Opsin is a phospholipid flippase

Polar lipids must flip-flop rapidly across biological membranes to sustain cellular life [1, 2], but flipping is energetically costly [3] and its intrinsic rate is low. To overcome this problem, cells have membrane proteins that function as lipid transporters (flippases) to accelerate flipping to a physiologically relevant rate. Flippases that operate at the plasma membrane of eukaryotes, coupling ATP hydrolysis to unidirectional lipid flipping, have been defined at a molecular level [2]. On the other hand, ATP-independent bidirectional flippases that translocate lipids in biogenic compartments, e.g., the endoplasmic reticulum, and specialized membranes, e.g., photoreceptor discs [4, 5], have not been identified even though their activity has been recognized for more than 30 years [1]. Here, we demonstrate that opsin is the ATP-independent phospholipid flippase of photoreceptor discs. We show that reconstitution of opsin into large unilamellar vesicles promotes rapid (τ<10 s) flipping of phospholipid probes across the vesicle membrane. This is the first molecular identification of an ATP-independent phospholipid flippase in any system. It reveals an unexpected activity for opsin and, in conjunction with recently available structural information on this G protein-coupled receptor [6, 7], significantly advances our understanding of the mechanism of ATP-independent lipid flip-flop.     

Flippase activity in proteoliposomes reconstituted with Spinacea oleracea endoplasmic reticulum membrane proteins: evidence of biogenic membrane flippase in plants

Phospholipid translocation (flip-flop) in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum of eukaryotic cells (rat liver) and bacterial cytoplasmic membranes is a fundamental step in membrane biogenesis. It is known that flip-flop in these membranes occurs without a metabolic energy requirement, bidirectionally with no specificity for phospholipid headgroup. In this study, we demonstrate for the first time ATP-independent flippase activity in endoplasmic reticulum membranes of plants using spinach as a model system. For this, we generated proteoliposomes from a Triton X-100 extract of endoplasmic reticulum membranes of spinach and assayed them for flippase activity using fluorescently labeled phospholipids. The half-time for flipping was found to be 0.7-1.0 min. We also show that (a) proteoliposomes can flip fluorescently labeled analogues of phosphatidylcholine and phosphatidylethanolamine, (b) flipping activity is protein-mediated, (c) more than one class of lipid translocator (flippase) is present in spinach membranes, based on the sensitivity to protease and protein-modifying reagents, and (d) translocation of PC and PE is affected differently upon treatment with protease and protein-modifying reagents. Ca (2+)-dependent scrambling activity was not observed in the vesicles reconstituted from plant ER membranes, ruling out the possibility of the involvement of scramblase in translocation of phospholipids. These results suggest the existence of biogenic membrane flippases in plants and that the mechanism of membrane biogenesis is similar to that found in animals.     

Distinct flippases translocate glycerophospholipids and oligosaccharide diphosphate dolichols across the endoplasmic reticulum

Transbilayer movement, or flip-flop, of lipids across the endoplasmic reticulum (ER) is required for membrane biogenesis, protein glycosylation, and GPI anchoring. Specific ER membrane proteins, flippases, are proposed to facilitate lipid flip-flop, but no ER flippase has been biochemically identified. The glycolipid Glc 3Man 9GlcNAc 2-PP-dolichol is the oligosaccharide donor for protein N-glycosylation reactions in the ER lumen. Synthesis of Glc 3Man 9GlcNAc 2-PP-dolichol is initiated on the cytoplasmic side of the ER and completed on the lumenal side, requiring flipping of the intermediate Man 5GlcNAc 2-PP-dolichol (M5-DLO) across the ER. Here we report the reconstitution of M5-DLO flipping in proteoliposomes generated from Triton X-100-extracted Saccharomyces cerevisiae microsomal proteins. Flipping was assayed by using the lectin Concanavalin A to capture M5-DLOs that had been translocated from the inner to the outer leaflet of the vesicles. M5-DLO flipping in the reconstituted system was ATP-independent and trypsin-sensitive and required a membrane protein(s) that sedimented at approximately 4 S. Man 7GlcNAc 2-PP-dolichol, a higher-order lipid intermediate, was flipped >10-fold more slowly than M5-DLO at 25 degrees C. Chromatography on Cibacron Blue dye resin enriched M5-DLO flippase activity approximately 5-fold and resolved it from both the ER glycerophospholipid flippase activity and the genetically identified flippase candidate Rft1 [Helenius, J., et al. (2002) Nature 415, 447-450]. The latter result indicates that Rft1 is not the M5-DLO flippase. Our data (i) demonstrate that the ER has at least two distinct flippase proteins, each specifically capable of translocating a class of phospholipid, and (ii) provide, for the first time, a biochemical means of identifying the M5-DLO flippase.     

The mystery of phospholipid flip-flop in biogenic membranes

Phospholipid flip-flop is required for bilayer assembly and the maintenance of biogenic (self-synthesizing) membranes such as the eukaryotic endoplasmic reticulum and the bacterial cytoplasmic membrane. Due to the membrane topology of phospholipid biosynthesis, newly synthesized phospholipids are initially located in the cytoplasmic leaflet of biogenic membranes and must be translocated to the exoplasmic leaflet to give uniform bilayer growth. It is clear from many studies that phospholipid flip-flop in biogenic membranes occurs very rapidly, within a period of a few minutes. These studies also reveal that phospholipid translocation in biogenic membranes occurs bi-directionally, independently of the phospholipid head group, via a facilitated diffusion process in the absence of metabolic energy input, and that this type of transport requires specific membrane proteins. These translocators have been termed biogenic membrane flippases, and they differ from metabolic energy-dependent transporters (ABC transporters and MDR proteins). No biogenic membrane flippases have been characterized. This review briefly discusses the importance of biogenic membrane flippases, the various assay methods used for measuring the rate of phospholipid flip-flop, and the progress that has been made towards identifying these proteins.     

Inhibition of biogenic membrane flippase activity in reconstituted ER proteoliposomes in the presence of low cholesterol levels

Biogenic membranes or self-synthesizing membranes are the site of synthesis of new lipids such as the endoplasmic reticulum (ER) in eukaryotes. Newly synthesized phospholipids (PLs) at the cytosolic leaflet of ER need to be translocated to the lumen side for membrane biogenesis and this is facilitated by a special class of lipid translocators called biogenic membrane flippase. Even though ER is the major site of cholesterol synthesis, it contains very low amounts of cholesterol, since newly synthesized cholesterol in ER is rapidly transported to other organelles and is highly enriched in plasma membrane. Thus, only low levels of cholesterol are present at the biosynthetic compartment (ER), which results in loose packing of ER lipids. We hypothesize that the prevalence of cholesterol in biogenic membranes might affect the rapid flip-flop. To validate our hypothesis, detergent solubilized ER membranes from both bovine liver and spinach leaves were reconstituted into proteoliposomes with varying mol% of cholesterol. Our results show that (i) with increase in the cholesterol/PL ratio, the half-life time of PL translocation increased, suggesting that cholesterol affects the kinetics of flipping, (ii) flipping activity was completely inhibited in proteoliposomes reconstituted with 1 mol% cholesterol, and (iii) FRAP and DSC experiments revealed that 1 mol% cholesterol did not alter the bilayer properties significantly and that flippase activity inhibition is probably mediated by interaction of cholesterol with the protein.     

Specific proteins are required to translocate phosphatidylcholine bidirectionally across the endoplasmic reticulum

BACKGROUND: A long-standing problem in understanding the mechanism by which the phospholipid bilayer of biological membranes is assembled concerns how phospholipids flip back and forth between the two leaflets of the bilayer. This question is important because phospholipid biosynthetic enzymes typically face the cytosol and deposit newly synthesized phospholipids in the cytosolic leaflet of biogenic membranes such as the endoplasmic reticulum (ER). These lipids must be transported across the bilayer to populate the exoplasmic leaflet for membrane growth. Transport does not occur spontaneously and it is presumed that specific membrane proteins, flippases, are responsible for phospholipid flip-flop. No biogenic membrane flippases have been identified and there is controversy as to whether proteins are involved at all, whether any membrane protein is sufficient, or whether non-bilayer arrangements of lipids support flip-flop. RESULTS: To test the hypothesis that specific proteins facilitate phospholipid flip-flop in the ER, we reconstituted transport-active proteoliposomes from detergent-solubilized ER vesicles under conditions in which protein-free liposomes containing ER lipids were inactive. Transport was measured using a synthetic, water-soluble phosphatidylcholine and was found to be sensitive to proteolysis and associated with proteins or protein-containing complexes that sedimented operationally at 3.8S. Chromatographic analyses indicated the feasibility of identifying the transporter(s) by protein purification approaches, and raised the possibility that at least two different proteins are able to facilitate transport. Calculations based on a simple reconstitution scenario suggested that the transporters represent approximately 0.2% of ER membrane proteins. CONCLUSIONS: Our results clearly show that specific proteins are required to translocate a phosphatidylcholine analogue across the ER membrane. These proteins are likely to be the flippases, which are required to translocate natural phosphatidylcholine and other phospholipids across the ER membrane. The methodology that we describe paves the way for identification of a flippase.     

Reconstitution of phospholipid flippase activity from E. coli inner membrane: a test of the protein translocon as a candidate flippase

Phospholipid flipping in biogenic membranes is a key feature of membrane bilayer assembly. Flipping is facilitated by proteinaceous transporters (flippases) that do not need metabolic energy to function. No flippase has yet been identified. The architecture of the E. coli protein translocon suggests that it could account for the flippase activity in the bacterial inner membrane. To test this possibility, we used E. coli cells depleted of SecYE or YidC to assay flipping in proteoliposomes reconstituted from detergent extracts of their inner membranes. We conclude that the protein translocon contributes minimally, if at all, to phospholipid flippase activity in the inner membrane.     

Homocysteine-induced cardiomyocyte apoptosis and plasma membrane flip-flop are independent of S-adenosylhomocysteine: a crucial role for nuclear p47phox

We previously found that homocysteine (Hcy) induced plasma membrane flip-flop, apoptosis, and necrosis in cardiomyocytes. Inactivation of flippase by Hcy induced membrane flip-flop, while apoptosis was induced via a NOX2-dependent mechanism. It has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in hyperhomocysteinemia (HHC)-induced pathogenesis of cardiovascular disease. Therefore, we evaluated whether the observed cytotoxic effect of Hcy in cardiomyocytes is SAH dependent. Rat cardiomyoblasts (H9c2 cells) were treated under different conditions: (1) non-treated control (1.5 nM intracellular SAH with 2.8 μM extracellular L -Hcy), (2) incubation with 50 μM adenosine-2,3-dialdehyde (ADA resulting in 83.5 nM intracellular SAH, and 1.6 μM extracellular L -Hcy), (3) incubation with 2.5 mM D, L -Hcy (resulting in 68 nM intracellular SAH and 1513 μM extracellular L -Hcy) with or without 10 μM reactive oxygen species (ROS)-inhibitor apocynin, and (4) incubation with 100 nM, 10 μM, and 100 μM SAH. We then determined the effect on annexin V/propodium iodide positivity, flippase activity, caspase-3 activity, intracellular NOX2 and p47(phox) expression and localization, and nuclear ROS production. In contrast to Hcy, ADA did not induce apoptosis, necrosis, or membrane flip-flop. Remarkably, both ADA and Hcy induced a significant increase in nuclear NOX2 expression. However, in contrast to ADA, Hcy additionally induced nuclear p47(phox) expression, increased nuclear ROS production, and inactivated flippase. Incubation with SAH did not have an effect on cell viability, nor on flippase activity, nor on nuclear NOX2-, p47phox expression or nuclear ROS production. HHC-induced membrane flip-flop and apoptosis in cardiomyocytes is due to increased Hcy levels and not primarily related to increased intracellular SAH, which plays a crucial role in nuclear p47(phox) translocation and subsequent ROS production.     

Transbilayer movement of dipalmitoylphosphatidylcholine in proteoliposomes reconstituted from detergent extracts of endoplasmic reticulum. Kinetics of transbilayer transport mediated by a single flippase and identification of protein fractions enriched in flippase activity

Phospholipid translocation (flip-flop) across membrane bilayers is typically assessed via assays utilizing partially water-soluble phospholipid analogs as transport reporters. These assays have been used in previous work to show that phospholipid translocation in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum is facilitated by specific membrane proteins (flippases). To extend these studies to natural phospholipids while providing a framework to guide the purification of a flippase, we now describe an assay to measure the transbilayer translocation of dipalmitoylphosphatidylcholine, a membrane-embedded phospholipid, in proteoliposomes generated from detergent-solubilized rat liver endoplasmic reticulum. Translocation was assayed using phospholipase A(2) under conditions where the vesicles were determined to be intact. Phospholipase A(2) rapidly hydrolyzed phospholipids in the outer leaflet of liposomes and proteoliposomes with a half-time of approximately 0.1 min. However, for flippase-containing proteoliposomes, the initial rapid hydrolysis phase was followed by a slower phase reflecting flippase-mediated translocation of phospholipids from the inner to the outer leaflet. The amplitude of the slow phase was decreased in trypsin-treated proteoliposomes. The kinetic characteristics of the slow phase were used to assess the rate of transbilayer equilibration of phospholipids. For 250-nm diameter vesicles containing a single flippase, the half-time was 3.3 min. Proportionate reductions in equilibration half-time were observed for preparations with a higher average number of flippases/vesicle. Preliminary purification steps indicated that flippase activity could be enriched approximately 15-fold by sequential adsorption of the detergent extract onto anion and cation exchange resins.     

Flippase activity detected with unlabeled lipids by shape changes of giant unilamellar vesicles

Transbilayer movement of phospholipids in biological membranes is mediated by energy-dependent and energy-independent flippases. Available methods for detection of flippase mediated transversal flip-flop are essentially based on spin-labeled or fluorescent lipid analogues. Here we demonstrate that shape change of giant unilamellar vesicles (GUVs) can be used as a new tool to study the occurrence and time scale of flippase-mediated transbilayer movement of unlabeled phospholipids. Insertion of lipids into the external leaflet created an area difference between the two leaflets that caused the formation of a bud-like structure. Under conditions of negligible flip-flop, the bud was stable. Upon reconstitution of the energy-independent flippase activity of the yeast endoplasmic reticulum into GUVs, the initial bud formation was reversible, and the shapes were recovered. This can be ascribed to a rapid flip-flop leading to relaxation of the monolayer area difference. Theoretical analysis of kinetics of shape changes provides self-consistent determination of the flip-flop rate and further kinetic parameters. Based on that analysis, the half-time of phospholipid flip-flop in the presence of endoplasmic reticulum proteins was found to be on the order of few minutes. In contrast, GUVs reconstituted with influenza virus protein formed stable buds. The results argue for the presence of specific membrane proteins mediating rapid flip-flop.     

Flip-flop of fluorescently labeled phospholipids in proteoliposomes reconstituted with Saccharomyces cerevisiae microsomal proteins

A phospholipid flippase activity from the endoplasmic reticulum (ER) of the model organism Saccharomyces cerevisiae has been characterized and functionally reconstituted into proteoliposomes. Analysis of the transbilayer movement of acyl-7-nitrobenz-2-oxa-1,3-diazol-4-yl (acyl-NBD)-labeled phosphatidylcholine in yeast microsomes using a fluorescence stopped-flow back exchange assay revealed a rapid, ATP-independent flip-flop (half-time, <2 min). Proteoliposomes prepared from a Triton X-100 extract of yeast microsomal membranes were also capable of flipping NBD-labeled phospholipid analogues rapidly in an ATP-independent fashion. Flippase activity was sensitive to the protein modification reagents N-ethylmaleimide and diethylpyrocarbonate. Resolution of the Triton X-100 extract by velocity gradient centrifugation resulted in the identification of a approximately 4S protein fraction enriched in flippase activity as well as of other fractions where flippase activity was depleted or undetectable. We estimate that flippase activity is due to a protein(s) representing approximately 2% (wt/wt) of proteins in the Triton X-100 extract. These results indicate that specific proteins are required to facilitate ATP-independent phospholipid flip-flop in the ER and that their identification is feasible. The architecture of the ER protein translocon suggests that it could account for the flippase activity in the ER. We tested this hypothesis using microsomes prepared from a temperature-sensitive yeast mutant in which the major translocon component, Sec61p, was quantitatively depleted. We found that the protein translocon is not required for transbilayer movement of phospholipids across the ER. Our work defines yeast as a promising model system for future attempts to identify the ER phospholipid flippase and to test and purify candidate flippases.     

Chemical modification identifies two populations of glycerophospholipid flippase in rat liver ER

Transbilayer flipping of glycerophospholipids in the endoplasmic reticulum (ER) is a key feature of membrane biogenesis. Flipping appears to be an ATP-independent, bidirectional process facilitated by specific proteins or flippases. Although a phospholipid flippase has yet to be identified, evidence supporting the existence of dedicated flippases was recently obtained through biochemical reconstitution studies showing that certain chromatographically resolved fractions of detergent-solubilized ER proteins were enriched in flippase activity, whereas others were inactive. We now extend these studies by describing two convenient assays of flippase activity utilizing fluorescent phospholipid analogues as transport reporters. We use these assays to show that (i) proteoliposomes generated from a flippase-enriched Triton X-100 extract of ER can flip analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine; (ii) flipping of all three phospholipids is likely due to the same flippase(s) rather than distinct, phospholipid-specific transport proteins; (iii) functional flippases represent approximately 1% (w/w) of ER membrane proteins in the Triton extract; and (iv) glycerophospholipid flippase activity in the ER can be attributed to two functionally distinct proteins (or classes of proteins) defined by their sensitivity to the cysteine and histidine modification reagents N-ethylmaleimide and diethylpyrocarbonate, respectively. Analyses of the N-ethylmaleimide-sensitive class of flippase activity revealed that the functionally critical sulfhydryl group in the flippase protein is buried in a hydrophobic environment in the membrane but becomes reactive on extraction of the protein into Triton X-100. This observation holds considerable promise for future attempts to isolate the flippase via an affinity approach.     

Flip-flop of phospholipids in proteoliposomes reconstituted from detergent extract of chloroplast membranes: kinetics and phospholipid specificity

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6 ± 1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents.     

Lipid specific activation of the murine P4-ATPase Atp8a1 (ATPase II)

The asymmetric transbilayer distribution of phosphatidylserine (PS) in the mammalian plasma membrane and secretory vesicles is maintained, in part, by an ATP-dependent transporter. This aminophospholipid "flippase" selectively transports PS to the cytosolic leaflet of the bilayer and is sensitive to vanadate, Ca(2+), and modification by sulfhydryl reagents. Although the flippase has not been positively identified, a subfamily of P-type ATPases has been proposed to function as transporters of amphipaths, including PS and other phospholipids. A candidate PS flippase ATP8A1 (ATPase II), originally isolated from bovine secretory vesicles, is a member of this subfamily based on sequence homology to the founding member of the subfamily, the yeast protein Drs2, which has been linked to ribosomal assembly, the formation of Golgi-coated vesicles, and the maintenance of PS asymmetry. To determine if ATP8A1 has biochemical characteristics consistent with a PS flippase, a murine homologue of this enzyme was expressed in insect cells and purified. The purified Atp8a1 is inactive in detergent micelles or in micelles containing phosphatidylcholine, phosphatidic acid, or phosphatidylinositol, is minimally activated by phosphatidylglycerol or phosphatidylethanolamine (PE), and is maximally activated by PS. The selectivity for PS is dependent upon multiple elements of the lipid structure. Similar to the plasma membrane PS transporter, Atp8a1 is activated only by the naturally occurring sn-1,2-glycerol isomer of PS and not the sn-2,3-glycerol stereoisomer. Both flippase and Atp8a1 activities are insensitive to the stereochemistry of the serine headgroup. Most modifications of the PS headgroup structure decrease recognition by the plasma membrane PS flippase. Activation of Atp8a1 is also reduced by these modifications; phosphatidylserine-O-methyl ester, lysophosphatidylserine, glycerophosphoserine, and phosphoserine, which are not transported by the plasma membrane flippase, do not activate Atp8a1. Weakly translocated lipids (PE, phosphatidylhydroxypropionate, and phosphatidylhomoserine) are also weak Atp8a1 activators. However, N-methyl-phosphatidylserine, which is transported by the plasma membrane flippase at a rate equivalent to PS, is incapable of activating Atp8a1 activity. These results indicate that the ATPase activity of the secretory granule Atp8a1 is activated by phospholipids binding to a specific site whose properties (PS selectivity, dependence upon glycerol but not serine, stereochemistry, and vanadate sensitivity) are similar to, but distinct from, the properties of the substrate binding site of the plasma membrane flippase.     

Translocation of phospholipids is facilitated by a subset of membrane-spanning proteins of the bacterial cytoplasmic membrane

The mechanism by which phospholipids are transported across biogenic membranes, such as the bacterial cytoplasmic membrane, is unknown. We hypothesized that this process is mediated by the presence of the membrane-spanning segments of inner membrane proteins, rather than by dedicated flippases. In support of the hypothesis, it was demonstrated that transmembrane alpha-helical peptides, mimicking the membrane-spanning segments, mediate flop of 2-6-(7-nitro-2,1,3-benzoxadiazol-4-yl) aminocaproyl (C6-NBD)-phospholipids (Kol, M. A., de Kroon, A. I., Rijkers, D. T., Killian, J. A., and de Kruijff, B. (2001) Biochemistry 40, 10500-10506). Here the dithionite reduction assay was used to measure transbilayer equilibration of C6-NBD-phospholipids in proteoliposomes, composed of Escherichia coli phospholipids and a subset of bacterial membrane proteins. It is shown that two well characterized integral proteins of the bacterial cytoplasmic membrane, leader peptidase and the potassium channel KcsA, induce phospholipid translocation, most likely by their transmembrane domains. In contrast, the ATP-binding cassette transporter from the E. coli inner membrane MsbA, a putative lipid flippase, did not mediate phospholipid translocation, irrespective of the presence of ATP. OmpT, an outer membrane protein from E. coli, did not facilitate flop either, demonstrating specificity of protein-mediated phospholipid translocation. The results are discussed in the light of phospholipid transport across the E. coli inner membrane.     

ATP-dependent transport of phosphatidylserine analogues in human erythrocytes

The plasma membrane of most cells contains a number of lipid transporters that catalyze the ATP-dependent movement of phospholipids across the membrane and assist in the maintenance of lipid asymmetry. The most well-characterized of these transporters is the erythrocyte aminophospholipid flippase, which selectively transports phosphatidylserine (PS) from the outer to the inner monolayer. Previous work has demonstrated that PS and to a lesser extent phosphatidylethanolamine (PE) are substrates for the flippase and that other phospholipids move across the membrane only by passive flip-flop. The present study re-evaluates these results. The incorporation and transbilayer movement of a number of short-chain (dilauroyl) phospholipid analogues in human erythrocytes was measured by observing lipid-induced changes in cell morphology, and the effect of an ATPase inhibitor (vanadate) and a sulfyhdryl reagent (N-ethylmaleimide) was determined. Incubation of cells with these lipids causes the rapid formation of echinocytes, because of the accumulation of the lipid in the outer monolayer. While dilauroylphosphatidylcholine-treated cells retained this shape, cells treated with sn-1,2-DLP-l-S, sn-1,2-DLP-d-S, or N-methyl-DLPS rapidly changed morphology to stomatocytes, which is consistent with the transport and accumulation of the lipid in the inner monolayer. A similar, although slower, stomatocytic shape change was induced by sn-2,3-DLP-l-S. Other lipids that were tested (dilauroylphosphatidylhydroxypropionate, dilauroylphosphatidylhomoserine, DLPS-methyl ester, or sn-2,3-DLP-d-S) reverted to discocytes only. In all cases, pretreatment with vanadate or N-ethylmaleimide inhibited the conversion of echinocytes to discocytes or stomatocytes. This is the first report of a protein- and energy-dependent pathway for the inwardly directed transbilayer movement of lipids other than PS and PE in the erythrocyte membrane and suggests that the flippase has broader specificity for substrates or that other lipid transporters are present.     

Phospholipid flip-flop modulated by transmembrane peptides WALP and melittin

Select transmembrane proteins found in biogenic membranes are known to facilitate rapid bidirectional flip-flop of lipids between the membrane leaflets, while others have no little or no effect. The particular characteristics which determine the extent to which a protein will facilitate flip-flop are still unknown. To determine if the relative polarity of the transmembrane protein segment influences its capacity for facilitation of flip-flop, we have studied lipid flip-flop dynamics for bilayers containing the peptides WALP₂₃ and melittin. WALP₂₃ is used as a model hydrophobic peptide, while melittin consists of both hydrophobic and hydrophilic residues. Sum-frequency vibrational spectroscopy (SFVS) was used to characterize the bilayers and determine the kinetics of flip-flop for the lipid component, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), within the mixed bilayers. The kinetic data were utilized to determine the activation thermodynamics for DSPC flip-flop in the presence of the peptides. Melittin was found to significantly reduce the free energy barrier to DSPC flip-flop when incorporated into the bilayer at 1 mol.%, while incorporation of WALP₂₃ at the same concentration led to a more modest reduction of the free energy barrier. The possible mechanisms by which these peptides facilitate flip-flop are analyzed and discussed in terms of the observed activation thermodynamics.     

Is lipid flippase activity of SNARE transmembrane domains required for membrane fusion?

It has been suggested that lipids translocate between the outer and inner leaflets of fusing membranes, or flip-flop, to facilitate changes in bilayer leaflet areas at various stages of fusion. Here, we investigated the lipid flip activity of synthetic peptides that mimic SNARE transmembrane domains (TMDs). These peptides indeed induce flip of marker lipids. However, mutations that reduce flip activity do not diminish fusogenicity and cholesterol blocks flip much more efficiently than fusion. Therefore, our data do not support a role for flip in membrane fusion. On the other hand, the ability of SNARE TMDs to catalyze flip is consistent with a role of SNAREs in biogenic lipid flip.     

Reconstitution of glucosylceramide flip-flop across endoplasmic reticulum: implications for mechanism of glycosphingolipid biosynthesis

Most glycosphingolipids are synthesized by the sequential addition of monosaccharides to glucosylceramide (GlcCer) in the lumen of the Golgi apparatus. Because GlcCer is synthesized on the cytoplasmic face of Golgi membranes, it must be flipped to the non-cytoplasmic face by a lipid flippase in order to nucleate glycosphingolipid synthesis. Halter et al. (Halter, D., Neumann, S., van Dijk, S. M., Wolthoorn, J., de Mazière, A. M., Vieira, O. V., Mattjus, P., Klumperman, J., van Meer, G., and Sprong, H. (2007) Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis. J. Cell Biol. 179, 101–115) proposed that this essential flipping step is accomplished via a complex trafficking itinerary; GlcCer is moved from the cytoplasmic face of the Golgi to the endoplasmic reticulum (ER) by FAPP2, a cytoplasmic lipid transfer protein, flipped across the ER membrane, then delivered to the lumen of the Golgi complex by vesicular transport. We now report biochemical reconstitution studies to analyze GlcCer flipping at the ER. Using proteoliposomes reconstituted from Triton X-100-solubilized rat liver ER membrane proteins, we demonstrate rapid (t_½ < 20 s), ATP-independent flip-flop of N-(6-((7-nitro-2–1,3-benzoxadiazol-4-yl)amino)hexanoyl)-d-glucosyl-β1–1′-sphingosine, a fluorescent GlcCer analog. Further studies involving protein modification, biochemical fractionation, and analyses of flip-flop in proteoliposomes reconstituted with ER membrane proteins from yeast indicate that GlcCer translocation is facilitated by well characterized ER phospholipid flippases that remain to be identified at the molecular level. By reason of their abundance and membrane bending activity, we considered that the ER reticulons and the related Yop1 protein could function as phospholipid-GlcCer flippases. Direct tests showed that these proteins have no flippase activity.