No abstract

Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner and sphingolipids in the outer leaflet. This asymmetry is maintained by ATP-driven lipid transporters whose identities are unknown. The yeast plasma membrane contains two P-type ATPases, Dnf1p and Dnf2p, with structural similarity to ATPase II, a candidate aminophospholipid translocase from bovine chromaffin granules. Loss of Dnf1p and Dnf2p virtually abolished ATP-dependent transport of NBD-labeled phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine from the outer to the inner plasma membrane leaflet, leaving transport of sphingolipid analogs unaffected. Labeling with trinitrobenzene sulfonic acid revealed that the amount of phosphatidylethanolamine exposed on the surface of ⌬dnf1⌬dnf2 cells increased twofold relative to wild-type cells. Phosphatidylethanolamine exposure by ⌬dnf1⌬dnf2 cells further increased upon removal of Drs2p, an ATPase II homolog in the yeast Golgi. These changes in lipid topology were accompanied by a cold-sensitive defect in the uptake of markers for bulk-phase and receptor-mediated endocytosis. Our findings demonstrate a requirement for Dnf1p and Dnf2p in lipid translocation across the yeast plasma membrane. Moreover, it appears that Dnf1p, Dnf2p and Drs2p each help regulate the transbilayer lipid arrangement in the plasma membrane, and that this regulation is critical for budding endocytic vesicles.

ATP-binding-cassette transporter 1 (ABC1) has been implicated in processes related to membrane-lipid turnover. Here, using in vivo loss-of-function and in vitro gain-of-function models, we show that ABC1 promotes Ca2+-induced exposure of phosphatidylserine at the membrane, as determined by a prothrombinase assay, membrane microvesiculation and measurement of transbilayer redistribution of spin-labelled phospholipids. That ABC1 promotes engulfment of dead cells is shown by the impaired ability of ABC1-deficient macrophages to engulf apoptotic preys and by the acquisition of phagocytic behaviour by ABC1 transfectants. Release of membrane phospholipids and cholesterol to apo-AI, the protein core of the cholesterol-shuttling high-density lipoprotein (HDL) particle, is also ABC1-dependent. We propose that both the efficiency of apoptotic-cell engulfment and the efflux of cellular lipids depend on ABC1-induced perturbation of membrane phosphatidylserine turnover. Transient local exposure of anionic phospholipids in the outer membrane leaflet may be sufficient to alter the general properties of the membrane and thus influence discrete physiological functions.

Spin-labeled analogs of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine have been used to study phospholipid transverse diffusion and asymmetry in the human erythrocyte membrane. Ascorbate reduction was used to assess the transbilayer distribution of the labels. All three spin-labeled phospholipids initially incorporated into the outer leaflet of the membrane. On fresh erythrocytes at 50C, the phosphatidylcholine label remained mainly in the outer leaflet. In contrast, the phosphatidylserine and phosphatidylethanolamine labels underwent rapid transverse diffusion that led to their asymmetric distribution in favor of the inner leaflet. The latter effect was reversibly inhibited after ATP depletion of the erythrocytes and could be reproduced on resealed erythrocyte ghosts only if hydrolyzable Mg-ATP was included in the internal medium. It is suggested that an ATPdriven transport of amino phospholipids toward the inner leaflet could be the major cause of the phospholipid asymmetry in the erythrocyte membrane. It is also proposed that the same mechanism could explain the ATP requirement of the maintenance of the erythrocyte membrane discoid shape. that this method can be used successfully in erythrocytes, and the transverse diffusion of phosphatidylcholine has been measured. In the present study, the following phospholipid spin labels have been used.These phospholipids possess an unmodified polar head group R that can be either choline, serine, or ethanolamine and areAn asymmetric distribution of phospholipids exists between the two halves of many biological membranes (for review, see ref. 1). The human erythrocyte membrane appears to be the best characterized in this regard. Numerous studies using chemical labeling (2, 3) and phospholipases (4-6) indicate that, in this membrane, phosphatidylcholine and sphingomyelin are distributed in favor of the outer leaflet, while phosphatidylethanolamine and phosphatidylserine are mainly located in the inner leaflet.These observations raise the problem of how this asymmetry is maintained during the 120-day life span of the erythrocyte. The asymmetric distribution could in principle be maintained if transbilayer diffusion of phospholipids was negligible. However, for phosphatidylcholine, transbilayer "flip-flop" has been shown to occur with a half-time of 8-15 hr in the native membrane (7,8), although no such measurements exist for the three other major phospholipid species. A possible role of cytoskeletal proteins in maintaining the asymmetry in spite of transbilayer diffusion has been proposed by Haest et al. (9,10), but no direct evidence for such a process has been observed in the intact native membrane.In the present study, the formation and maintenance of phospholipid asymmetry in the erythrocyte membrane has been addressed by measuring the transverse diffusion of spin-labeled phospholipids bearing different polar head groups. The method, introduced by Kornberg and McConnell (11), is based on the accessibility of membrane-associated spin labe...

Mixtures of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC) in water form disks also called bicelles and different bilayer organizations when the mol ratio of the two lipids and the temperature are varied. The spontaneous alignment in a magnetic field of these bilayers above the transition temperature T(m) of DMPC is an attractive property that was successfully used to investigate protein structure by NMR. In this article, we have attempted to give an overview of all structural transformations of DMPC/DHPC mixtures that can be inferred from broad band (31)P-NMR spectroscopy between 5 and 60 degrees C. We show that above a critical temperature, T(v), perforated vesicles progressively replace alignable structures. The holes in these vesicles disappear above a new temperature threshold, T(h). The driving force for these temperature-dependent transformations that has been overlooked in previous studies is the increase of DHPC miscibility in the bilayer domain above T(m). Accordingly, we propose a new model (the "mixed bicelle" model) that emphasizes the consequence of the mixing. This investigation shows that the various structures of DMPC in the presence of increasing mol ratios of the short-chain DHPC is reminiscent of the observation put forward by several laboratories investigating solubilization and reconstitution of biological membranes.