Showing papers on "Membrane lipids published in 2002"

Cholesterol, lipid rafts, and disease

Abstract: Lipid rafts are dynamic assemblies of proteins and lipids that float freely within the liquid-disordered bilayer of cellular membranes but can also cluster to form larger, ordered platforms. Rafts are receiving increasing attention as devices that regulate membrane function in eukaryotic cells. In this Perspective, we briefly summarize the structure and regulation of lipid rafts before turning to their evident medical importance. Here, we will give some examples of how rafts contribute to our understanding of the pathogenesis of different diseases. For more information on rafts, the interested reader is referred to recent reviews (1, 2). Composition of lipid rafts Lipid rafts have changed our view of membrane organization. Rafts are small platforms, composed of sphingolipids and cholesterol in the outer exoplasmic leaflet, connected to phospholipids and cholesterol in the inner cytoplasmic leaflet of the lipid bilayer. These assemblies are fluid but more ordered and tightly packed than the surrounding bilayer. The difference in packing is due to the saturation of the hydrocarbon chains in raft sphingolipids and phospholipids as compared with the unsaturated state of fatty acids of phospholipids in the liquid-disordered phase (3). Thus, the presence of liquid-ordered microdomains in cells transforms the classical membrane fluid mosaic model of Singer and Nicholson into a more complex system, where proteins and lipid rafts diffuse laterally within a two-dimensional liquid. Membrane proteins are assigned to three categories: those that are mainly found in the rafts, those that are present in the liquid-disordered phase, and those that represent an intermediate state, moving in and out of rafts. Constitutive raft residents include glycophosphatidylinositol-anchored (GPI-anchored) proteins; doubly acylated proteins, such as tyrosine kinases of the Src family, Gα subunits of heterotrimeric G proteins, and endothelial nitric oxide synthase (eNOS); cholesterol-linked and palmitate-anchored proteins like Hedgehog (see Jeong and McMahon, this Perspective series, ref. 4); and transmembrane proteins, particularly palmitoylated proteins such as influenza virus hemagglutinin and β-secretase (BACE) (1). Some membrane proteins are regulated raft residents and have a weak affinity for rafts in the unliganded state. After binding to a ligand, they undergo a conformational change and/or become oligomerized. When proteins oligomerize, they increase their raft affinity (5). A peripheral membrane protein, such as a nonreceptor tyrosine kinase, can be reversibly palmitoylated and can lose its raft association after depalmitoylation (6). By these means, the partitioning of proteins in and out of rafts can be tightly regulated.

Lipid rafts are enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization/mass spectrometric analysis.

Abstract: Lipid rafts are specialized cholesterol-enriched membrane domains that participate in cellular signaling processes. Caveolae are related domains that become invaginated due to the presence of the structural protein, caveolin-1. In this paper, we use electrospray ionization mass spectrometry (ESI/MS) to quantitatively compare the phospholipids present in plasma membranes and nondetergent lipid rafts from caveolin-1-expressing and nonexpressing cells. Lipid rafts are enriched in cholesterol and sphingomyelin as compared to the plasma membrane fraction. Expression of caveolin-1 increases the amount of cholesterol recovered in the lipid raft fraction but does not affect the relative proportions of the various phospholipid classes. Surprisingly, ESI/MS demonstrated that lipid rafts are enriched in plasmenylethanolamines, particularly those containing arachidonic acid. While the total content of anionic phospholipids was similar in plasma membranes and nondetergent lipid rafts, the latter were highly enriched in phosphatidylserine but relatively depleted in phosphatidylinositol. Detergent-resistant membranes made from the same cells showed a higher cholesterol content than nondetergent lipid rafts but were depleted in anionic phospholipids. In addition, these detergent-resistant membranes were not enriched in arachidonic acid-containing ethanolamine plasmalogens. These data provide insight into the structure of lipid rafts and identify potential new roles for these domains in signal transduction.

Linearly concatenated cyclobutane lipids form a dense bacterial membrane

Abstract: Lipid membranes are essential to the functioning of cells, enabling the existence of concentration gradients of ions and metabolites. Microbial membrane lipids can contain three-, five-, six- and even seven-membered aliphatic rings, but four-membered aliphatic cyclobutane rings have never been observed. Here we report the discovery of cyclobutane rings in the dominant membrane lipids of two anaerobic ammonium-oxidizing (anammox) bacteria. These lipids contain up to five linearly fused cyclobutane moieties with cis ring junctions. Such 'ladderane' molecules are unprecedented in nature but are known as promising building blocks in optoelectronics. The ladderane lipids occur in the membrane of the anammoxosome, the dedicated intracytoplasmic compartment where anammox catabolism takes place. They give rise to an exceptionally dense membrane, a tight barrier against diffusion. We propose that such a membrane is required to maintain concentration gradients during the exceptionally slow anammox metabolism and to protect the remainder of the cell from the toxic anammox intermediates. Our results further illustrate that microbial membrane lipid structures are far more diverse than previously recognized.

Small heat-shock proteins regulate membrane lipid polymorphism

Abstract: Thermal stress in living cells produces multiple changes that ultimately affect membrane structure and function. We report that two members of the family of small heat-shock proteins (sHsp) (α-crystallin and Synechocystis HSP17) have stabilizing effects on model membranes formed of synthetic and cyanobacterial lipids. In anionic membranes of dimyristoylphosphatidylglycerol and dimyristoylphosphatidylserine, both HSP17 and α-crystallin strongly stabilize the liquid-crystalline state. Evidence from infrared spectroscopy indicates that lipid/sHsp interactions are mediated by the polar headgroup region and that the proteins strongly affect the hydrophobic core. In membranes composed of the nonbilayer lipid dielaidoylphosphatidylethanolamine, both HSP17 and α-crystallin inhibit the formation of inverted hexagonal structure and stabilize the bilayer liquid-crystalline state, suggesting that sHsps can modulate membrane lipid polymorphism. In membranes composed of monogalactosyldiacylglycerol and phosphatidylglycerol (both enriched with unsaturated fatty acids) isolated from Synechocystis thylakoids, HSP17 and α-crystallin increase the molecular order in the fluid-like state. The data show that the nature of sHsp/membrane interactions depends on the lipid composition and extent of lipid unsaturation, and that sHsps can regulate membrane fluidity. We infer from these results that the association between sHsps and membranes may constitute a general mechanism that preserves membrane integrity during thermal fluctuations.

Lipid rafts: structure, function and role in HIV, Alzheimer's and prion diseases.

Abstract: The fluid mosaic model of the plasma membrane has evolved considerably since its original formulation 30 years ago. Membrane lipids do not form a homogeneous phase consisting of glycerophospholipids (GPLs) and cholesterol, but a mosaic of domains with unique biochemical compositions. Among these domains, those containing sphingolipids and cholesterol, referred to as membrane or lipid rafts, have received much attention in the past few years. Lipid rafts have unique physicochemical properties that direct their organisation into liquid-ordered phases floating in a liquid-crystalline ocean of GPLs. These domains are resistant to detergent solubilisation at 4 degrees C and are destabilised by cholesterol- and sphingolipid-depleting agents. Lipid rafts have been morphologically characterised as small membrane patches that are tens of nanometres in diameter. Cellular and/or exogenous proteins that interact with lipid rafts can use them as transport shuttles on the cell surface. Thus, rafts act as molecular sorting machines capable of co-ordinating the spatiotemporal organisation of signal transduction pathways within selected areas ('signalosomes') of the plasma membrane. In addition, rafts serve as a portal of entry for various pathogens and toxins, such as human immunodeficiency virus 1 (HIV-1). In the case of HIV-1, raft microdomains mediate the lateral assemblies and the conformational changes required for fusion of HIV-1 with the host cell. Lipid rafts are also preferential sites of formation for pathological forms of the prion protein (PrPSc) and of the [beta]-amyloid peptide associated with Alzheimer's disease. The possibility of modulating raft homeostasis, using statins and synthetic sphingolipid analogues, offers new approaches for therapeutic interventions in raft-associated diseases.

A polytopic membrane protein displays a reversible topology dependent on membrane lipid composition.

Abstract: To address the role of phospholipids in the topological organization of polytopic membrane proteins, the function and assembly of lactose permease (LacY) was studied in mutants of Escherichia coli lacking phosphatidylethanolamine (PE). PE is required for the proper conformation and active transport function of LacY. The N-terminal half of LacY assembled in PE-lacking cells adopts an inverted topology in which normally non-translocated domains are translocated and vice versa. Post-assembly synthesis of PE triggers a conformational change, resulting in a lipid-dependent recovery of normal conformation and topology of at least one LacY subdomain accompanied by restoration of active transport. These results demonstrate that membrane protein topology once attained can be changed in a reversible manner in response to alterations in phospholipid composition, and may be subject to post-assembly proofreading to correct misfolded structures.

Role of membrane fluidity in pressure resistance of Escherichia coli NCTC 8164.

Abstract: The relationship among growth temperature, membrane fatty acid composition, and pressure resistance was examined in Escherichia coli NCTC 8164. The pressure resistance of exponential-phase cells was maximal in cells grown at 10°C and decreased with increasing growth temperatures up to 45°C. By contrast, the pressure resistance of stationary-phase cells was lowest in cells grown at 10°C and increased with increasing growth temperature, reaching a maximum at 30 to 37°C before decreasing at 45°C. The proportion of unsaturated fatty acids in the membrane lipids decreased with increasing growth temperature in both exponential- and stationary-phase cells and correlated closely with the melting point of the phospholipids extracted from whole cells examined by differential scanning calorimetry. Therefore, in exponential-phase cells, pressure resistance increased with greater membrane fluidity, whereas in stationary-phase cells, there was apparently no simple relationship between membrane fluidity and pressure resistance. When exponential-phase or stationary-phase cells were pressure treated at different temperatures, resistance in both cell types increased with increasing temperatures of pressurization (between 10 and 30°C). Based on the above observations, we propose that membrane fluidity affects the pressure resistance of exponential- and stationary-phase cells in a similar way, but it is the dominant factor in exponential-phase cells whereas in stationary-phase cells, its effects are superimposed on a separate but larger effect of the physiological stationary-phase response that is itself temperature dependent.

Membrane lipids, EGF receptors, and intracellular signals colocalize and are polarized in epithelial cells moving directionally in a physiological electric field

Abstract: Directed cell migration is essential for tissue formation, inflammation, and wound healing. Chemotaxis plays a major role in these situations and is underpinned by asymmetric intracellular signaling. Endogenous electric fields (EFs) are common where cell movement occurs, such as in wound healing, and cells respond to electric field gradients by reorienting and migrating directionally (galvanotaxis/electrotaxis). We show that a physiological EF redistributed both EGF (epidermal growth factor) receptors and detergent-insoluble membrane lipids asymmetrically, leading to cathodal polarization and enhanced activation of the MAP kinase, ERK1/2. This induced leading-edge actin polymerization in directionally migrating mammalian epithelial cells. Inhibiting the EGF receptor-MAP kinase signaling pathway significantly decreased leading edge actin asymmetry and directional migration. We propose a model in which EF-polarized membrane lipid domains and EGF receptors cause asymmetric signaling through MAP kinase, which drives directional cell migration. A comparison is made with the mechanisms underpinning chemotaxis.

Drug susceptibilities of yeast cells are affected by membrane lipid composition

Abstract: In the present study we have exploited isogenic erg mutants of Saccharomyces cerevisiae to examine the contribution of an altered lipid environment on drug susceptibilities of yeast cells. It is observed that erg mutants, which possess high levels of membrane fluidity, were hypersensitive to the drugs tested, i.e., cycloheximide (CYH), o-phenanthroline, sulfomethuron methyl, 4-nitroquinoline oxide, and methotrexate. Most of the erg mutants except mutant erg4 were, however, resistant to fluconazole (FLC). By using the fluorophore rhodamine-6G and radiolabeled FLC to monitor the passive diffusion, it was observed that erg mutant cells elicited enhanced diffusion. The addition of a membrane fluidizer, benzyl alcohol (BA), to S. cerevisiae wild-type cells led to enhanced membrane fluidity. However, a 10 to 12% increase in BA-induced membrane fluidity did not alter the drug susceptibilities of the S. cerevisiae wild-type cells. The enhanced diffusion observed in erg mutants did not seem to be solely responsible for the observed hypersensitivity of erg mutants. In order to ascertain the functioning of drug extrusion pumps encoding the genes CDR1 (ATP-binding cassette family) and CaMDR1 (MFS family) of Candida albicans in a different lipid environment, they were independently expressed in an S. cerevisiae erg mutant background. While the fold change in drug resistance mediated by CaMDR1 remained the same or increased in erg mutants, susceptibility to FLC and CYH mediated by CDR1 was increased (decrease in fold resistance). Our results demonstrate that between the two drug extrusion pumps, Cdr1p appeared to be more adversely affected by the fluctuations in the membrane lipid environment (particularly to ergosterol). By using 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino-hexanoyl] sphingosyl phosphocholine (a fluorescent analogue of sphingomyelin), a close interaction between membrane ergosterol and sphingomyelin which appears to be disrupted in erg mutants is demonstrated. Taken together it appears that multidrug resistance in yeast is closely linked to the status of membrane lipids, wherein the overall drug susceptibility phenotype of a cell appears to be an interplay among drug diffusion, extrusion pumps, and the membrane lipid environment.

Interactions between lipids and bacterial reaction centers determined by protein crystallography.

Abstract: The structure of the reaction center from Rhodobacter sphaeroides has been solved by using x-ray diffraction at a 2.55-Å resolution limit. Three lipid molecules that lie on the surface of the protein are resolved in the electron density maps. In addition to a cardiolipin that has previously been reported [McAuley, K. E., Fyfe, P. K., Ridge, J. P., Isaacs, N. W., Cogdell, R. J. & Jones, M. R. (1999) Proc. Natl. Acad. Sci. USA 96, 14706–14711], two other major lipids of the cell membrane are found, a phosphatidylcholine and a glucosylgalactosyl diacylglycerol. The presence of these three lipids has been confirmed by laser mass spectroscopy. The lipids are located in the hydrophobic region of the protein surface and interact predominately with hydrophobic amino acids, in particular aromatic residues. Although the cardiolipin is over 15 Å from the cofactors, the other two lipids are in close contact with the cofactors and may contribute to the difference in energetics for the two branches of cofactors that is primarily responsible for the asymmetry of electron transfer. The glycolipid is 3.5 Å from the active bacteriochlorophyll monomer and shields this cofactor from the solvent in contrast to a much greater exposed surface evident for the inactive bacteriochlorophyll monomer. The phosphate atom of phosphatidylcholine is 6.5 Å from the inactive bacteriopheophytin, and the associated electrostatic interactions may contribute to electron transfer rates involving this cofactor. Overall, the lipids span a distance of ≈30 Å, which is consistent with a bilayer-like arrangement suggesting the presence of an “inner shell” of lipids around membrane proteins that is critical for membrane function.

Eicosapentaenoic acid regulates scallop (Placopecten magellanicus) membrane fluidity in response to cold

Abstract: The lipid core of a biological membrane requires a certain degree of structural rigidity, but it must also be sufficiently fluid to permit lateral movement of the constituent lipids and embedded proteins. Ectotherms can counteract the ordering effects of reduced temperature by changing the structure of their membranes, a process known as homeoviscous adaptation (1). Although the content of unsaturated fatty acids in the membranes of ectothermic animals is generally known to increase in response to cold (2), no clear and direct relationship between unsaturated fatty acids and membrane fluidity has been established in marine organisms. For example, phospholipid molecular species containing docosahexaenoic acid (22:6 3) are believed to be important in controlling finfish membrane fluidity (3–6), but a direct correlation between 22:6 3 and membrane fluidity has not been found (4, 5, 7, 8). In contrast, we show here a simple but very strong relationship between fluidity and a single polyunsaturated fatty acid, eicosapentaenoic acid (20:5 3), in gill membranes from a marine bivalve mollusc, the sea scallop Placopecten magellanicus. Phospholipids are the main structural elements of biological membranes, and their physical characteristics are key determinants of membrane structure and function. Many vital cell activities that depend on the optimal functioning of membranes are therefore sensitive to the chemistry of the membrane lipids (9) and to environmental conditions, such as temperature and pressure, that perturb the phase behavior and dynamics of lipids in membranes (10). Under extreme or variable conditions, organisms can exploit the tremendous chemical diversity among membrane lipids to defend the physical properties of the membrane (10). Thus in ectotherms, where changes in temperature cause important membrane perturbations, the usual adaptive response includes a modification of lipid composition (11). Sessile animals living in Newfoundland waters must maintain membrane structure and function in the face of extreme cold in deep waters (as low as 1.4°C) or seasonally highly variable conditions in surface waters (as much as 22°C in 6 months) (12). In the present study, we exposed sea scallops to a 10°C decrease in temperature for up to 3 weeks and then examined the relationship between the fatty acid composition of branchial phospholipids and membrane fluidity. Vesicles were prepared from the gills of scallops acclimated to temperatures of 15 and 5°C. After three weeks of thermal acclimation, the structural order of the phospholipids was measured by electron spin resonance (ESR) spectroscopy at five temperatures (0–20°C) that span the physiological range of Placopecten magellanicus (Fig. 1). The vesicles prepared from gills of 5°C-acclimated scallops were significantly (ANCOVA, P 0.03) less ordered than vesicles from 15°C-acclimated scallops. Temperature acclimation had shifted the order parameter curve 1–2°C toward lower assay temperatures, giving a homeoviscous efficacy (13) of 14%. Such a partial adjustment towards an ideal or complete homeoviscous response has also been found in crabs (14) and crayfish (15). In these invertebrates, the costs of perfect compensation may be too high, or the benefits too low. On the other hand, the ESR measurements in this study were made with the spin probe 5-doxyl stearic acid, reflecting the homeoviscous response in the outer region of the purified lipid bilayer. It is possible that the response deeper in the bilayer, in the actual region of alkenyl chain unsaturation, would have been greater (16). Received 30 November 2001; accepted 22 March 2002. 1 Present address: Department of Physiology and Experimental Medicine, The George Washington University Medical Center, Ross Hall Room 402, 2300 Eye Street, NW, Washington, DC 20037. 2 To whom correspondence should be addressed. E-mail: cparrish@ mun.ca Reference: Biol. Bull. 202: 201–203. (June 2002)

Lipid interaction differentiates the constitutive and stress-induced heat shock proteins Hsc70 and Hsp70.

Abstract: Heat shock proteins play a major role in the process of protein folding, and they have been termed molecular chaperones. Two members of the Hsp70 family, Hsc70 and Hsp70, have a high degree of sequence homology. But they differ in their expression pattern. Hsc70 is constitutively expressed, whereas Hsp70 is stress inducible. These 2 proteins are localized in the cytosol and the nucleus. In addition, they have also been observed in close proximity to cellular membranes. We have recently reported that Hsc70 is capable of interacting with a lipid bilayer forming ion-conductance channels. In the present study, we found that both Hsc70 and Hsp70 interact with lipids and can be differentiated by their characteristic induction of liposome aggregation. These proteins promote the aggregation of phosphatidylserine liposomes in a time- and protein concentration-dependent manner. Although both proteins are active in this process, the level and kinetics of aggregation are different between them. Calcium ions enhance Hsc70 and Hsp70 liposome aggregation, but the effect is more dramatic for Hsc70 than for Hsp70. Addition of adenosine triphosphate blocks liposome aggregation induced by both proteins. Adenosine diphosphate (ADP) also blocks Hsp70-mediated liposome aggregation. Micromolar concentrations of ADP enhance Hsc70-induced liposome aggregation, whereas at millimolar concentrations the nucleotide has an inhibitory effect. These results confirm those of previous studies indicating that the Hsp70 family can interact with lipids directly. It is possible that the interaction of Hsp70s with lipids may play a role in the folding of membrane proteins and the translocation of polypeptides across membranes.

The Fusion Peptide of Semliki Forest Virus Associates with Sterol-Rich Membrane Domains

Abstract: Enveloped viruses use cellular membranes during viral entry into the cell via membrane fusion and during virus exit as the source of the virus envelope proteins and lipid bilayer. Viruses have clearly evolved to take advantage of specific cell surface proteins as receptors during entry and/or fusion. Recent reports suggest that some viruses also make use of specific membrane lipids during budding, particularly the laterally segregating cholesterol- and sphingolipid-enriched membrane domains known as rafts (35, 40, 41, 51). These membrane domains are known by a variety of terms, including detergent-resistant membranes (DRMs), due to their relative resistance to solubilization by Triton X-100 (TX-100) in the cold (6). Within cells, DRMs are involved in a variety of important cellular processes, including membrane sorting, signal transduction, and apical targeting (for review, see references 4, 5, 33, and 46). The property of detergent resistance is a function of the lipid composition of the domains (1, 5) and is widely used as an operational definition and an experimental tool. The enveloped alphavirus Semliki Forest virus (SFV) infects cells via endocytic uptake in clathrin-coated vesicles and low-pH-dependent fusion within the endosome and buds from the plasma membrane (for review, see references 18 and 47). Alphaviruses are icosahedrally symmetrical viruses containing a nucleocapsid composed of the capsid protein and the positive-sense RNA. The nucleocapsid is surrounded by a lipid bilayer containing 80 trimers (E1/E2/E3)3 of the E1 and E2 transmembrane (TM) polypeptides, each of about 50 kDa, and a peripheral E3 polypeptide of about 10 kDa. Virus fusion is mediated by the E1 protein, which contains a highly conserved hydrophobic internal region proposed to be the virus fusion peptide (11). During fusion, E1 interacts with the target bilayer and undergoes specific low-pH-triggered conformational changes that result in exposure of previously masked epitopes and formation of a highly stable E1 homotrimer that appears to be required for fusion (22). A proteolytically truncated ectodomain form of E1, E1*, has been used to follow E1's membrane interactions and conformational changes in the absence of virus fusion (12, 13, 20, 25). The SFV E1 ectodomain has recently been crystallized and characterized structurally (27). The native E1 structure is remarkably similar to that of the fusion protein of the flavivirus tick-borne encephalitis (TBE) virus (39), with the protein lying down on the surface of the virus and the putative fusion peptide located at the tip of the molecule. An interesting feature of the alphavirus membrane fusion reaction is its requirement for specific lipids in the target membrane (19, 53). Fusion is greatly promoted by the presence of cholesterol and sphingolipid in target liposomes, and fusion and infection are strongly inhibited by depleting cells of cholesterol. Cholesterol and sphingolipids act to promote the low-pH-dependent conformational changes in either E1 or E1*, including the protein's membrane interaction, acid epitope exposure, and homotrimer formation. srf-3 (sterol requirement in function), an SFV mutant with a decreased requirement for cholesterol in fusion, is less dependent on cholesterol for the conformational changes in E1 (8). The decrease in srf-3's cholesterol requirement is due to a single point mutation on the E1 protein, P226→S (50), which lies outside of the putative fusion peptide. A variety of evidence thus indicates that SFV requires both cholesterol and sphingolipid for fusion. Although this dual requirement is reminiscent of the involvement of these two lipids in the formation of cellular raft domains, no evidence has been presented for raft involvement in SFV fusion. For example, srf-3 has a decreased cholesterol requirement but is unchanged in its sphingolipid requirement, suggesting their independent control (8). Moreover, galactosylceramide, a sphingolipid that does not interact with cholesterol in model monolayer studies, is nonetheless fully active in SFV fusion (36). Given the efficient insertion of the E1 ectodomain into target liposomes at low pH, however, we used this system to examine the association of membrane-bound E1 with the DRM fraction. Our results indicated that the E1 fusion peptide is strongly associated with cholesterol- and sphingolipid-dependent membrane rafts and that it is specifically released following reduction in the cholesterol content of the membrane. (The data in Fig. ​Fig.88 are from a thesis to be submitted by D. L. Gibbons in partial fulfillment of the requirements for the Doctor of Philosophy degree from the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y.) FIG. 8. E1* associates with rafts via its fusion peptide. Radiolabeled ectodomains were mixed with 1 mM complete liposomes (A) or buffer (B), and the mixtures were acid treated at pH 5.5 for 3 min at 37°C. The sample for panel A was then floated ...

Plasma membrane lipids in the resurrection plant Ramonda serbica following dehydration and rehydration.

Abstract: Plants of Ramonda serbica were dehydrated to 3.6% relative water content (RWC) by withholding water for 3 weeks, afterwards the plants were rehydrated for 1 week to 93.8% RWC. Plasma membranes were isolated from leaves using a two-phase aqueous polymer partition system. Compared with well-hydrated (control) leaves, dehydrated leaves suffered a reduction of about 75% in their plasma membrane lipid content, which returned to the control level following rewatering. Also the lipid to protein ratio decreased after dehydration, almost regaining the initial value after rehydration. Lipids extracted from the plasma membrane of fully-hydrated leaves were characterized by a high level of free sterols and a much lower level of phospholipids. Smaller amounts of cerebrosides, acylated steryl glycosides and steryl glycosides were also detected. The main phospholipids of control leaves were phosphatidylcholine and phosphatidylethanolamine, whereas sitosterol was the free sterol present in the highest amount. Following dehydration, leaf plasma membrane lipids showed a constant level of free sterols and a reduction in phospholipids compared with the well-hydrated leaves. Both phosphatidylcholine and phosphatidylethanolamine decreased following dehydration, their molar ratio remaining unchanged. Among free sterols, the remarkably high cholesterol level present in the control leaves (about 14 mol%) increased 2-fold as a result of dehydration. Dehydration caused a general decrease in the unsaturation level of individual phospholipids and total lipids as well. Upon rehydration the lipid composition of leaf plasma membranes restored very quickly approaching the levels of well-hydrated leaves.