Seminal plasma proteins regulate the association of lipids and proteins within detergent-resistant membrane domains of bovine spermatozoa

Maturing spermatozoa acquire full fertilization competence by undergoing major changes in membrane fluidity and protein composition and localization. In epididymal spermatozoa, several proteins are associated with cholesterol- and sphingolipid-enriched detergent-resistant membrane (DRM) domains. These proteins dissociate from DRM in capacitated sperm cells, suggesting that DRM may play a role in the redistribution of integral and peripheral proteins in response to cholesterol removal. Since seminal plasma regulates sperm cell membrane fluidity, we hypothesized that seminal plasma factors could be involved in DRM disruption and redistribution of DRM-associated proteins. Our results indicate that: 1) the sperm-associated proteins, P25b and adenylate kinase 1, are linked to DRM of epididymal spermatozoa, but were exclusively associated with detergent-soluble material in ejaculated spermatozoa; 2) seminal plasma treatment of cauda epididymal spermatozoa significantly lowered the content of cholesterol and the ganglioside, GM1, in DRM; and 3), seminal plasma dissociates P25b from DRM in epididymal spermatozoa. We found that the seminal plasma protein, Niemann-Pick C2 protein, is involved in cholesterol and GM1 depletion within DRM, then leading to membrane redistribution of P25b that occurs in a very rapid and capacitation-independent manner. Together, these data suggest that DRM of ejaculated spermatozoa are reorganized by specific seminal plasma proteins, which induce lipid efflux as well as dissociation of DRM-anchored proteins. This process could be physiologically relevant in vivo to allow sperm survival and attachment within the female reproductive tract and to potentiate recognition, binding, and penetration of the oocyte.     

T cell glycolipid-enriched membrane domains are constitutively assembled as membrane patches that translocate to immune synapses

In T cells, glycolipid-enriched membrane (GEM) domains, or lipid rafts, are assembled into immune synapses in response to Ag presentation. However, the properties of T cell GEM domains in the absence of stimulatory signals, such as their size and distribution in the plasma membrane, are less clear. To address this question, we used confocal microscopy to measure GEM domains in unstimulated T cells expressing a GEM-targeted green fluorescent protein molecule. Our experiments showed that the GEM domains were assembled into membrane patches that were micrometers in size, as evidenced by a specific enrichment of GEM-associated molecules and resistance of the patches to extraction by Triton X-100. However, treatment of cells with latrunculin B disrupted the patching of the GEM domains and their resistance to Triton X-100. Similarly, the patches were coenriched with F-actin, and actin occurred in the detergent-resistant GEM fraction of T cells. Live-cell imaging showed that the patches were mobile and underwent translocation in the plasma membrane to immune synapses in stimulated T cells. Targeting of GEM domains to immune synapses was found to be actin-dependent, and required phosphatidylinositol 3-kinase activity and myosin motor proteins. We conclude from our results that T cell GEM domains are constitutively assembled by the actin cytoskeleton into micrometer-sized membrane patches, and that GEM domains and the GEM-enriched patches can function as a vehicle for targeting molecules to immune synapses.     

Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domains

p56lck (Lck) is a lymphoid-specific Src family tyrosine kinase that is critical for T-cell development and activation. Lck is also a membrane protein, and approximately half of the membrane-associated Lck is associated with a glycolipid-enriched membrane (GEM) fraction that is resistant to solubilization by Triton X-100 (TX-100). To compare the membrane-associated Lck present in the GEM and TX-100-soluble fractions of Jurkat cells, Lck from each fraction was immunoblotted with antibody to phosphotyrosine. Lck in the GEM fraction was found to be hyperphosphorylated on tyrosine, and this correlated with a lower kinase specific activity relative to the TX-100-soluble Lck. Peptide mapping and phosphatase diagests showed that the hyperphosphorylation and lower kinase activity of GEM-associated Lck was due to phosphorylation of the regulatory COOH-terminal Tyr505. In addition, we determined that the membrane-bound tyrosine phosphatase CD45 was absent from the GEM fraction. Cells lacking CD45 showed identical phosphorylation of Lck in GEM and TX-100-soluble membranes. We propose that the GEM fraction represents a specific membrane domain present in T-cells, and that the hyperphosphorylation of tyrosine and lower kinase activity of GEM-associated Lck is due to exclusion of CD45 from these domains. Lck associated with the GEM domains may therefore consitute a reservoir of enzyme that can be readily activated.     

Visualization of antigen presentation by actin-mediated targeting of glycolipid-enriched membrane domains to the immune synapse of B cell APCs

Glycolipid-enriched membrane (GEM) domains, or lipid rafts, function in signaling in immune cells, but their properties during Ag presentation are less clear. To address this question, GEM domains were studied using fluorescence cell imaging of mouse CH27 B cells presenting Ag to D10 T cells. Our experiments showed that APCs were enriched with GEM domains in the immune synapse, and this occurred in an actin-dependent manner. This enrichment was specific to GEM domains, because a marker for non-GEM regions of the membrane was excluded from the immune synapse. Furthermore, fluorescence photobleaching experiments showed that protein in the immune synapse was dynamic and rapidly exchanged with that in other compartments of CH27 cells. To identify the signals for targeting GEM domains to the immune synapse in APCs, capping of the domains was measured in cells after cross-linking surface molecules. This showed that co-cross-linking CD48 with MHC class II was required for efficient capping and intracellular signaling. Capping of GEM domains by co-cross-linking CD48 and MHC class II occurred with co-capping of filamentous actin, and both domain capping and T cell-CH27 cell conjugation were inhibited by pretreating CH27 cells with latrunculin B. Furthermore, disruption of the actin cytoskeleton of the CH27 cells also inhibited formation of a mature immune synapse in those T cells that did conjugate to APCs. Thus, Ag presentation and efficient T cell stimulation occur by an actin-dependent targeting of GEM domains in the APC to the site of T cell engagement.     

Proteomic analysis of a detergent-resistant membrane skeleton from neutrophil plasma membranes

Plasma membranes are organized into functional domains both by liquid-ordered packing into "lipid rafts," structures that resist Triton extraction, and by attachments to underlying cytoskeletal proteins in assemblies called "membrane skeletons." Although the actin cytoskeleton is implicated in many lipid raft-mediated signaling processes, little is known about the biochemical basis for actin involvement. We show here that a subset of plasma membrane skeleton proteins from bovine neutrophils co-isolates with cholesterol-rich, detergent-resistant membrane fragments (DRMs) that exhibit a relatively high buoyant density in sucrose (DRM-H; d approximately 1.16 g/ml). By using matrix-assisted laser desorption/ionization time of flight and tandem mass spectrometry, we identified 19 major DRM-H proteins. Membrane skeleton proteins include fodrin (nonerythroid spectrin), myosin-IIA, myosin-IG, alpha-actinin 1, alpha-actinin 4, vimentin, and the F-actin-binding protein, supervillin. Other DRM-H components include lipid raft-associated integral membrane proteins (stomatin, flotillin 1, and flotillin 2), extracellular surface-bound and glycophosphatidylinositol-anchored proteins (IgM, membrane-type 6 matrix metalloproteinase), and intracellular dually acylated signaling proteins (Lyn kinase, Galpha(i-2)). Consistent with cytoskeletal association, most DRM-H-associated flotillin 2, Lyn, and Galpha(i-2) also resist extraction with 0.1 m octyl glucoside. Supervillin, myosin-IG, and myosin-IIA resist extraction with 0.1 m sodium carbonate, a treatment that removes all detectable actin, suggesting that these cytoskeletal proteins are proximal to the DRM-H bilayer. Binding of supervillin to the DRM-H fragments is confirmed by co-immunoaffinity purification. In spreading neutrophils, supervillin localizes with F-actin in cell extensions and in discrete basal puncta that partially overlap with Galpha(i) staining. We suggest that the DRM-H fraction represents a membrane skeleton-associated subset of leukocyte signaling domains.     

CD24 induces apoptosis in human B cells via the glycolipid-enriched membrane domains/rafts-mediated signaling system

The glycosylphosphatidylinositol-anchored CD24 protein is a B cell differentiation Ag that is expressed on mature resting B cells but disappears upon Ag stimulation. We used Burkitt's lymphoma (BL) cells, which are thought to be related to germinal center B cells, to examine the biological effect of Ab-mediated CD24 cross-linking on human B cells and observed 1) induction of apoptosis in BL cells mediated by cross-linking of CD24; and 2) synergism between the cross-linking of CD24 and that of the B cell receptor for Ag in the effect on apoptosis induction. We also observed activation of mitogen-activated protein kinases following CD24 cross-linking, suggesting that CD24 mediates the intracellular signaling that leads to apoptosis in BL cells. Although CD24 has no cytoplasmic portion to transduce signals intracellularly, analysis of biochemically separated glycolipid-enriched membrane (GEM) fractions indicated enhanced association of CD24 and Lyn protein tyrosine kinase in GEM as well as increased Lyn kinase activity after CD24 cross-linking, suggesting that CD24 mediates intracellular signaling via a GEM-dependent mechanism. Specific microscopic cocapping of CD24 and Lyn, but not of other kinases, following CD24 cross-linking supported this idea. We further observed that apoptosis induction by cross-linking is a common feature shared by GEM-associated molecules expressed on BL cells, including GPI-anchored proteins and glycosphingolipids. CD24-mediated apoptosis in BL cells may provide a model for the cell death mechanism initiated by GEM-associated molecules, which is closely related to B cell receptor for Ag-mediated apoptosis.     

Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway.

Clathrin-dependent endocytosis has long been presented as the only efficient mechanism by which transmembrane receptors are internalized. We selectively blocked this process using dominant-negative mutants of Eps15 and showed that clathrin-mediated endocytosis of transferrin was inhibited, while endocytosis of interleukin 2 (IL2) receptors proceeded normally. Ultrastructural and biochemical experiments showed that clathrin-independent endocytosis of IL2 receptors exists constitutively in lymphocytes and is coupled to their association with detergent-resistant membrane domains. Finally, clathrin-independent endocytosis requires dynamin and is specifically regulated by Rho family GTPases. These results define novel properties of receptor-mediated endocytosis and establish that the IL2 receptor is efficiently internalized through this clathrin-independent pathway.     

Insights into the association of FcgammaRII and TCR with detergent-resistant membrane domains: isolation of the domains in detergent-free density gradients facilitates membrane fragment reconstitution

Plasma membrane rafts are routinely isolated as detergent-resistant membranes (DRMs) floating in detergent-free density gradients. Here we show that both the presence and exclusion of TX-100 during the density gradient fractionation have profound effects on the location of FcgammaRII and TCR in DRM fractions. The presence of TX-100 during fractionation promoted solubilization of non-cross-linked FcgammaRII when the receptor was insufficiently dissolved upon cell lysis. In the detergent-supplemented gradients, TX-100 micelles floated, further enhancing dissociation of FcgammaRII and TCR from DRMs and promoting a shift of the receptors toward higher-density fractions. Hence, fractionation of cell lysates over the detergent-containing gradients enables isolation of DRMs devoid of weakly associated proteins, like nonactivated FcgammaRII and TCR. On the other hand, in a detergent-free gradient, non-cross-linked FcgammaRII, fully soluble in 0.2% TX-100, was recovered in DRM fractions. Moreover, employment of the TX-100-free gradient for refractionation of intermediate-density fractions, derived from detergent-supplemented gradients and containing FcgammaRII and TCR, resulted in flotation of the receptors to buoyant fractions. An analysis of the TX-100 concentration revealed that after fractionation of 0.2% TX-100 cell lysates in the absence of detergent, the level of TX-100 in DRM fractions was reduced to 0.01%, below the critical micelle concentration. Therefore, fractionation of detergent cell lysates over detergent-free gradients can mimic conditions for a membrane reconstitution, evoking association of a distinct subset of membrane proteins, including FcgammaRII and TCR, with DRMs.     

Dynamic compositional changes of detergent-resistant plasma membrane microdomains during plant cold acclimation

Plants increase their freezing tolerance upon exposure to low, non-freezing temperatures, which is known as cold acclimation. Cold acclimation results in a decrease in the proportion of sphingolipids in the plasma membrane in many plants including Arabidopsis thaliana. The decrease in sphingolipids has been considered to contribute to the increase in the cryostability of the plasma membrane through regulating membrane fluidity. Recently we have proposed a possibility of another important sphingolipid function associated with cold acclimation.¹ In animal cells, it has been known that the plasma membrane contains microdomains due to the characteristics of sphingolipids and sterols, and the sphingolipid- and sterol-enriched microdomains are thought to function as platforms for cell signaling, membrane trafficking and pathogen response. In our research on characterization of microdomain-associated lipids and proteins in Arabidopsis, a cold-acclimation-induced decrease in sphingolipids resulted in a decrease of microdomains in the plasma membrane and there were considerable changes in membrane transport-, cytoskeleton- and endocytosis-related proteins in the microdomains during cold acclimation. Based on these results, we discuss a functional relationship between the changes in microdomain components and plant cold acclimation.Key words: Arabidopsis, cold acclimation, detergent-resistant plasma membrane, plasma membrane lipid, plasma membrane protein, microdomain, proteome analysisIn fall or early winter, plants recognize the decrease in temperature and change cellular metabolism to survive against freezing stress. This phenomenon is termed as cold acclimation.² Because the plasma membrane is the critical site in cell survival during freezing, diverse cold-acclimation-induced changes are believed to ultimately protect the plasma membrane from the irreversible damage under freezing stress.³ One of the notable changes during cold acclimation is a decrease in sphingolipids, a characteristic plasma membrane lipid.⁴ Sphingolipids have melting temperatures higher than do phosphsolipids, major plasma membrane lipids. Thus, quantitative decreases in sphonglipids are considered to increase in membrane fluidity at low temperatures.⁴ Some 20 years ago, however, experimental results that sphinglipids form lipid microdomains in the plasma membrane were reported in mammalian and yeast cells.^5–7 Sphingolipids are heterogeneously distributed and self-associated with sterols and specific proteins in the plasma membrane. The sphingolipid/sterol-enriched microdomains in the plasma membrane are sometime called “membrane (lipid) raft” or “caveolae” in mammalian cells, and similar domains have been proposed later in plant cells.^8–11 The microdomains are biochemically isolated as low-density detergent-resistant plasma membrane (DRM) fractions and contain specific proteins associated with membrane trafficking, signal transduction, membrane transport, cytoskeleton interaction and pathogen infection.¹² Consequently, the microdomains are suspected to function as platform for assembly of these functional protein complexes and temporal interaction between protein-protein or protein-lipid.⁷ The microdomains change not only in domain size by coalescence of individual domains but also in protein and lipid compositions by physiological stimulus.^12–15We hypothesized that a decrease of sphingolipids in the plant plasma membrane during cold acclimation might not only increase membrane fluidity but also change microdomain formation and/or function. Our recent paper characterized cold-responsiveness of lipid and protein components in plant DRMs.¹ Arabidopsis thaliana is able to increase in freezing tolerance after few days of cold treatment [the temperature of 50% survival is −7°C before cold treatment at 2°C and decreases to −15°C after 7-d-treatment]. We first isolated plasma membrane-enriched fractions using aqueous two-phase partition system from Arabidopsis seedlings before and after cold acclimation. Next, plasma membrane fractions were subjected to 1% (w/v) Triton X-100 on ice for 30 min and then sucrose density gradient centrifugation. DRM fractions appeared as two white bands at about 40% (w/w) sucrose. DRMs in plants are generally recovered as heavier fractions than those in animals.^16–18 This is probably because the ratio of protein to lipid is greater in plants than in animals. Arabidopsis DRM fractions were enriched in sphingolipids (glucocerebrosides) and sterols (free sterols, acylated sterylglucosides and sterylglucosides).¹ Figure 1 shows the protein and lipid amounts in DRM during cold acclimation. DRM protein recovery rate from the plasma membrane was less than 10% and cold treatment resulted in a gradual decrease of the recovery: the recovery rate of DRM lipids from the plasma membrane rapidly decreased by half only after 2 days of cold acclimation. These data suggest a decrease in the proportion of microdomains in the plasma membrane and temporal changes in proteins and lipids in DRM during cold acclimation.Open in a separate windowFigure 1Changes in the protein and lipid amount in DRM recovered from plasma membrane fractions during cold acclimation. NA, non-acclimated; CA 2, CA 4 and CA 7, cold-acclimated for 2, 4 and 7 days, respectively. (Modified from Minami et al.)We found that there were significant differences in lipid alterations in plasma membrane and DRM fractions in cold acclimation (Fig. 2). The amount of total lipids (per mg of protein) in the plasma membrane fraction greatly increased after cold acclimation but not in the DRM fraction. In the plasma membrane fraction, cold acclimation for 2 days resulted in an increase in the proportions of phospholipids and free sterols and a decrease in the proportion of sphingolipids. In contrast, in the DRM fractions, free sterols increased after 2 days of cold acclimation but the proportion of phospholipids and sphingolipids did not change significantly. These results suggest that the changes in lipid classes in DRM differ from the changes in the whole plasma membrane. Our lipid analysis suggests that the decrease in sphingolipids in the plasma membrane affects the quantitative decrease of microdomains in the plasma membrane during cold acclimation (see Fig. 1). However, the lipid changes in the whole plasma membrane are unlikely to affect proportional changes in DRM-localized lipids except for free sterols.Open in a separate windowFigure 2Lipid changes in DRM and plasma membrane fractions during cold acclimation. NA, non-acclimated; CA 2, CA 4 and CA 7, cold-acclimated for 2, 4 and 7 days, respectively. FS, free sterols; ASG, acylated sterylglucosides; SG, sterylglucosides; GlcCer, glucocerebrosides; PL, phospholipids. (Modified from Minami et al.¹)We demonstrated quantitative changes of DRM-localized proteins during cold acclimation using two-dimensional differential gel electrophoresis (2D-DIGE) and western blot analyses.¹ 2D-DIGE analysis showed that one-third of the DRM-localized proteins quantitatively changed during cold acclimation. Subsequent mass spectrometric analysis of DRM proteins revealed significant changes in various proteins including increases in aquaporin, P-type H⁺-ATPase and endocytosis-related proteins and decreases in cytoskeletal proteins (tubulins and actins) and V-type H⁺-ATPase subunits during cold acclimation. The changes were first detected after 2 days of cold acclimation. Based on these results of protein analyses, Figure 3 illustrates changes in distribution patterns of DRM-localized proteins in the plasma membrane during cold acclimation. Cold acclimation induces the decrease in the amount of DRM proteins and lipids in the plasma membrane (Fig. 1), suggesting that component in microdomains decreases in the plasma membrane during cold acclimation. Furthermore, the proportion of some functional proteins changes in DRM during cold acclimation. Qualitative and quantitative changes of DRM proteins during cold acclimation are possibly associated with the plasma membrane functions. Plant cells at low temperature suffer from changes in membrane fluidity and cytoplasmic pH.^19–21 Upon freezing occurs, plant cells are subjected to severe dehydration and deformation stresses induced by extracellular ice formation.²² To avoid the occurrence of damages from these stresses, plants change plasma membrane components during cold acclimation.²³ H⁺-ATPase or aquaporins are thought to function in regulation of cytoplasmic pH or water transfer across the plasma membrane, respectively.^24,25 Cytoskeleton regulates cell structure and intracellular vesicle-trafficking processes reconstruct plasma membrane itself. Thus, the quantitative changes of these proteins in microdomains are likely associated with protective functions against freezing stress in cold acclimation.Open in a separate windowFigure 3Our hypothesis on changes in microdomains during plant cold acclimation. Cold acclimation results in a decrease in microdomains in the plasma membrane (see Fig. 1) and differential changes in various protein compositions in microdomains. We categorized DRM proteins as (1) membrane transport, (2) vesicle trafficking, (3) cytoskeleton, (4) microdomain-associated proteins and (5) others (e.g., plasma membrane and cell-wall reconstruction). Aquaporin, P-type H⁺-ATPase (1) and endocytosis-related proteins (2) increased and cytoskeletal proteins (3) and V-type H⁺-ATPase subunits (1) decreased in DRM during cold acclimation.We clearly demonstrated that cold acclimation decreased the amount of DRM and changed both lipid and protein compositions in plant DRM. Our study represents a first step towards elucidation of functions of plant microdomains in cold acclimation, strongly suggesting that microdomains, which function as a platform of membrane transport, membrane trafficking and cytoskeleton interaction, are associated with plant cold acclimation. Changes in microdomain lipids may also affect the protein activities during cold acclimation because sterols or sphingolipids are known to regulate activities of membrane transport or endocytosis. Thus, we suspect that the quantitative changes in microdomain lipids and proteins may correlate with development of freezing tolerance during cold acclimation. The hypothesis that the changes in microdomain components are functionally associated with plant cold acclimation should be reinforced by various approaches such as genetics, biochemistry or physical chemistry.     

Defining raft domains in the plasma membrane

Many plasma membrane (PM) functions depend on the cholesterol concentration in the PM in strikingly nonlinear, cooperative ways: fully functional in the presence of physiological cholesterol levels (35~45 mol%), and nonfunctional below 25 mol% cholesterol; namely, still in the presence of high concentrations of cholesterol. This suggests the involvement of cholesterol‐based complexes/domains formed cooperatively. In this review, by examining the results obtained by using fluorescent lipid analogs and avoiding the trap of circular logic, often found in the raft literature, we point out the fundamental similarities of liquid‐ordered (Lo)‐phase domains in giant unilamellar vesicles, Lo‐phase‐like domains formed at lower temperatures in giant PM vesicles, and detergent‐resistant membranes: these domains are formed by cooperative interactions of cholesterol, saturated acyl chains, and unsaturated acyl chains, in the presence of >25 mol% cholesterol. The literature contains evidence, indicating that the domains formed by the same basic cooperative molecular interactions exist and play essential roles in signal transduction in the PM. Therefore, as a working definition, we propose that raft domains in the PM are liquid‐like molecular complexes/domains formed by cooperative interactions of cholesterol with saturated acyl chains as well as unsaturated acyl chains, due to saturated acyl chains' weak multiple accommodating interactions with cholesterol and cholesterol's low miscibility with unsaturated acyl chains and TM proteins. Molecules move within raft domains and exchange with those in the bulk PM. We provide a logically established collection of fluorescent lipid probes that preferentially partition into raft and non‐raft domains, as defined here, in the PM.     

Transferrin uptake may occur through detergent-resistant membrane domains at the cytopharynx of Trypanosoma cruzi epimastigote forms

Uptake of transferrin by epimastigote forms of the protozoan Trypanosoma cruzi occurs mainly through a cytostome/ cytopharynx, via uncoated endocytic vesicles that bud off from the bottom of the cytopharynx. We have here examined whether detergent-resistant membrane (DRM) domains might be involved in this process. Purified whole cell membrane fractions were assayed for cholesterol levels and used in dot blot analyses. Detergent-resistant membrane markers (cholera B toxin and anti-flotillin-1 antibody) presented positive reaction by dot blots in cholesterol-rich/ protein-poor membrane sub-fractions. The positive dot blot fraction was submitted to lipid composition analysis, showing composition similar to that of raft fractions described for other eukaryotic cells. Immunofluorescence assays allowed the localization of punctual positive signal for flotillin-1, matching the precise cytostome/ cytopharynx location. These data were confirmed by immunofluorescence assays with the co-localization of flotillin-1 and the transferrin uptake site. Our data suggest that DRM domains occur and are integrated at the cytostome/ cytopharynx of T. cruzi epimastigotes, being the main route for transferrin uptake.     

Sortilin and prosaposin localize to detergent-resistant membrane microdomains

Most soluble lysosomal hydrolases are sorted in the trans-Golgi network (TGN) and delivered to the lysosomes by the mannose 6-phosphate receptor (M6PR). However, the non-enzymic sphingolipid activator protein (SAP), prosaposin, as well as certain soluble lysosomal hydrolases, is sorted and trafficked to the lysosomes by sortilin. Based on previous results demonstrating that prosaposin requires sphingomyelin to be targeted to the lysosomes, we hypothesized that sortilin and its ligands are found in detergent-resistant membranes (DRMs). To test this hypothesis we have analyzed DRM fractions and demonstrated the presence of sortilin and its ligand, prosaposin. Our results showed that both the M6PR and its cargo, cathepsin B, were also present in DRMs. Cathepsin H has previously been demonstrated to interact with sortilin, while cathepsin D interacts with both sortilin and the M6PR. Both of these soluble lysosomal proteins were also found in DRM fractions. Using sortilin shRNA we have showed that prosaposin is localized to DRM fractions only in the presence of sortilin. These observations suggest that in addition to interacting with the same adaptor proteins, such as GGAs, AP-1 and retromer, both sortilin and the M6PR localize to similar membrane platforms, and that prosaposin must interact with sortilin to be recruited to DRMs.     

Detergent-free domain isolated from Xenopus egg plasma membrane with properties similar to those of detergent-resistant membranes

Microdomains known as "rafts" have been isolated from many cell types as detergent-resistant membranes (DRMs) and are enriched in sphingolipids and cholesterol. However, there has been considerable controversy over whether such domains are found in native membranes or are artificially generated by the purification procedure. This controversy is based at least in part on the fact that raft membranes were first detected following detergent extraction in the cold. We isolated two plasma membrane fractions, without detergent treatment, using a discontinuous sucrose density gradient. One fraction was designated "light" and the other "heavy." These fractions were compared with DRMs, which were isolated in the presence of 1% Triton X-100. We found that Xenopus DRMs are enriched with sphingomyelin and cholesterol and exhibit a phase state similar to the liquid-ordered phase. Comparison of DRM complexes with the light and heavy plasma membrane fractions revealed some physical and biochemical similarities between the light fraction of the plasma membrane and the DRM complexes, based on (1) the phosphatidylcholine/sphingomyelin ratio and (2) the protein composition visualized on a two-dimensional gel. These two fractions are also quite similar in their thermotropic phase behavior, and their high levels of ganglioside GM1. We conclude that the light membrane fraction isolated in a detergent-free environment has many of the characteristics normally associated with DRMs.     

HIV-1 entry into T-cells is not dependent on CD4 and CCR5 localization to sphingolipid-enriched,detergent-resistant,raft membrane domains

The contribution of raft domains to human immunodeficiency virus (HIV) 1 entry was assessed. In particular, we asked whether the CD4 and CCR5 HIV-1 receptors need to associate with sphingolipid-enriched, detergent-resistant membrane domains (rafts) to allow viral entry into primary and T-cell lines. Based on Triton X-100 solubilization and confocal microscopy, CD4 was shown to distribute partially to rafts. In contrast, CCR5 did not associate with rafts and localized in nonraft plasma membrane domains. HIV-1-receptor partitioning remained unchanged upon viral adsorption, suggesting that viral entry probably takes place outside rafts. To directly investigate this possibility, we targeted CD4 to nonraft domains of the membrane by preventing CD4 palmitoylation and interaction with p56(lck). Directed mutagenesis of both targeting signals significantly prevented association of CD4 with rafts, but did not suppress the HIV-1 receptor function of CD4. Collectively, these results strongly suggest that the presence of HIV-1 receptors in rafts is not required for viral infection. We show, however, that depleting plasma membrane cholesterol inhibits HIV-1 entry. We therefore propose that cholesterol modulates the HIV-1 entry process independently of its ability to promote raft formation.     

Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts

The trafficking and function of cell surface proteins in eukaryotic cells may require association with detergent-resistant sphingolipid- and sterol-rich membrane domains. The aim of this work was to obtain evidence for lipid domain phenomena in plant membranes. A protocol to prepare Triton X-100 detergent-resistant membranes (DRMs) was developed using Arabidopsis (Arabidopsis thaliana) callus membranes. A comparative proteomics approach using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry revealed that the DRMs were highly enriched in specific proteins. They included eight glycosylphosphatidylinositol-anchored proteins, several plasma membrane (PM) ATPases, multidrug resistance proteins, and proteins of the stomatin/prohibitin/hypersensitive response family, suggesting that the DRMs originated from PM domains. We also identified a plant homolog of flotillin, a major mammalian DRM protein, suggesting a conserved role for this protein in lipid domain phenomena in eukaryotic cells. Lipid analysis by gas chromatography-mass spectrometry showed that the DRMs had a 4-fold higher sterol-to-protein content than the average for Arabidopsis membranes. The DRMs were also 5-fold increased in sphingolipid-to-protein ratio. Our results indicate that the preparation of DRMs can yield a very specific set of membrane proteins and suggest that the PM contains phytosterol and sphingolipid-rich lipid domains with a specialized protein composition. Our results also suggest a conserved role of lipid modification in targeting proteins to both the intracellular and extracellular leaflet of these domains. The proteins associated with these domains provide important new experimental avenues into understanding plant cell polarity and cell surface processes.     

Characterization of functional domains of the lymphocyte plasma membrane

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (fraction 1) eluted freely from the affinity column, the second (fraction 2) adhered specifically to concanavalin A-Sepharose. Previous analysis showed that both subfractions were right-side-out (Resch, K., Schneider, S. and Szamel, M. (1981) Anal. Biochem. 117, 282-292). The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. After enzymatic radioiodination of thymocytes, the relative distribution of labelled proteins and externally exposed phospholipids was very similar in isolated plasma membranes and in both membrane subfractions, indicating the plasma membrane nature of the subfractions separated by affinity chromatography on concanavalin A-Sepharose. This finding was further substantiated by the nearly identical specific activities of some membrane-bound enzymes, Mg2+-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase. The specific activities of (Na+ + K+)-ATPase and of lysolecithin acyltransferase were several-fold enriched in fraction 2 compared to fraction 1, especially after rechromatography of fraction 1 on concanavalin A-Sepharose. Unseparated membrane vesicles contained two types of binding site for concanavalin A. In contrast, isolated subfractions showed a linear Scatchard plot; fraction 2 exhibited fewer binding sites for concanavalin A: the association constant was, however, 3.5-times higher than that measured in fraction 1. When plasma membranes isolated from concanavalin A-stimulated lymphocytes were separated by affinity chromatography, the yield of the two subfractions was similar to that of membranes from unstimulated lymphocytes. Upon stimulation with concanavalin A, Mg2+-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase were suppressed in their activities in both membrane subfractions. In contrast, the specific activities of (Na+ + K+)-ATPase and lysolecithin acyltransferase were enhanced preferentially in the adherent fraction (fraction 2). The data suggest the existence of domains in the plasma membrane of lymphocytes which are formed by a spatial and functional coupling of receptors with high affinity for concanavalin A, and certain membrane-bound enzymes, implicated in the initiation of lymphocyte activation.     

Isolation of domains of the plasma membrane of hepatocytes

Several recent studies have demonstrated the ability of techniques based on immunoadsorption to selectively isolate specialized subregions of membranes, termed domains, which are derived from a larger more complex parent membrane like the plasma membrane. The immunoadsorbent is directed against a specific antigen that resides exclusively or predominantly in the membrane domain to be isolated. Thus, a monospecific antibody to the domain-specific antigen is required. In the present study we developed a method employing a modified immunoblotting strategy which could utilize polyspecific antibodies to isolate membrane vesicles derived from a specific membrane domain of the hepatocyte plasma membrane. We also used specific cell surface labeling of the hepatocyte plasma membrane by lactoperoxidase-catalyzed iodination at 4 degrees C and preparation of different sized vesicles by sonication to facilitate isolation of the specific domain. For this study, polyspecific antisera were raised in goats against a membrane fraction, denoted N2u, which is enriched in bile canalicular proteins. This antiserum recognizes, among other antigens, a 110,000 Mr polypeptide previously shown to be localized in the bile canaliculus (J. Cook et al. (1983) J. Cell. Biol. 97, 1823-1833). A monospecific antiserum was raised in rabbits against the rat hepatocyte asialoglycoprotein receptor, a sinusoidal domain-specific set of glycoproteins whose major form has a Mr of 43,000. These antisera were each coupled indirectly to different pieces of nitrocellulose by the immunoblotting protocol and were used to isolate membrane vesicles from a crude extract of liver plasma membrane prepared by sonication. The ratio of iodinated asialoglycoprotein receptor to the 110,000 Mr polypeptide in vesicles isolated by the affinity nitrocellulose immunoadsorbent method indicate a 10- to 15-fold enrichment of sinusoidal-derived vesicles relative to bile canalicular-derived membrane vesicles. These results show that the affinity nitrocellulose immunoadsorbent method can be used to isolate domain-specific vesicles. Further, the affinity immunoadsorbent method described here for the isolation of domains of the plasma membrane is an integrative one allowing isolation of vesicles present in relatively small concentration in crude cell extracts and it requires minimal ultracentrifugation time.     

Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiae GAS1 protein to the plasma membrane.

A 125-kDa glycoprotein exposed on the surface of Saccharomyces cerevisiae cells belongs to a class of eucaryotic membrane proteins anchored to the lipid bilayer by covalent linkage to an inositol-containing glycophospholipid. We have cloned the gene (GAS1) encoding the 125-kDa protein (Gas1p) and found that the function of Gas1p is not essential for cell viability. The nucleotide sequence of GAS1 predicts a 60-kDa polypeptide with a cleavable N-terminal signal sequence, potential sites for N- and O-linked glycosylation, and a C-terminal hydrophobic domain. Determination of the anchor attachment site revealed that the C-terminal hydrophobic domain of Gas1p is removed during anchor addition. However, this domain is essential for addition of the glycophospholipid anchor, since a truncated form of the protein failed to become attached to the membrane. Anchor addition was also abolished by a point mutation affecting the hydrophobic character of the C-terminal sequence. We conclude that glycophospholipid anchoring of Gas1p depends on the integrity of the C-terminal hydrophobic domain that is removed during anchor attachment.