Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile

Nonbilayer lipids can be defined as cone-shaped lipids with a preference for nonbilayer structures with a negative curvature, such as the hexagonal phase. All membranes contain these lipids in large amounts. Yet, the lipids in biological membranes are organized in a bilayer. This leads to the question: what is the physiological role of nonbilayer lipids? Different models are discussed in this review, with a focus on the lateral pressure profile within the membrane. Based on this lateral pressure model, predictions can be made for the effect of nonbilayer lipids on peripheral and integral membrane proteins. Recent data on the catalytic domain of Leader Peptidase and the potassium channel KcsA are discussed in relation to these predictions and in relation to the different models on the function of nonbilayer lipids. The data suggest a general mechanism for the interaction between nonbilayer lipids and membrane proteins via the membrane lateral pressure.     

Phase equilibria of membrane lipids from Acholeplasma laidlawii: importance of a single lipid forming nonlamellar phases

A basis for the reorganization of the bilayer structure in biological membranes is the different aggregate structures formed by lipids in water. The phase equilibria of all individual lipids and several in vivo polar lipid mixtures from acyl chain modified membranes of Acholeplasma laidlawii were investigated with different NMR techniques. All dioleoyl (DO) polar lipids, except monoglucosyldiglyceride (MGDG), form lamellar liquid crystalline (L alpha) phases only. The phase diagram of DOMGDG reveals reversed cubic (III), reversed hexagonal (HII), and L alpha phases. In mixtures of DOMGDG and dioleoyldiglycosyldiglyceride (DODGDG), the formation of an III (or HII) phase is enhanced by DOMGDG and low hydration or high temperatures. For in vivo mixtures of all polar DO lipids, a transition from an L alpha to an III phase is promoted by low hydration or high temperatures (50 degrees C). The phospholipids are incorporated in this III phase. Likewise, III and HII phases are formed at similar temperatures in a series of in vivo mixtures with different extents of acyl chain unsaturation. However, their melting temperatures (Tm) vary in an expected manner. All cubic and hexagonal phases, except the III phase with DOMGDG, exist in equilibrium with excess water. The maximum hydration of MGDG and DGDG is similar and increases with acyl chain unsaturation but is substantially lower than that for, e.g., phosphatidylcholine. The translational diffusion of the lipids in the cubic phases is rapid, implying bicontinuous structures. However, their appearances in freeze-fracture electron microscope pictures are different. The III phase of DOMGDG belongs to the Ia3d space group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles

The structural basis for organizational heterogeneity of lipids and proteins underlies fundamental questions about the plasma membrane of eukaryotic cells. A current hypothesis is the participation of liquid ordered (Lo) membrane domains (lipid rafts) in dynamic compartmentalization of membrane function, but it has been difficult to demonstrate the existence of these domains in live cells. Recently, giant plasma membrane vesicles (GPMVs) obtained by chemically induced blebbing of cultured cells were found to phase separate into optically resolvable, coexisting fluid domains containing Lo-like and liquid disordered (Ld)-like phases as identified by fluorescent probes. In the present study, we used these GPMVs to investigate the structural bases for partitioning of selected lipids and proteins between coexisting Lo-like/Ld-like fluid phases in compositionally complex membranes. Our results with lipid probes show that the structure of the polar headgroups, in addition to acyl chain saturation, can significantly affect partitioning. We find that the membrane anchor of proteins and the aggregation state of proteins both significantly influence their distributions between coexisting fluid phases in these biological membranes. Our results demonstrate the value of GPMVs for characterizing the phase preference of proteins and lipid probes in the absence of detergents and other perturbations of membrane structure.     

Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles

The structural basis for organizational heterogeneity of lipids and proteins underlies fundamental questions about the plasma membrane of eukaryotic cells. A current hypothesis is the participation of liquid ordered (Lo) membrane domains (lipid rafts) in dynamic compartmentalization of membrane function, but it has been difficult to demonstrate the existence of these domains in live cells. Recently, giant plasma membrane vesicles (GPMVs) obtained by chemically induced blebbing of cultured cells were found to phase separate into optically resolvable, coexisting fluid domains containing Lo-like and liquid disordered (Ld)-like phases as identified by fluorescent probes. In the present study, we used these GPMVs to investigate the structural bases for partitioning of selected lipids and proteins between coexisting Lo-like/Ld-like fluid phases in compositionally complex membranes. Our results with lipid probes show that the structure of the polar headgroups, in addition to acyl chain saturation, can significantly affect partitioning. We find that the membrane anchor of proteins and the aggregation state of proteins both significantly influence their distributions between coexisting fluid phases in these biological membranes. Our results demonstrate the value of GPMVs for characterizing the phase preference of proteins and lipid probes in the absence of detergents and other perturbations of membrane structure.     

Spiroplasma membrane lipids.

Membranes of six spiroplasma strains belonging to different Spiroplasma species and subgroups were isolated by a combination of osmotic lysis and sonication in the presence of EDTA to block endogenous phospholipase activity. Analysis of membrane lipids showed that in addition to free and esterified cholesterol the spiroplasmas incorporated exogenous phospholipids from the growth medium. Sphingomyelin was preferentially incorporated from phosphatidylcholine-sphingomyelin vesicles or from the serum used to supplement the growth medium. Palmitate was incorporated better than oleate into membrane lipids synthesized by the organisms during growth. The major phospholipid synthesized by the spiroplasmas was phosphatidylglycerol. The positional distribution of the fatty acids in phosphatidylglycerol of Spiroplasma floricola resembled that found in Mycoplasma species, in which the saturated fatty acids prefer position 2 in the glycerol backbone and not position 1 as found in Acholeplasma species and elsewhere in nature. Electron paramagnetic resonance analysis of spin-labeled fatty acids incorporated into S. floricola membranes exhibited homogeneous single-component spectra without immobilized regions. The S. floricola membranes were more rigid than those of Acholeplasma laidlawii and less rigid than those of Mycoplasma gallisepticum.     

Lysosomal degradation of membrane lipids

The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes.     

Non-covalent binding of membrane lipids to membrane proteins

Polar lipids and membrane proteins are major components of biological membranes, both cell membranes and membranes of enveloped viruses. How these two classes of membrane components interact with each other to influence the function of biological membranes is a fundamental question that has attracted intense interest since the origins of the field of membrane studies. One of the most powerful ideas that driven the field is the likelihood that lipids bind to membrane proteins at specific sites, modulating protein structure and function. However only relatively recently has high resolution structure determination of membrane proteins progressed to the point of providing atomic level structure of lipid binding sites on membrane proteins. Analysis of X-ray diffraction, electron crystallography and NMR data over 100 specific lipid binding sites on membrane proteins. These data demonstrate tight lipid binding of both phospholipids and cholesterol to membrane proteins. Membrane lipids bind to membrane proteins by their headgroups, or by their acyl chains, or binding is mediated by the entire lipid molecule. When headgroups bind, binding is stabilized by polar interactions between lipid headgroups and the protein. When acyl chains bind, van der Waals effects dominate as the acyl chains adopt conformations that complement particular sites on the rough protein surface. No generally applicable motifs for binding have yet emerged. Previously published biochemical and biophysical data link this binding with function. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Structures of archaebacterial membrane lipids

Structural data on archaebacterial lipids is presented with emphasis on the ether lipids of the methanogens. These ether lipids normally account for 80–95% of the membrane lipids with the remaining 5–20% of neutral squalenes and other isoprenoids. Genus-specific combinations of various lipid core structures found in methanogens include diether-tetraether, diether-hydroxydiether, or diether-macrocyclic diether-tetraether lipid moieties. Some species have only the standard diether core lipid, but none are known with predominantly tetraether lipids as found in certain sulfur-dependent archaebacteria. The relative proportions of these lipid cores are known to vary in relation to growth conditions inMethanococcus jannaschii andMethanobacterium thermoautotrophicum. Polar headgroups in glycosidic or phosphodiester linkage to thesn-1 orsn-1 carbons of glycerol consist of polyols, carbohydrates, and amino compounds. The available structural data indicate a close similarity among the polar lipids synthesized within the species of the same genus. Detection of lipid molecular ions by mass spectrometry of total polar lipid extracts is a promising technique to provide valuable comparative data. Since these lipid structures are stable within the extreme environments that many archaebacteria inhabit, there may be specific applications for their use in biotechnology.     

Fourier transform infrared assay of membrane lipids immobilized to silica: leaching and stability of immobilized artificial membrane-bonded phases

A nondestructive, sensitive assay to monitor the hydrocarbon content of silica-based chromatography particles has been developed. The assay requires a microscope accessory interfaced with a Fourier transform infrared (FTIR) spectrometer. For determining hydrocarbon content, undiluted alkyl-silica-bonded phases were pressed into a thin wafer. Hydrocarbon content was quantitated using the integrated hydrocarbon band intensity between 2995 and 2825 cm-1 [i.e., band area C-H] and the integrated silica oxide band intensity between 1945 and 1780 cm-1 [i.e., band area Si-O]. Plotting the [band area C-H]/[band area Si-O] ratio vs the carbon content determined by elemental analysis gave a correlation coefficient of r = 0.997. The FTIR assay was validated on 5-, 7-, and 12-microns silica particles using three different immobilized artificial membrane (IAM) silica-bonded phases. The utility of the FTIR assay in determining hydrocarbon content was demonstrated by evaluating hydrocarbon leaching from IAM phases exposed to mobile-phase solvents. The ability of organic solvents to leach hydrocarbon from IAM phases containing phosphatidylcholine (PC) as the immobilized ligand was chloroform greater than ethanol approximately methanol greater than ethyl acetate greater than methylene chloride greater than acetonitrile greater than acetone. Acetone and acetonitrile cause very little hydrocarbon leaching from HPLC-IAM.PC columns. When challenged with different mobile phases, IAM.PC columns perfused with mobile phase are more stable than IAM.PC-bonded phases stirred in mobile phases. IAM.PC contains lecithin linked to silica by amide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excimer-forming lipids in membrane research

Pyrenedecanoic acid and pyrene lecithin are optical probes well suited to investigate lipid bilayer membranes. The method is based on the determination of the formation of excited dimers or excimers. The rate of excimer formation yields information on the dynamic molecular properties of artificial as well as of natural membranes. This article will review applications of the excimer-forming probes.Pyrene lipid probes are used to determine the coefficient of the lateral diffusion in fluid lipid membranes. Results in artificial membranes are comparable to the values obtained in erythrocyte membranes.Moreover, the excimer formation rate is a very sensitive measure of changes in membrane fluidity. Membrane fluidity is an important regulator of membrane functional proteins. For example, there is a correlation between membrane fluidity and enzyme activities of the adenylate cyclase system.The excimer formation technique is not restricted to the measurement of lateral mobility in membranes. It can also be used to determine the transversal mobility, that is, the lipid exchange between the lipid layers of one bilayer or between bilayers of different vesicles. Again, artificial as well as natural membranes can be investigated by this technique.Another important area of investigation in membrane research is the interaction between lipids and proteins. Lipids, in the presence of a protein, show a different dynamic behavior from free lipids. Because of changes in fluidity and a modified solubility of the pyrene probes within different membrane regions, our methods could also be applied to the examination of phase separation phenomena and to lipid-protein interactions.     

Subcellular targeting by membrane lipids

The reversible localization of signaling proteins to both the plasma and the internal membranes of cells is critical for the selective activation of downstream functions and depends on interactions with both proteins and membrane lipids. New structural and biochemical analyses of C1, C2, PH, FYVE, FERM and other domains have led to an unprecedented amount of information on the molecular interactions of these signaling proteins with regulatory lipids. A wave of studies using GFP-tagged membrane binding domains as reporters has led to new quantitative insights into the kinetics of these signaling mechanisms.     

The membrane lipids of Halobacterium halobium

The lipid content of the cell membrane of Halobacterium halobium increased from about 15% to 21% during exponential growth of the organism. Total lipid phosphorus more than doubled during the growth cycle. The mixture of membrane lipids from stationary-phase organisms was similar to lipid mixtures from whole cells of other halobacteria inasmuch as 80% of the lipid phosphorus occurred in a diether analogue of phosphatidylglycerophosphate and an additional 7.5% occurred in the ether analogue of phosphatidylglycerol. The lipid mixture was more complex than those reported for other halophils, however, 12 components being recognized in the acetone-insoluble fraction and 17 in the acetone-soluble fraction. There were major changes in the proportions of some minor components of the acetone-insoluble fraction during a growth cycle. Three nitrogenous lipids were recognized in the acetone-insoluble fraction, but all were present in relatively low proportion. One, which was not a phospholipid, contained a bound peptide. Of the 17 acetonesoluble compounds, 15 were pigments. The major carotenoids were alpha- and beta-bacteriorubrin. The carotenoid pigments occurred at maximal concentration after 6-7 days' growth.     

Phagosomal membrane lipids of LM fibroblasts

Summary Murine fibroblasts, LM cells, were cultured in suspension or monolayer in a chemically defined medium without serum and exposed to polystyrene beads. The LM cells endocytized the beads in direct proportion to the bead/cell ratio and the bead surface area. However, equal volumes of beads irrespective of size or surface area were internalized. The lipid composition of the phagosome membrane differed significantly from the parent primary membrane in having higher contents of phosphatidylcholine, phosphatidylserine, and sterol but lower contents of sphingomyelin and lysophosphatidylcholine. When phagosomes isolated from suspension-cultured LM fibroblasts were exposed to trinitrobenzenesulfonic acid at 4°C, 55±1.6% of the phagosomal membrane phosphatidylethanolamine was trinitrophenylated. The asymmetric distribution of phosphatidylethanolamine across the phagosomal membrane was not affected by the bead/cell ratio, bead diameter, or exposure time of LM fibroblasts to the beads. When cells were reacted with trinitrobenzenesulfonic acid at 4°C prior to phagocytosis, the amount of trinitrophenylphosphatidylethanolamine was greater in the isolated phagosomes than in the parent primary plasma membrane. Culturing LM fibroblasts in suspension or monolayer had no effect on the asymmetric distribution of phosphatidylethanolamine across primary plasma membrane bilayers. The data are consistent with the observation that LM fibroblasts grown either in suspension or monolayer internalize polystyrene beads at selective sites in the surface membrane.     

Lactoperoxidase-catalyzed iodination of surface membrane lipids

Lactoperoxidase-catalyzed iodination of intact cells, is known to label predominantly, if not exclusively, the exposed tyrosine residues of cell surface proteins. The present study demonstrates that during this iodination process surface membrane lipids are also iodinated through an enzyme-dependent step. Phosphatidylcholine, cholesterol-phosphatidylcholine liposomes and confluent secondary cultures of chick embryo cells were iodinated by the lactoperoxidase-glucose oxidase-glucose [¹²⁵I] procedure. Liposomes were efficiently labeled. In the cells, 20–30% of the radioactivity was found in proteins and 20–30% in the lipids. Both neutral and polar lipids were found to bind [¹²⁵I] covalently. Controls in which lactoperoxidase was omitted showed < 6% of the radioactivity found in liposomes or cells labeled with the enzyme.