Showing papers on "Membrane lipids published in 2000"
TL;DR: A model for the budding of HIV virions through lipid rafts is proposed whereby host cell cholesterol, sphingolipids, and GPI-linked proteins within these domains are incorporated into the viral envelope, perhaps as a result of preferential sorting of HIV Gag to lipid rafting.
Abstract: A number of recent studies have demonstrated the significance of detergent-insoluble, glycolipid-enriched membrane domains or lipid rafts, especially in regard to activation and signaling in T lymphocytes. These domains can be viewed as floating rafts composed of sphingolipids and cholesterol which sequester glycosylphosphatidylinositol (GPI)-linked proteins, such as Thy-1 and CD59. CD45, a 200-kDa transmembrane phosphatase protein, is excluded from these domains. We have found that human immunodeficiency virus type 1 (HIV-1) particles produced by infected T-cell lines acquire the GPI-linked proteins Thy-1 and CD59, as well as the ganglioside GM1, which is known to partition preferentially into lipid rafts. In contrast, despite its high expression on the cell surface, CD45 was poorly incorporated into virus particles. Confocal fluorescence microscopy revealed that HIV-1 proteins colocalized with Thy-1, CD59, GM1, and a lipid raft-specific fluorescent lipid, DiIC(16)(3), in uropods of infected Jurkat cells. CD45 did not colocalize with HIV-1 proteins and was excluded from uropods. Dot immunoassay of Triton X-100-extracted membrane fractions revealed that HIV-1 p17 matrix protein and gp41 were present in the detergent-resistant fractions and that [(3)H]myristic acid-labeled HIV Gag showed a nine-to-one enrichment in lipid rafts. We propose a model for the budding of HIV virions through lipid rafts whereby host cell cholesterol, sphingolipids, and GPI-linked proteins within these domains are incorporated into the viral envelope, perhaps as a result of preferential sorting of HIV Gag to lipid rafts.
786 citations
TL;DR: Detergent insolubility is an invaluable tool for studying cellular rafts and characterizing their composition and can depend on lipid composition, detergent and temperature.
689 citations
TL;DR: Both the liquid‐ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent‐resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts.
Abstract: The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membranes on a millisecond time scale and with positional accuracy of ∼50 nm, using ‘single dye tracing’. This first application of single molecule microscopy to living cells rendered possible the direct observation of lipid-specific membrane domains. These domains were sensed by a lipid probe with saturated acyl chains as small areas in a liquid-ordered phase: the probe showed confined but fast diffusion, with high partitioning (∼100-fold) and long residence time (∼13 s). The analogous probe with mono-unsaturated chains diffused predominantly unconfined within the membrane. With ∼15 saturated probes per domain, the locations, sizes, shapes and motions of individual domains became clearly visible. Domains had a size of 0.7 μm (0.2–2 μm), covering ∼13% of total membrane area. Both the liquid-ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent-resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts.
577 citations
TL;DR: The results suggest that lipid rafts are involved in the biosynthetic delivery of proteins to the yeast plasma membrane.
Abstract: Lipid rafts, formed by lateral association of sphingolipids and cholesterol, have been implicated in membrane traffic and cell signaling in mammalian cells. Sphingolipids also have been shown to play a role in protein sorting in yeast. Therefore, we wanted to investigate whether lipid rafts exist in yeast and whether these membrane microdomains have an analogous function to their mammalian counterparts. We first developed a protocol for isolating detergent-insoluble glycolipid-enriched complexes (DIGs) from yeast cells. Sequencing of the major protein components of the isolated DIGs by mass spectrometry allowed us to identify, among others, Gas1p, Pma1p, and Nce2p. Using lipid biosynthetic mutants we could demonstrate that conditions that impair the synthesis of sphingolipids and ergosterol also disrupt raft association of Gas1p and Pma1p but not the secretion of acid phosphatase. That endoplasmic reticulum (ER)-to-Golgi transport of Gas1p is blocked in the sphingolipid mutant lcb1-100 raised the question of whether proteins associate with lipid rafts in the ER or later as shown in mammalian cells. Using the sec18-1 mutant we found that DIGs are present already in the ER. Taken together, our results suggest that lipid rafts are involved in the biosynthetic delivery of proteins to the yeast plasma membrane.
564 citations
TL;DR: The results suggest that α-Syn membrane interactions are physiologically important and the lipid composition of the cellular membranes may affect these interactions in vivo.
555 citations
TL;DR: The data suggest that lipid rafts are important in controlling appropriate protein interactions in resting and activated T cells, and that aggregation of rafts following receptor ligation may be a general mechanism for promoting immune cell signalling.
457 citations
TL;DR: The discovery of extraplastidic DGDG biosynthesis induced by phosphate deprivation has revealed a biochemical mechanism for plants to conserve phosphate and apparently, plants replace phospholipid with nonphosphorous galactolipids if environmental conditions such as phosphate deprivation require this for survival.
Abstract: The galactolipids, mono- and digalactosyldiacylglycerol (DGDG), are the most common nonphosphorous lipids in the biosphere and account for 80% of the membrane lipids found in green plant tissues. These lipids are major constituents of photosynthetic membranes (thylakoids), and a large body of evidence suggests that galactolipids are associated primarily with plastid membranes in seed plants. A null-mutant of Arabidopsis (dgd1), which lacks the DGDG synthase (DGD1) resulting in a 90% reduction in the amount of DGDG under normal growth conditions, accumulated DGDG after phosphate deprivation up to 60% of the amount present in the wild type. This observation suggests the existence of a DGD1-independent pathway of galactolipid biosynthesis. The fatty acid composition of the newly formed DGDG was distinct, showing an enrichment of 16-carbon fatty acids in the C-1 position of the glycerol backbone of DGDG. Roots with their rudimentary plastids accumulated large amounts of DGDG after phosphate deprivation, suggesting that this galactolipid may be located in extraplastidic membranes. Corroborating evidence for this hypothesis was obtained directly by fractionation of subcellular membranes from leaf tissue and indirectly by lipid analysis of the phosphate-deprived fad3 mutant primarily deficient in extraplastidic fatty acid desaturation. The discovery of extraplastidic DGDG biosynthesis induced by phosphate deprivation has revealed a biochemical mechanism for plants to conserve phosphate. Apparently, plants replace phospholipids with nonphosphorous galactolipids if environmental conditions such as phosphate deprivation require this for survival.
359 citations
TL;DR: A crucial role for cholesterol is indicated in the regulation of signaling pathways in T cells, which is likely to reflect its importance in the formation of plasma membrane lipid rafts.
Abstract: Lipid rafts are specialized plasma membrane microdomains, in which glycosphingolipids and cholesterol are major structural components. In T lymphocytes, several signaling proteins are associated with lipid rafts including the protein tyrosine kinase LCK and the adapter protein LAT. To investigate their importance in T cell signaling, lipid rafts were disrupted by depleting cholesterol with methyl-beta-cyclodextrin (MbetaCD). This transiently induced tyrosine phosphorylation of multiple proteins, including the ZAP-70 tyrosine kinase, its associated T cell antigen receptor zeta chain, LAT and phospholipase Cgamma1. Tyrosine phosphorylation was dependent on expression of LCK in lipid rafts. Depletion of cholesterol also resulted in activation of the Ras-ERK pathway. This was largely dependent on phorbol ester-sensitive protein kinase C (PKC) and the PKC-theta isoform translocated to the plasma membrane following MbetaCD treatment. MbetaCD did not stimulate intracellular Ca2+ fluxes; however, consistent with its ability to stimulate Ras, MbetaCD synergized with a Ca2+ ionophore to induce formation of the transcription factor NF-AT. These data indicate a crucial role for cholesterol in the regulation of signaling pathways in T cells, which is likely to reflect its importance in the formation of plasma membrane lipid rafts.
340 citations
TL;DR: Characterization of intracellular lipid particles of three different eukaryotic species, namely mammalian cells, plants and yeast are described and models for the biogenesis of lipid particles are presented and compared among the different species.
323 citations
TL;DR: The evidence reviewed here shows that the MDR1 P-glycoprotein and the multidrug resistance (-associated) transporter 1 (MRP1) are able to transport lipid analogues, but probably not major natural membrane lipids.
308 citations
TL;DR: It is shown that the associations of annexins II, V, and VI with smooth muscle microsomal membranes exhibit a high degree of Ca2+ sensitivity, and that the extraction of annexin II and VI by detergent is prevented by elevated Ca2 + concentrations.
Abstract: The sarcolemma of smooth muscle cells is composed of alternating stiff actin-binding, and flexible caveolar domains. In addition to these stable macrodomains, the plasma membrane contains dynamic glycosphingolipid- and cholesterol-enriched microdomains, which act as sorting posts for specific proteins and are involved in membrane trafficking and signal transduction. We demonstrate that these lipid rafts are neither periodically organized nor exclusively confined to the actin attachment sites or caveolar regions. Changes in the Ca2+ concentration that are affected during smooth muscle contraction lead to important structural rearrangements within the sarcolemma, which can be attributed to members of the annexin protein family. We show that the associations of annexins II, V, and VI with smooth muscle microsomal membranes exhibit a high degree of Ca2+ sensitivity, and that the extraction of annexins II and VI by detergent is prevented by elevated Ca2+ concentrations. Annexin VI participates in the formation of a reversible, membrane–cytoskeleton complex (Babiychuk, E.B., R.J. Palstra, J. Schaller, U. Kampfer, and A. Draeger. 1999. J. Biol. Chem. 274:35191–35195). Annexin II promotes the Ca2+-dependent association of lipid raft microdomains, whereas annexin V interacts with glycerophospholipid microcompartments. These interactions bring about a new configuration of membrane-bound constituents, with potentially important consequences for signaling events and Ca2+ flux.
TL;DR: Structural and biochemical analysis of protein kinase C homology-1 and -2, FYVE, and pleckstrin homology domains has shown that their molecular mechanisms of membrane binding depend on a combination of specific and nonspecific interactions with membrane lipids.
Abstract: Protein kinase C homology-1 and -2, FYVE, and pleckstrin homology domains are ubiquitous in eukaryotic signal transduction and membrane-trafficking proteins. These domains regulate subcellular localization and protein function by binding to lipid ligands embedded in cell membranes. Structural and biochemical analysis of these domains has shown that their molecular mechanisms of membrane binding depend on a combination of specific and nonspecific interactions with membrane lipids. In vivo studies of green fluorescent protein fusions have highlighted the key roles of these domains in regulating protein localization to plasma and internal membranes in cells.
TL;DR: The hypothesis that lysosomal storage diseases can be caused by the accumulation of lipid rafts in late endosomes/lysosomes is forward.
TL;DR: How the biophysical properties of the membrane bilayer and the chemistry of individual lipid molecules play a role in the intracellular trafficking of the lipids themselves, as well as influencing the trafficking of transmembrane proteins is discussed.
Abstract: Lipid compositions vary greatly among organelles, and specific sorting mechanisms are required to establish and maintain these distinct compositions. In this review, we discuss how the biophysical properties of the membrane bilayer and the chemistry of individual lipid molecules play a role in the intracellular trafficking of the lipids themselves, as well as influencing the trafficking of transmembrane proteins. The large diversity of lipid head groups and acyl chains lead to a variety of weak interactions, such as ionic and hydrogen bonding at the lipid/water interfacial region, hydrophobic interactions, and van-der-Waals interactions based on packing density. In simple model bilayers, these weak interactions can lead to large-scale phase separations, but in more complex mixtures, which mimic cell membranes, such phase separations are not observed. Nevertheless, there is growing evidence that domains (i.e., localized regions with non-random lipid compositions) exist in biological membranes, and it is likely that the formation of these domains are based on interactions similar to those that lead to phase separations in model systems. Sorting of lipids appears to be based in part on the inclusion or exclusion of certain types of lipids in vesicles or tubules as they bud from membrane organelles.
TL;DR: The results support the idea that the uptake mechanism involves only the interaction of the peptide with the membrane lipids and conclude that the translocation does not involve pore formation.
TL;DR: Novel mechanisms regulated by the DRM lipids, sphingomyelin and cholesterol, mediate the uptake of host DRM proteins and maintenance of the intracellular vacuole in the non‐endocytic red cell, which may have implications for intrACEllular parasitism and pathogenesis.
Abstract: Erythrocytes, which are incapable of endocytosis or phagocytosis, can be infected by the malaria parasite Plasmodium falciparum. We find that a transmembrane protein (Duffy), glycosylphosphatidylinositol (GPI)-anchored and cytoplasmic proteins, associated with detergent-resistant membranes (DRMs) that are characteristic of microdomains in host cell membranes, are internalized by vacuolar parasites, while the major integral membrane and cytoskeletal proteins are not. The internalized host proteins and a plasmodial transmembrane resident parasitophorous vacuolar membrane (PVM) protein are detected in DRMs associated with vacuolar parasites. This is the first report of a host transmembrane protein being recruited into an apicomplexan vacuole and of the presence of vacuolar DRMs; it establishes that integral association does not preclude protein internalization into the P.FALCIPARUM: vacuole. Rather, as shown for Duffy, intracellular accumulation occurs at the same rate as that seen for a DRM-associated GPI-anchored protein. Furthermore, novel mechanisms regulated by the DRM lipids, sphingomyelin and cholesterol, mediate (i) the uptake of host DRM proteins and (ii) maintenance of the intracellular vacuole in the non-endocytic red cell, which may have implications for intracellular parasitism and pathogenesis.
TL;DR: It is demonstrated that during the course of measles virus replication, a significant proportion of MV structural proteins were selectively enriched in the detergent-resistant glycosphingolipids and cholesterol-rich membranes (rafts), indicating that raft membranes could provide a platform for MV assembly.
Abstract: The process of measles virus (MV) assembly and subsequent budding is thought to occur in localized regions of the plasma membrane, to favor specific incorporation of viral components, and to facilitate the exclusion of host proteins. We demonstrate that during the course of virus replication, a significant proportion of MV structural proteins were selectively enriched in the detergent-resistant glycosphingolipids and cholesterol-rich membranes (rafts). Isolated rafts could infect the cell through a membrane fusion step and thus contained all of the components required to create a functional virion. However, they could be distinguished from the mature virions with regards to density and Triton X-100 resistance behavior. We further show that raft localization of the viral internal nucleoprotein and matrix protein was independent of the envelope glycoproteins, indicating that raft membranes could provide a platform for MV assembly. Finally, at least part of the raft MV components were included in the viral particle during the budding process. Taken together, these results strongly suggest a role for raft membranes in the processes of MV assembly and budding.
TL;DR: The function of vitamin E in membranes is described and the current state of knowledge of the effect ofitamin E on the structure and phase behaviour of phospholipid model membranes is reviewed.
Abstract: Summary Vitamin E is a fat-soluble vitamin that consists of a group of tocols and tocotrienols with hydrophobic character, butpossessing a hydroxyl substituent that confers an amphipathic character on them. The isomers of biological importance are the tocopherols, of which a-tocopherol is the most potent vitamin. Vitamin E partitions into lipoproteins and cell membranes, where it represents a minor constituent of most membranes. It has a major function in its action as a lipid antioxidant to protect the polyunsaturated membrane lipids against free radical attack. Other functions are believed to be to act as membrane stabilizers by forming complexes with the products of membrane lipid hydrolysis, such as lysophospholipids and free fatty acids. The main experimental approach to explain the functions of vitamin E in membranes has been to study its effects on the structure and stability of model phospholipid membranes. This review describes the function of vitamin E in membranes and reviews the current state of knowledge of the effect of vitamin E on the structure and phase behaviour of phospholipid model membranes.
01 Jan 2000
TL;DR: In this paper, the authors present a synopsis of the physical properties of lipid systems and indicate how they may relate to the functional capacities of biological membranes, including the major role of membrane lipids.
Abstract: Biological membranes contain an astonishing variety of lipids. As detailed throughout this book, generation of this diversity requires elaborate metabolic pathways. The lipid compounds representing the end products of these pathways must bestow significant evolutionary advantages to the cellular or multicellular systems in which they reside, implying particular functional roles for each component. However, clarification of the functional roles of individual lipid species has proven a difficult problem. Here we present a synopsis of the physical properties of lipid systems and indicate how they may relate to the functional capacities of biological membranes. The major role of membrane lipids has been understood in broad outline since the early experiments of Gorter and Grendell [l], who extracted lipids from the erythrocyte membrane and measured the areas these lipids were able to cover as a monolayer at an air-water interface. This work led to the conclusion that the erythrocytes contained sufficient lipid to provide a bilayer lipid matrix surrounding the red blood cell. This bilayer lipid organization, which provides a permeability barrier between exterior and interior compartments, has remained a dominant theme in our understanding of the organization and function of biological membranes. Subsequent observations that such bilayers are fluid, allowing rapid lateral diffusion of lipid and protein in the plane of the membrane, and that membrane proteins are often inserted into and through the lipid matrix, have further contributed to our present understanding of membranes, resulting in the Singer and Nicholson [2] fluid mosaic model, a refined version of which is shown in Fig.1
TL;DR: Recent research that has given new insights into how plants prevent the accumulation of unusual fatty acids in membrane lipids are discussed, and how strict this censorship of membrane composition is is described.
TL;DR: The lipid phase of the membrane cannot be overlooked while investigating the MDR phenotype and taking into account these aspects should be useful in the search of ways to obviate MDR and could also be relevant to the study of other multidrug transporters.
Abstract: P-glycoprotein (Pgp), the so-called multidrug transporter, is a plasma membrane glycoprotein often involved in the resistance of cancer cells towards multiple anticancer agents in the multidrug-resistant (MDR) phenotype. It has long been recognized that the lipid phase of the plasma membrane plays an important role with respect to multidrug resistance and Pgp because: the compounds involved in the MDR phenotype are hydrophobic and diffuse passively through the membrane; Pgp domains involved in drug binding are located within the putative transmembrane segments; Pgp activity is highly sensitive to its lipid environment; and Pgp may be involved in lipid trafficking and metabolism. Unraveling the different roles played by the membrane lipid phase in MDR is relevant, not only to the evaluation of the precise role of Pgp, but also to the understanding of the mechanism of action and function of Pgp. With this aim, I review the data from different fields (cancer research, medicinal chemistry, membrane biophysics, pharmaceutical research) concerning drug-membrane, as well as Pgp-membrane, interactions. It is emphasized that the lipid phase of the membrane cannot be overlooked while investigating the MDR phenotype. Taking into account these aspects should be useful in the search of ways to obviate MDR and could also be relevant to the study of other multidrug transporters.
TL;DR: It is shown that the rate of movement of cholesterol from lysosomes to plasma membranes in NP-C cells is at least as great as normal, as was also found previously for amphiphile-treated cells, and the lysOSomes in these cells filled with plasma membrane cholesterol in the absence of lipoproteins.
TL;DR: It was found that the cholesterol induced changes on the membrane properties such as lipid order, linewidth broadening, efflux rates, bilayer widths, etc., did not depend on the ability of the lipids to participate in the hydrogen bonding interactions with the 3beta-OH of cholesterol.
TL;DR: The results suggest that permeabilization of the lipid bilayer with saponin to admit the access of antibodies to the cytoplasmic surface of cells can aggregate transmembrane proteins and affect the immunocytochemical localization of associated proteins of the cytoskeleton.
TL;DR: The interaction of DNA with a novel cationic phospholipid transfection reagent, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), was investigated, revealing significant differences in the DNA-lipid particle, depending upon whether complexes were formed by the addition of DNA to lipid or vice versa.
TL;DR: The submicron clusters of IL-2Ralpha chains and their coclustering with HLA and CD48, presumably associated with lipid rafts, could underlie the efficiency of signaling in lymphoid cells.
Abstract: Immunogold staining and electron microscopy show that IL-2 receptor α-subunits exhibit nonrandom surface distribution on human T lymphoma cells. Analysis of interparticle distances reveals that this clustering on the scale of a few hundred nanometers is independent of the presence of IL-2 and of the expression of the IL-2R β-subunit. Clustering of IL-2Rα is confirmed by confocal microscopy, yielding the same average cluster size, ≈600–800 nm, as electron microscopy. HLA class I and II and CD48 molecules also form clusters of the same size. Disruption of cholesterol-rich lipid rafts with filipin or depletion of membrane cholesterol with methyl-β-cyclodextrin results in the blurring of cluster boundaries and an apparent dispersion of clusters for all four proteins. Interestingly, the transferrin receptor, which is thought to be located outside lipid rafts, exhibits clusters that are only 300 nm in size and are less affected by modifying the membrane cholesterol content. Furthermore, transferrin receptor clusters hardly colocalize with IL-2Rα, HLA, and CD48 molecules (crosscorrelation coefficient is 0.05), whereas IL-2Rα colocalizes with both HLA and CD48 (crosscorrelation coefficient is between 0.37 and 0.46). This coclustering is confirmed by electron microscopy. The submicron clusters of IL-2Rα chains and their coclustering with HLA and CD48, presumably associated with lipid rafts, could underlie the efficiency of signaling in lymphoid cells.
TL;DR: Comparing several parameters of membrane structure and function in three tPMP-1-resistant (tPMp-1r) strains and their genetically related, t PMP- 1-susceptible (tPGM-1s) counterpart strains provides further support for the concept that specific alterations in the cytoplasmic membrane of S. aureus strains are associated with tPMC-1 resistance in vitro.
Abstract: Platelet microbicidal proteins (PMPs) are small, cationic peptides which possess potent microbicidal activities against common bloodstream pathogens, such as Staphylococcus aureus. We previously showed that S. aureus strains exhibiting resistance to thrombin-induced PMP (tPMP-1) in vitro have an enhanced capacity to cause human and experimental endocarditis (T. Wu, M. R. Yeaman, and A. S. Bayer, Antimicrob. Agents Chemother. 38:729‐732, 1994; A. S. Bayer et al., Antimicrob. Agents Chemother. 42:3169‐3172, 1998; V. K. Dhawan et al., Infect. Immun. 65:3293‐3299, 1997). However, the mechanisms mediating tPMP-1 resistance in S. aureus are not fully delineated. The S. aureus cell membrane appears to be a principal target for the action of tPMP-1. To gain insight into the basis of tPMP-1 resistance, we compared several parameters of membrane structure and function in three tPMP-1-resistant (tPMP-1 r ) strains and their genetically related, tPMP-1susceptible (tPMP-1 s ) counterpart strains. The tPMP-1 r strains were derived by three distinct methods: transposon mutagenesis, serial passage in the presence of tPMP-1 in vitro, or carriage of a naturally occurring multiresistance plasmid (pSK1). All tPMP-1 r strains were found to possess elevated levels of longer-chain, unsaturated membrane lipids, in comparison to their tPMP-1 s counterparts. This was reflected in corresponding differences in cell membrane fluidity in the strain pairs, with tPMP-1 r strains exhibiting significantly higher degrees of fluidity as assessed by fluorescence polarization. These data provide further support for the concept that specific alterations in the cytoplasmic membrane of S. aureus strains are associated with tPMP-1 resistance in vitro.
TL;DR: Membrane microdomains (lipid rafts) are enriched in selected signaling molecules and may compartmentalize receptor-mediated signals, and BCR stimulation induced rapid and transient translocation of the Src homology 2 domain-containing inositol phosphatase, SHIP, into lipid rafts.
Abstract: Membrane microdomains (lipid rafts) are enriched in selected signaling molecules and may compartmentalize receptor-mediated signals. Here, we report that in primary human B lymphocytes and in Ramos B cells B cell receptor (BCR) stimulation induces rapid and transient redistribution of a subset of engaged BCRs to lipid rafts and phosphorylation of raft-associated tyrosine kinase substrates. Cholesterol sequestration disrupted the lipid rafts, preventing BCR redistribution, but did not inhibit tyrosine kinase activation or phosphorylation of mitogen-activated protein kinase/extracellular regulated kinase. However, raft disruption enhanced the release of calcium from intracellular stores, suggesting that rafts may sequester early signaling events that down-regulate calcium flux. Consistent with this, BCR stimulation induced rapid and transient translocation of the Src homology 2 domain-containing inositol phosphatase, SHIP, into lipid rafts.
TL;DR: This study establishes that each module is an independent membrane-targeting module with each, independently of the other, containing determinants for membrane recognition.
Abstract: Protein kinase C is specifically activated by binding two membrane lipids: the second messenger, diacylglycerol, and the amino phospholipid, phosphatidylserine. This binding provides the energy to release an autoinhibitory pseudosubstrate from the active site. Interaction with these lipids recruits the enzyme to the membrane by engaging two membrane-targeting modules: the C1 domain (present as a tandem repeat in most protein kinase Cs) and the C2 domain. Here we dissect the contribution of each domain in recruiting protein kinase C betaII to membranes. Binding analyses of recombinant domains reveal that the C2 domain binds anionic lipids in a Ca(2+)-dependent, but diacylglycerol-independent, manner, with little selectivity for phospholipid headgroup beyond the requirement for negative charge. The C1B domain binds membranes in a diacylglycerol/phorbol ester-dependent, but Ca(2+)-independent manner. Like the C2 domain, the C1B domain preferentially binds anionic lipids. However, in striking contrast to the C2 domain, the C1B domain binds phosphatidylserine with an order of magnitude higher affinity than other anionic lipids. This preference for phosphatidylserine is, like that of the full-length protein, stereoselective for sn-1, 2-phosphatidyl-L-serine. Quantitative analysis of binding constants of individual domains and that of full-length protein reveals that the full-length protein binds membranes with lower affinity than expected based on the binding affinity of isolated domains. In addition to entropic and steric considerations, the difference in binding energy may reflect the energy required to expel the pseudosubstrate from the substrate binding cavity. This study establishes that each module is an independent membrane-targeting module with each, independently of the other, containing determinants for membrane recognition. The presence of each of these modules, separately, in a number of other signaling proteins epitomizes the use of these modules as discreet membrane targets.
TL;DR: It is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin is not purely electrostatic.