentration was monitored after bovine serum albumin (BSA) extracti...

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Section: Discussionmentioning

confidence: 98%

“…The washed erythrocytes were stored on ice and used within 6 hr. All samples contained 5 mM diisopropyl fluorophosphate (6) to minimize hydrolysis of spin-labeled phospholipids.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Measurement of outward translocation of phospholipids across human erythrocyte membrane.

Bitbol

Devaux

1988

Proc. Natl. Acad. Sci. U.S.A.

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“…9 This lipid asymmetry is maintained by a P-type ATPase, 10 called the aminophospholipid translocase, which transports the anionic phospholipids PS and phosphatidylethanolamine from the outer to the inner leaflet of the bilayer. 11,12 However, the resulting asymmetric distribution can be randomized by activation of a second membrane protein, termed the scramblase, that catalyzes rapid, bidirectional transbilayer movement of all classes of phospholipids across the membrane. 13 ± 16 Coordinated inhibition of the translocase and activation of the scramblase occur in T lymphocytes undergoing apoptosis.…”

Section: Introductionmentioning

confidence: 99%

Regulation of phosphatidylserine exposure and phagocytosis of apoptotic T lymphocytes

et al. 1999

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In lymphocytes, an asymmetric distribution of phospholipids across the plasma membrane is maintained by an ATPdependent translocase which specifically transports aminophospholipids from the outer to the inner leaflet of the bilayer. During apoptosis, this enzyme is down-regulated and a lipid flipsite, termed the scramblase, is activated. Together, these events lead to the appearance of phosphatidylserine (PS) on the cell surface. In DO11.10 T lymphocyte hybridoma cells undergoing apoptosis, the kinetics of PS externalization are paralleled by the development of PS-sensitive phagocytosis by macrophages. This parallel is also observed when PS externalization is effected directly by application of a Ca 2+ ionophore, suggesting that PS externalization is not only necessary, but sufficient, to generate a recognition signal. The broad spectrum aspartate-directed cysteine protease (caspase) inhibitor zVAD-fmk blocks externalization of PS and terminal cell lysis after induction of apoptosis by anti-CD3 antibody, but is ineffective when apoptosis is induced in the same cells by treatment with glucocorticoid. These results suggest that apoptosis induced by glucocorticoid does not require the same zVAD-sensitive caspase steps which are required for Fas/FasL-dependent death induced by anti-CD3 antibody, and that the action of these proteases is also not required for PS externalization. Extracellular Ca 2+ is required to complete the later stages of apoptosis in DO11.10 cells, and its removal restores normal transport of PS, suggesting that down-regulation of the aminophospholipid translocase and up-regulation of the scramblase are not effected by irreversible protease cleavage.

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“…Such proteins, named "phospholipid flippases", were postulated by Bretscher (1974). Their existence in human erythrocytes were shown in 1984 using spin-labeled lipids (Seigneuret and Devaux, 1984) and confirmed later with many different techniques in erythrocytes as well as in other mammalian cells (Daleke and Huestis, 1985;Martin and Pagano, 1987;Tilley et al, 1987;Connor and Schroit, 1987;Tannert et al, 2003;Zachowski et al, 1987a). In fact, in all eukaryotic cells where a flippase activity was assayed, the ATP-dependent translocation of aminophospholipids (PS and PE) from the outer to the inner monolayer was demonstrated, which shows the ubiquitous character of this lipid pumping system.…”

Section: Introduction: the Quest Of Lipid Translocasesmentioning

confidence: 99%

Proteins involved in lipid translocation in eukaryotic cells

Devaux

López‐Montero

Bryde

2006

Chemistry and Physics of Lipids

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Since the first discovery of ATP-dependent translocation of lipids in the human erythrocyte membrane in 1984, there has been much evidence of the existence of various ATPases translocating lipids in eukaryotic cell membranes. They include Ptype ATPases involved in inwards lipid transport from the exoplasmic leaflet to the cytosolic leaflet and ABC proteins involved in outwards transport. There are also ATP-independent proteins that catalyze the passage of lipids in both directions. Five Ptype ATPase involved in lipid transport have been genetically characterized in yeast cells, suggesting a pool of several proteins with partially redundant activities responsible for the regulation of lipid asymmetry. However, expression and purification of individual yeast proteins is still insufficient to allow reconstitution experiments in liposomes. In this review, we want to give an overview over current investigation efforts about the identification and purification of proteins that may be involved in lipid translocation.

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Phospholipid outside-inside translocation in lymphocyte plasma membranes is a protein-mediated phenomenon

Cited by 89 publications

References 16 publications

Measurement of outward translocation of phospholipids across human erythrocyte membrane.

Measurement of outward translocation of phospholipids across human erythrocyte membrane.

Regulation of phosphatidylserine exposure and phagocytosis of apoptotic T lymphocytes

Proteins involved in lipid translocation in eukaryotic cells

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