Membrane rafts in plant cells

Over the past five years, the structure, composition and possible functions of membrane raft-like domains on plant plasma membranes (PM) have been described. Proteomic analyses have indicated that a high proportion of proteins associated with detergent-insoluble membranes (DIMs), supposed to contain raft-like domains isolated from the PM, might be involved in signalling pathways. Recently, the dynamic association of specific proteins with the DIM fraction upon environmental stress has been reported. Innovative imaging methods have shown that lateral segregation of lipids and proteins exists at the nanoscale level in the plant PM, correlating detergent insolubility and membrane-domain localization of presumptive raft proteins. These data suggest a role for plant rafts as signal transduction platforms, similar to those documented for mammalian cells.     

Lipids of plant membrane rafts

Lipids tend to organize in mono or bilayer phases in a hydrophilic environment. While they have long been thought to be incapable of coherent lateral segregation, it is now clear that spontaneous assembly of these compounds can confer microdomain organization beyond spontaneous fluidity. Membrane raft microdomains have the ability to influence spatiotemporal organization of protein complexes, thereby allowing regulation of cellular processes. In this review, we aim at summarizing briefly: (i) the history of raft discovery in animals and plants, (ii) the main findings about structural and signalling plant lipids involved in raft segregation, (iii) imaging of plant membrane domains, and their biochemical purification through detergent-insoluble membranes, as well as the existing debate on the topic. We also discuss the potential involvement of rafts in the regulation of plant physiological processes, and further discuss the prospects of future research into plant membrane rafts.     

Detergent-insoluble glycosphingolipid/cholesterol-rich membrane domains, lipid rafts and caveolae (review).

Within the cell membrane glycosphingolipids and cholesterol cluster together in distinct domains or lipid rafts, along with glycosyl-phosphatidylinositol (GPI)-anchored proteins in the outer leaflet and acylated proteins in the inner leaflet of the bilayer. These lipid rafts are characterized by insolubility in detergents such as Triton X-100 at 4 degrees C. Studies on model membrane systems have shown that the clustering of glycosphingolipids and GPI-anchored proteins in lipid rafts is an intrinsic property of the acyl chains of these membrane components, and that detergent extraction does not artefactually induce clustering. Cholesterol is not required for clustering in model membranes but does enhance this process. Single particle tracking, chemical cross-linking, fluorescence resonance energy transfer and immunofluorescence microscopy have been used to directly visualize lipid rafts in membranes. The sizes of the rafts observed in these studies range from 70-370 nm, and depletion of cellular cholesterol levels disrupts the rafts. Caveolae, flask-shaped invaginations of the plasma membrane, that contain the coat protein caveolin, are also enriched in cholesterol and glycosphingolipids. Although caveolae are also insoluble in Triton X-100, more selective isolation procedures indicate that caveolae do not equate with detergent-insoluble lipid rafts. Numerous proteins involved in cell signalling have been identified in caveolae, suggesting that these structures may function as signal transduction centres. Depletion of membrane cholesterol with cholesterol binding drugs or by blocking cellular cholesterol biosynthesis disrupts the formation and function of both lipid rafts and caveolae, indicating that these membrane domains are involved in a range of biological processes.     

Plant Lipid Rafts: Fluctuat Nec Mergitur*

Lipid rafts in plasma membranes are hypothesized to play key roles in many cellular processes including signal transduction, membrane trafficking and entry of pathogens. We recently documented the biochemical characterization of lipid rafts, isolated as detergent-insoluble membranes, from Medicago truncatula root plasma membranes. We evidenced that the plant-specific lipid steryl-conjugates are among the main lipids of rafts together with free sterols and sphingolipids. An extensive proteomic analysis showed the presence of a specific set of proteins common to other lipid rafts, plus the presence of a redox system around a cytochrome b₅₆₁ not previously identified in lipid rafts of either plants or animals. Here, we discuss the similarities and differences between the lipids and proteins of plant and animal lipid rafts. Moreover we describe the potential biochemical functioning of the M. truncatula root lipid raft redox proteins and question whether they may play a physiological role in legume-symbiont interactions.Key Words: plasma membrane, Medicago, root, legume-Rhizobium symbiosis, redox, sterol, sphingolipid     

Flotillins functionally organize the bacterial membrane

Proteins and lipids are heterogeneously distributed in biological membranes. The correct function of membrane proteins depends on spatiotemporal organization into defined membrane areas, called lipid domains or rafts. Lipid microdomains are therefore thought to assist compartmentalization of membranes. However, how lipid and protein assemblies are organized and whether proteins are actively involved in these processes remains poorly understood. We now have identified flotillins to be responsible for lateral segregation of defined membrane domains in the model organism Bacillus subtilis. We show that flotillins form large, dynamic assemblies that are able to influence membrane fluidity and prevent condensation of Laurdan stained membrane regions. Absence of flotillins in vivo leads to coalescence of distinct domains of high membrane order and, hence, loss of flotillins in the bacterial plasma‐membrane reduces membrane heterogeneity. We show that flotillins interact with various proteins involved in protein secretion, cell wall metabolism, transport and membrane‐related signalling processes. Importantly, maintenance of membrane heterogeneity is critical for vital cellular processes such as protein secretion.     

MHC class II molecules traffic into lipid rafts during intracellular transport

There have been many studies demonstrating that a portion of MHC class II molecules reside in detergent-insoluble membrane domains (commonly referred to as lipid rafts). We have proposed that the function of raft association is to concentrate specific MHC class II-peptide complexes in plasma membrane microdomains that can facilitate efficient T cell activation. We now show that MHC class II becomes lipid raft associated before binding antigenic peptides. Using pulse-chase radiolabeling techniques, we find that newly synthesized MHC class II and MHC class II-invariant chain complexes initially reside in a detergent-soluble membrane fraction and acquire detergent insolubility as they traffic to lysosomal Ag processing compartments. Monensin, an inhibitor of protein transport through the Golgi apparatus, blocks association of newly synthesized MHC class II with lipid rafts. Treatment of cells with leupeptin, which inhibits invariant chain degradation, leads to the accumulation of MHC class II in lipid rafts within the lysosome-like Ag-processing compartments. Raft fractionation of lysosomal membranes confirmed the presence of MHC class II in detergent-insoluble microdomains in Ag-processing compartments. These findings indicate that newly synthesized MHC class II complexes are directed to detergent-insoluble lipid raft microdomains before peptide loading, a process that may facilitate the loading of similar peptides on MHC class II complexes in these microdomains.     

Evolution and development of model membranes for physicochemical and functional studies of the membrane lateral heterogeneity

One of the main questions in the membrane biology is the functional roles of membrane heterogeneity and molecular localization. Although segregation and local enrichment of protein/lipid components (rafts) have been extensively studied, the presence and functions of such membrane domains still remain elusive. Along with biochemical, cell observation, and simulation studies, model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information on the physicochemical properties of membrane proteins and lipids. Segregation of fluid lipid bilayer into liquid-ordered (Lo) and liquid-disordered (Ld) phases has been studied as a simplified model of raft in model membranes, including giant unilamellar vesicles (GUVs), giant plasma membrane vesicles (GPMVs), and supported lipid bilayers (SLB). Partition coefficients of membrane proteins between Lo and Ld phases were measured to gauze their affinities to lipid rafts (raftophilicity). One important development in model membrane is patterned SLB based on the microfabrication technology. Patterned Lo/Ld phases have been applied to study the partition and function of membrane-bound molecules. Quantitative information of individual molecular species attained by model membranes is critical for elucidating the molecular functions in the complex web of molecular interactions. The present review gives a short account of the model membranes developed for studying the lateral heterogeneity, especially focusing on patterned model membranes on solid substrates.     

VIP36, a novel component of glycolipid rafts and exocytic carrier vesicles in epithelial cells.

In simple epithelial cells, apical and basolateral proteins and lipids in transit to the cell surface are sorted in the trans-Golgi network. We have recently isolated detergent-insoluble complexes from Madin-Darby canine kidney cells that are enriched in glycosphingolipids, apical cargo and a subset of the proteins of the exocytic carrier vesicles. The vesicular proteins are thought to be involved in protein sorting and include VIP21-caveolin. The vesicular protein VIP36 (36 kDa vesicular integral membrane protein) has been purified from a CHAPS-insoluble residue and a cDNA encoding VIP36 has been isolated. The N-terminal 31 kDa luminal/exoplasmic domain of the encoded protein shows homology to leguminous plant lectins. The transiently expressed protein is localized to the Golgi apparatus, endosomal and vesicular structures and the plasma membrane, as predicted for a protein involved in transport between the Golgi and the cell surface. It is diffusely localized on the plasma membrane but can be redistributed by antibody modulation into caveolae and clathrin-coated pits. We speculate that VIP36 binds to sugar residues of glycosphingolipids and/or glycosylphosphatidyl-inositol anchors and might provide a link between the extracellular/luminal face of glycolipid rafts and the cytoplasmic protein segregation machinery.     

Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts

Gaining an understanding of the structural and functional roles of cholesterol in membrane lipid rafts is a critical issue in studies on cellular signaling and because of the possible involvement of lipid rafts in various diseases. We have focused on the potential of perfringolysin O (theta-toxin), a cholesterol-binding cytolysin produced by Clostridium perfringens, as a probe for studies on membrane cholesterol. We prepared a protease-nicked and biotinylated derivative of perfringolysin O (BCtheta) that binds selectively to cholesterol in cholesterol-rich microdomains of cell membranes without causing membrane lesions. Since the domains fulfill the criteria of lipid rafts, BCtheta can be used to detect cholesterol-rich lipid rafts. This is in marked contrast to filipin, another cholesterol-binding reagent, which binds indiscriminately to cell cholesterol. Using BCtheta, we are now searching for molecules that localize specifically in cholesterol-rich lipid rafts. Recently, we demonstrated that the C-terminal domain of perfringolysin O, domain 4 (D4), possesses the same binding characteristics as BCtheta. BIAcore analysis showed that D4 binds specifically to cholesterol with the same binding affinity as the full-size toxin. Cell-bound D4 is recovered predominantly from detergent-insoluble, low-density membrane fractions where raft markers, such as cholesterol, flotillin and Src family kinases, are enriched, indicating that D4 also binds selectively to lipid rafts. Furthermore, a green fluorescent protein-D4 fusion protein (GFP-D4) was revealed to be useful for real-time monitoring of cholesterol in lipid rafts in the plasma membrane. In addition, the expression of GFP-D4 in the cytoplasm might allow the investigations of intracellular trafficking of lipid rafts. The simultaneous visualization of lipid rafts in plasma membranes and inside cells might help in gaining a total understanding of the dynamic behavior of lipid rafts.     

High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes

"Lipid rafts" enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.     

Bile Acids Modulate Signaling by Functional Perturbation of Plasma Membrane Domains

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.     

Dynamics of raft molecules in the cell and artificial membranes: approaches by pulse EPR spin labeling and single molecule optical microscopy

Lipid rafts in the plasma membrane, domains rich in cholesterol and sphingolipids, have been implicated in a number of important membrane functions. Detergent insolubility has been used to define membrane "rafts" biochemically. However, such an approach does not directly contribute to the understanding of the size and the lifetime of rafts, dynamics of the raft-constituent molecules, and the function of rafts in the membrane in situ. To address these issues, we have developed pulse EPR spin labeling and single molecule tracking optical techniques for studies of rafts in both artificial and cell membranes. In this review, we summarize our results and perspectives obtained by using these methods. We emphasize the importance of clearly distinguishing small/unstable rafts (lifetime shorter than a millisecond) in unstimulated cells and stabilized rafts induced by liganded and oligomerized (GPI-anchored) receptor molecules (core receptor rafts, lifetime over a few minutes). We propose that these stabilized rafts further induce temporal, greater rafts (signaling rafts, lifetime on the order of a second) for signaling by coalescing other small/unstable rafts, including those in the inner leaflet of the membrane, each containing perhaps one molecule of the downstream effector molecules. At variance with the general view, we emphasize the importance of cholesterol segregation from the liquid-crystalline unsaturated bulk-phase membrane for formation of the rafts, rather than the affinity of cholesterol and saturated alkyl chains. In the binary mixture of cholesterol and an unsaturated phospholipid, cholesterol is segregated out from the bulk unsaturated liquid-crystalline phase, forming cholesterol-enriched domains or clustered cholesterol domains, probably due to the lateral nonconformability between the rigid planar transfused ring structure of cholesterol and the rigid bend of the unsaturated alkyl chain at C9-C10. However, such cholesterol-rich domains are small, perhaps consisting of only several cholesterol molecules, and are short-lived, on the order of 1-100 ns. We speculate that these cholesterol-enriched domains may be stabilized by the presence of saturated alkyl chains of sphingomyelin or glycosphingolipids, and also by clustered raft proteins. In the influenza viral membrane, one of the simplest forms of a biological membrane, the lifetime of a protein and cholesterol-rich domain was evaluated to be on the order of 100 micro, again showing the short lifetime of rafts in an unstimulated state. Finally, we propose a thermal Lego model for rafts as the basic building blocks for signaling pathways in the plasma membrane.     

A requirement for lipid rafts in B cell receptor induced Ca(2+) flux

Although the major biochemical events triggered by ligation of the B-cell receptor (BCR) have been well defined [1] [2], little is known about the spatio-temporal organization of BCR signaling components within the cell membrane and the mechanisms by which signaling specificity is achieved. Partitioning of signaling complexes into specialized domains in the plasma membrane may provide a mechanism for channeling specific stimuli into distinct signaling pathways. Here, we report that multiple tyrosine-phosphorylated proteins accumulate transiently upon BCR activation in detergent-insoluble membrane microdomains known as lipid rafts. We found an activation-dependent translocation to the rafts of the BCR itself, as well as phospholipase Cgamma2 (PLCgamma2), an enzyme critical for BCR-induced Ca(2+) flux in B cells. An intact raft structure was required for BCR-induced tyrosine phosphorylation of PLCgamma2 and the induction of Ca(2+) flux. Taken together, these data provide a functional role for lipid rafts in BCR signaling.     

Dynamics of raft molecules in the cell and artificial membranes: approaches by pulse EPR spin labeling and single molecule optical microscopy

Lipid rafts in the plasma membrane, domains rich in cholesterol and sphingolipids, have been implicated in a number of important membrane functions. Detergent insolubility has been used to define membrane “rafts” biochemically. However, such an approach does not directly contribute to the understanding of the size and the lifetime of rafts, dynamics of the raft-constituent molecules, and the function of rafts in the membrane in situ. To address these issues, we have developed pulse EPR spin labeling and single molecule tracking optical techniques for studies of rafts in both artificial and cell membranes. In this review, we summarize our results and perspectives obtained by using these methods. We emphasize the importance of clearly distinguishing small/unstable rafts (lifetime shorter than a millisecond) in unstimulated cells and stabilized rafts induced by liganded and oligomerized (GPI-anchored) receptor molecules (core receptor rafts, lifetime over a few minutes). We propose that these stabilized rafts further induce temporal, greater rafts (signaling rafts, lifetime on the order of a second) for signaling by coalescing other small/unstable rafts, including those in the inner leaflet of the membrane, each containing perhaps one molecule of the downstream effector molecules. At variance with the general view, we emphasize the importance of cholesterol segregation from the liquid-crystalline unsaturated bulk-phase membrane for formation of the rafts, rather than the affinity of cholesterol and saturated alkyl chains. In the binary mixture of cholesterol and an unsaturated phospholipid, cholesterol is segregated out from the bulk unsaturated liquid-crystalline phase, forming cholesterol-enriched domains or clustered cholesterol domains, probably due to the lateral nonconformability between the rigid planar transfused ring structure of cholesterol and the rigid bend of the unsaturated alkyl chain at C9-C10. However, such cholesterol-rich domains are small, perhaps consisting of only several cholesterol molecules, and are short-lived, on the order of 1-100 ns. We speculate that these cholesterol-enriched domains may be stabilized by the presence of saturated alkyl chains of sphingomyelin or glycosphingolipids, and also by clustered raft proteins. In the influenza viral membrane, one of the simplest forms of a biological membrane, the lifetime of a protein and cholesterol-rich domain was evaluated to be on the order of 100 μs, again showing the short lifetime of rafts in an unstimulated state. Finally, we propose a thermal Lego model for rafts as the basic building blocks for signaling pathways in the plasma membrane.     

A Pore-forming Toxin Interacts with a GPI-anchored Protein and Causes Vacuolation of the Endoplasmic Reticulum

In this paper, we have investigated the effects of the pore-forming toxin aerolysin, produced by Aeromonas hydrophila, on mammalian cells. Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid “rafts.” We show that the protoxin is then processed to its mature form by host cell proteases. We propose that the preferential association of the toxin with rafts, through binding to GPI-anchored proteins, is likely to increase the local toxin concentration and thereby promote oligomerization, a step that it is a prerequisite for channel formation. We show that channel formation does not lead to disruption of the plasma membrane but to the selective permeabilization to small ions such as potassium, which causes plasma membrane depolarization. Next we studied the consequences of channel formation on the organization and dynamics of intracellular membranes. Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments. Concomitantly we find that the COPI coat is released from biosynthetic membranes and that biosynthetic transport of newly synthesized transmembrane G protein of vesicular stomatitis virus is inhibited. Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.     

Insolubility of lipids in triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts)

The insolubility of lipids in detergents is a useful method for probing the structure of biological membranes. Insolubility in detergents like Triton X-100 is observed in lipid bilayers that exist in physical states in which lipid packing is tight. The Triton X-100-insoluble lipid fraction obtained after detergent extraction of eukaryotic cells is composed of detergent-insoluble membranes rich in sphingolipids and cholesterol. These insoluble membranes appear to arise from sphingolipid- and cholesterol-rich membrane domains (rafts) in the tightly packed liquid ordered state. Because the degree of lipid insolubility depends on the stability of lipid-lipid interactions relative to lipid-detergent interactions, the quantitative relationship between rafts and detergent-insoluble membranes is complex, and can depend on lipid composition, detergent and temperature. Nevertheless, when used conservatively detergent insolubility is an invaluable tool for studying cellular rafts and characterizing their composition.     

Mesoscale organization of domains in the plasma membrane – beyond the lipid raft

The plasma membrane is compartmentalized into several distinct regions or domains, which show a broad diversity in both size and lifetime. The segregation of lipids and membrane proteins is thought to be driven by the lipid composition itself, lipid–protein interactions and diffusional barriers. With regards to the lipid composition, the immiscibility of certain classes of lipids underlies the “lipid raft” concept of plasmalemmal compartmentalization. Historically, lipid rafts have been described as cholesterol and (glyco)sphingolipid-rich regions of the plasma membrane that exist as a liquid-ordered phase that are resistant to extraction with non-ionic detergents. Over the years the interest in lipid rafts grew as did the challenges with studying these nanodomains. The term lipid raft has fallen out of favor with many scientists and instead the terms “membrane raft” or “membrane nanodomain” are preferred as they connote the heterogeneity and dynamic nature of the lipid-protein assemblies. In this article, we will discuss the classical lipid raft hypothesis and its limitations. This review will also discuss alternative models of lipid-protein interactions, annular lipid shells, and larger membrane clusters. We will also discuss the mesoscale organization of plasmalemmal domains including visible structures such as clathrin-coated pits and caveolae.     

Lipid rafts and assembly of enveloped viruses

The plasma membrane, late secretory pathway and endosomal compartments contain detergent-insoluble raft microdomains that are enriched in sphingolipids and cholesterol. Rafts are currently an intensively studied topic of cell biology, and raft involvement has been implicated in numerous cellular processes. A number of recent reports have localized structural proteins of several enveloped viruses to rafts, thus raising the possibility that rafts also play a role in the assembly and budding of viruses, but what exactly that role might be is still unknown.     

Plasma Membranes Are Subcompartmentalized into a Plethora of Coexisting and Diverse Microdomains in Arabidopsis and Nicotiana benthamiana

Eukaryotic plasma membranes are highly compartmentalized structures. So far, only a few individual proteins that function in a wide range of cellular processes have been shown to segregate into microdomains. However, the biological roles of most microdomain-associated proteins are unknown. Here, we investigated the heterogeneity of distinct microdomains and the complexity of their coexistence. This diversity was determined in living cells of intact multicellular tissues using 20 different marker proteins from Arabidopsis thaliana, mostly belonging to the Remorin protein family. These proteins associate with microdomains at the cytosolic leaflet of the plasma membrane. We characterized these membrane domains and determined their lateral dynamics by extensive quantitative image analysis. Systematic colocalization experiments with an extended subset of marker proteins tested in 45 different combinations revealed the coexistence of highly distinct membrane domains on individual cell surfaces. These data provide valuable tools to study the lateral segregation of membrane proteins and their biological functions in living plant cells. They also demonstrate that widely used biochemical approaches such as detergent-resistant membranes cannot resolve this biological complexity of membrane compartmentalization in vivo.     

Interactions of Ras proteins with the plasma membrane and their roles in signaling

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.