Domain formation induced by the adsorption of charged proteins on mixed lipid membranes

Peripheral proteins can trigger the formation of domains in mixed fluid-like lipid membranes. We analyze the mechanism underlying this process for proteins that bind electrostatically onto a flat two-component membrane, composed of charged and neutral lipid species. Of particular interest are membranes in which the hydrocarbon lipid tails tend to segregate owing to nonideal chain mixing, but the (protein-free) lipid membrane is nevertheless stable due to the electrostatic repulsion between the charged lipid headgroups. The adsorption of charged, say basic, proteins onto a membrane containing anionic lipids induces local lipid demixing, whereby charged lipids migrate toward (or away from) the adsorption site, so as to minimize the electrostatic binding free energy. Apart from reducing lipid headgroup repulsion, this process creates a gradient in lipid composition around the adsorption zone, and hence a line energy whose magnitude depends on the protein's size and charge and the extent of lipid chain nonideality. Above a certain critical lipid nonideality, the line energy is large enough to induce domain formation, i.e., protein aggregation and, concomitantly, macroscopic lipid phase separation. We quantitatively analyze the thermodynamic stability of the dressed membrane based on nonlinear Poisson-Boltzmann theory, accounting for both the microscopic characteristics of the proteins and lipid composition modulations at and around the adsorption zone. Spinodal surfaces and critical points of the dressed membranes are calculated for several different model proteins of spherical and disk-like shapes. Among the models studied we find the most substantial protein-induced membrane destabilization for disk-like proteins whose charges are concentrated in the membrane-facing surface. If additional charges reside on the side faces of the proteins, direct protein-protein repulsion diminishes considerably the propensity for domain formation. Generally, a highly charged flat face of a macroion appears most efficient in inducing large compositional gradients, hence a large and unfavorable line energy and consequently lateral macroion aggregation and, concomitantly, macroscopic lipid phase separation.     

Lipid Sorting and Multivesicular Endosome Biogenesis

Intracellular organelles, including endosomes, show differences not only in protein but also in lipid composition. It is becoming clear from the work of many laboratories that the mechanisms necessary to achieve such lipid segregation can operate at very different levels, including the membrane biophysical properties, the interactions with other lipids and proteins, and the turnover rates or distribution of metabolic enzymes. In turn, lipids can directly influence the organelle membrane properties by changing biophysical parameters and by recruiting partner effector proteins involved in protein sorting and membrane dynamics. In this review, we will discuss how lipids are sorted in endosomal membranes and how they impact on endosome functions.It is now well established that membranes along the endocytic and secretory pathway show differences not only in protein but also in lipid composition. For example, lipid gradients exist along the biosynthetic pathway with increasing density of cholesterol and sphingolipids from the endoplasmic reticulum (ER) to the plasma membrane (Maxfield and van Meer 2010). Also, phosphoinositides show distributions restricted to relatively well-characterized membrane territories (Di Paolo and De Camilli 2006). Given the facts that lipids are small and contain little structural information when compared with proteins, that they can diffuse rapidly within membranes, and that membranes are connected by membrane flow during transport, it is not always obvious how different lipids are segregated from each other.In this article, we will evoke different mechanisms that may contribute to the heterogeneous lipid composition of endocytic membranes, including physicochemical properties of the membrane, interactions with other proteins or lipids, and synthesis or degradation. In addition, it has also become apparent that peripheral membrane proteins often interact with membranes via diverse lipid-binding motifs, and thus that lipids directly contribute to the distribution of many peripheral membrane proteins. For example, phosphatidylinositol 3-phosphate (PI(3)P) is detected predominantly on early endosomes, where most characterized PI(3)P-binding proteins encoded by the human genome are found as well (Raiborg et al. 2013). We will also discuss how some lipids may regulate protein sorting and membrane transport within the endosomal system.     

Interaction of fibronectin type II proteins with membranes: the stallion seminal plasma protein SP-1/2

Seminal plasma of mammalians contains, among others, proteins that are characterized by the fibronectin (Fn) type II module. Our knowledge about the structure and the physiological function of seminal Fn type II proteins mainly originates from studies on PDC-109, the bovine representative of this protein family. The present work focuses on the equine protein SP-1/2 (also named HSP-1/2) with particular emphasis on its interaction with lipid membranes by employing the intrinsic protein fluorescence and a number of spin-labeled and fluorescent lipid analogues. The results indicate that the interaction of SP-1/2 with (lipid) membranes is similar to that of PDC-109 which can be explained by homologous amino acid sequences of both proteins. Like PDC-109, SP-1/2 has a specificity for phospholipids with the phosphocholine headgroup. Upon binding to lipid vesicles, the protein intercalates into the hydrophobic membrane core, resulting in a rigidification of the lipid phase and, at higher concentration, in a perturbation of membrane structure. However, compared with PDC-109, the impact of SP-1/2 on membranes is less intense in that the degree of protein-mediated immobilization of lipids was lower. Furthermore, different to PDC-109, SP-1/2 was not able to extract lipids from human red blood cells. The data are discussed with regard to similarities and species-specific differences of the function of seminal Fn type II proteins in the genesis of sperm cells.     

Interaction of a model apolipoprotein,apoLp-III,with an oil-phospholipid interface

Lipid droplets are “small” organelles that play an important role in de novo synthesis of new membrane, and steroid hormones, as well as in energy storage. The way proteins interact specifically with the oil-(phospho-)lipid monolayer interface of lipid droplets is a relatively unexplored but crucial question. Here, we use our home built liquid droplet tensiometer to mimic intracellular lipid droplets and study protein-lipid interactions at this interface. As model neutral lipid binding protein, we use apoLp-III, an amphipathic α-helix bundle protein. This domain is also found in proteins from the perilipin family and in apoE. Protein binding to the monolayer is studied by the decrease in the oil/water surface tension. Previous work used POPC (one of the major lipids found on lipid droplets) to form the phospholipid monolayer on the triolein surface. Here we expand this work by incorporating other lipids with different physico-chemical properties to study the effect of charge and lipid head-group size. This study sheds light on the affinity of this important protein domain to interact with lipids.     

Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes

The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.     

Interactions between misfolded protein oligomers and membranes: A central topic in neurodegenerative diseases?

The deposition of amyloid material has been associated with many different diseases. Although these diseases are very diverse the amyloid material share many common features such as cross-β-sheet structure of the backbone of the proteins deposited. Another common feature of the aggregation process for a wide variety of proteins is the presence of prefibrillar oligomers. These oligomers are linked to the cytotoxicity occurring during the aggregation of proteins. These prefibrillar oligomers interact extensively with lipid membranes and in some cases leads to destabilization of lipid membranes. This interaction is however highly dependent on the nature of both the oligomer and the lipids. Anionic lipids are often required for interaction with the lipid membrane while increased exposure of hydrophobic patches from highly dynamic protein oligomers are structural determinants of cytotoxicity of the oligomers. To explore the oligomer lipid interaction in detail the interaction between oligomers of α-synuclein and the 4th fasciclin-1 domain of TGFBIp with lipid membranes will be examined here. For both proteins the dynamic species are the ones causing membrane destabilization and the membrane interaction is primarily seen when the lipid membranes contain anionic lipids. Hence the dynamic nature of oligomers with exposed hydrophobic patches alongside the presence of anionic lipids could be essential for the cytotoxicity observed for prefibrillar oligomers in general. This article is part of a Special Issue entitled: Lipid–protein interactions.     

A Monte Carlo simulation study of protein-induced heat capacity changes and lipid-induced protein clustering.

Monte Carlo simulations were used to describe the interaction of peripheral and integral proteins with lipids in terms of heat capacity profiles and protein distribution. The simulations were based on a two-state model for the lipid, representing the lipid state as being either gel or fluid. The interaction between neighboring lipids has been taken into account through an unlike nearest neighbor free energy term delta omega, which is a measure of the cooperativity of the lipid transition. Lipid/protein interaction was considered using the experimental observation that the transition midpoints of lipid membranes are shifted upon protein binding, a thermodynamic consequence of different binding constants of protein with fluid or gel lipids. The difference of the binding free energies was used as an additional parameter to describe lipid-protein interaction. The heat capacity profiles of lipid/protein complexes could be well described for both peripheral and integral proteins. Binding of proteins results in a shift and an asymmetric broadening of the melting profile. The model results in a coexistence of gel and fluid lipid domains in the proximity of the thermotropic transition. As a consequence, bound peripheral proteins aggregate in the temperature range of the lipid transition. Integral proteins induce calorimetric melting curves that are qualitatively different from that of peripheral proteins and aggregate in either gel or liquid crystalline lipid phase. The results presented here are in good agreement with calorimetric experiments on lipid-protein complexes and have implementations for the functional control of proteins.     

The sticholysin family of pore-forming toxins induces the mixing of lipids in membrane domains

Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.     

Protein modulation of lipids, and vice-versa, in membranes

This review describes: (i) perturbations of the membrane lipids that are induced by integral membrane proteins, and reciprocally, (ii) the effects that the lipids may have on the function of membrane-associated proteins. Topics of the first category that are covered include: stoichiometry and selectivity of the first shell of lipids associated at the intramembranous perimeter of transmembrane proteins; the chain configuration and exchange rates of the first-shell lipids; the effects of transmembrane peptides on transbilayer movement of lipids (flip-flop); the effects of membrane proteins on lipid polymorphism and formation of non-lamellar phases; and the effects of hydrophobic mismatch on lipid chain configuration, phase stability and selectivity of lipid-protein association. Topics of the second category are: the influence of lipid selectivity on conformational changes in the protein; the effects of elastic fluctuations of the lipid bilayer on protein insertion and orientation in membranes; the effects of hydrophobic matching on intramembrane protein-protein association; and the effects of intrinsic lipid curvature on membrane integration, oligomer formation and activity of membrane proteins.     

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins

Lipid rafts are microdomains present in the membrane of eukaryotic organisms and bacterial pathogens. They are characterized by having tightly packed lipids and a subset of specific proteins. Lipid rafts are associated with a variety of important biological processes including signaling and lateral sorting of proteins. To determine whether lipid rafts exist in the inner membrane of Borrelia burgdorferi, we separated the inner and outer membranes and analyzed the lipid constituents present in each membrane fraction. We found that both the inner and outer membranes have cholesterol and cholesterol glycolipids. Fluorescence anisotropy and FRET showed that lipids from both membranes can form rafts but have different abilities to do so. The analysis of the biochemically defined proteome of lipid rafts from the inner membrane revealed a diverse set of proteins, different from those associated with the outer membrane, with functions in protein trafficking, chemotaxis and signaling.     

Lipid-protein interactions in thylakoid membranes of chilling-resistant and -sensitive plants studied by spin label electron spin resonance spectroscopy

Lipid-protein interactions in thylakoid membranes from lettuce, pea, tomato, and cucumber have been studied using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyl diglyceride and phosphatidylglycerol. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted lipids interacting with the integral membrane proteins. Comparison of the spectra from the same spin label in thylakoid membranes from different plants shows that the overall lipid fluidity in the membranes decreases with chilling sensitivity. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with integral membrane proteins. Thylakoid membranes of cucumber, a typical chilling-sensitive plant, have been found to have a higher proportion of motionally restricted lipids and a different lipid selectivity for lipid-protein interaction, as compared with those of pea, a typical chilling-resistant plant. This correlation with chilling sensitivity holds generally for the different plants studied. It seems likely that the chilling sensitivity in thylakoid membranes is not determined by lipid fluidity alone, but also by the lipid-protein interactions which could affect protein function in a more direct manner.     

Association of alpha-synuclein and mutants with lipid membranes: spin-label ESR and polarized IR

Alpha-synuclein is a presynaptic protein, the A53T and A30P mutants of which are linked independently to early-onset familial Parkinson's disease. The association of wild-type alpha-synuclein with lipid membranes was characterized previously by electron spin resonance (ESR) spectroscopy with spin-labeled lipids [Ramakrishnan, M., Jensen, P. H., and Marsh, D. (2003) Biochemistry 42, 12919-12926]. Here, we study the interaction of the A53T and A30P alpha-synuclein mutants and a truncated form that lacks the acidic C-terminal domain with phosphatidylglycerol bilayer membranes, using anionic phospholipid spin labels. The strength of the interaction with phosphatidylglycerol membranes lies in the order: wild type approximately truncated > A53T > A30P > fibrils approximately 0, and only the truncated form interacts with phosphatidylcholine membranes. The selectivity of the interaction of the mutant alpha-synucleins with different spin-labeled lipid species is reduced considerably, relative to the wild-type protein, whereas that of the truncated protein is increased. Polarized infrared (IR) spectroscopy is used to study the interactions of the wild-type and truncated proteins with aligned lipid membranes and additionally to characterize the fibrillar form. Wild-type alpha-synuclein is natively unfolded in solution and acquires secondary structure upon binding to membranes containing phosphatidylglycerol. Up to 30-40% of the amide I band intensity of the membrane-bound wild-type and truncated proteins is attributable to beta-sheet structure, at the surface densities used for IR spectroscopy. The remainder is alpha-helix and residual unordered structure. Fibrillar alpha-synuclein contains 62% antiparallel beta-sheet and is oriented on the substrate surface but does not interact with deposited lipid membranes. The beta-sheet secondary-structural elements of the wild-type and truncated proteins are partially oriented on the surface of membranes with which they interact.     

Bridging gaps in phospholipid transport

Phospholipid transport between membranes is a fundamental aspect of organelle biogenesis in eukaryotes; however, little is know about this process. A significant body of data demonstrates that newly synthesized phospholipids can move between membranes by routes that are independent of the vesicular traffic that carries membrane proteins. Evidence continues to accumulate in support of a system for phospholipid transport that occurs at zones of apposition and contact between donor membranes - the source of specific phospholipids - and acceptor membranes that are unable to synthesize the necessary lipids. Recent findings identify some of the lipids and proteins that must be present on membranes for inter-organelle phospholipid transport to occur between the endoplasmic reticulum and mitochondria or Golgi. These data suggest that protein and lipid assemblies on donors and acceptors promote membrane docking and facilitate lipid movement.     

Protein Arcs May Form Stable Pores in Lipid Membranes

Electron microscopy and atomic force microscopy images of cholesterol-dependent cytolysins and related proteins that form large pores in lipid membranes have revealed the presence of incomplete rings, or arcs. Some evidence indicates that these arcs are inserted into the membrane and induce membrane leakage, but other experiments seem to refute that. Could such pores, only partially lined by protein, be kinetically and thermodynamically stable? How would the lipids be structured in such a pore? Using the antimicrobial peptide protegrin-1 as a model, we test the stability of pores only partially lined by peptide using all-atom molecular dynamics simulations in POPC and POPE/POPG membranes. The data show that, whereas pure lipid pores close rapidly, pores partially lined by protegrin arcs are stable for at least 300 ns. Estimates of the thermodynamic stability of these arcs using line tension data and implicit solvent calculations show that these arcs can be marginally stable in both zwitterionic and anionic membranes. Arcs provide an explanation for the observed ion selectivity in protegrin electrophysiology experiments and could possibly be involved in other membrane permeabilization processes where lipids are thought to participate, such as those induced by antimicrobial peptides and colicins, as well as the Bax apoptotic pore.     

Protein Arcs May Form Stable Pores in Lipid Membranes

Electron microscopy and atomic force microscopy images of cholesterol-dependent cytolysins and related proteins that form large pores in lipid membranes have revealed the presence of incomplete rings, or arcs. Some evidence indicates that these arcs are inserted into the membrane and induce membrane leakage, but other experiments seem to refute that. Could such pores, only partially lined by protein, be kinetically and thermodynamically stable? How would the lipids be structured in such a pore? Using the antimicrobial peptide protegrin-1 as a model, we test the stability of pores only partially lined by peptide using all-atom molecular dynamics simulations in POPC and POPE/POPG membranes. The data show that, whereas pure lipid pores close rapidly, pores partially lined by protegrin arcs are stable for at least 300 ns. Estimates of the thermodynamic stability of these arcs using line tension data and implicit solvent calculations show that these arcs can be marginally stable in both zwitterionic and anionic membranes. Arcs provide an explanation for the observed ion selectivity in protegrin electrophysiology experiments and could possibly be involved in other membrane permeabilization processes where lipids are thought to participate, such as those induced by antimicrobial peptides and colicins, as well as the Bax apoptotic pore.     

Effect of line tension on the lateral organization of lipid membranes

The principles of organization and functioning of cellular membranes are currently not well understood. The raft hypothesis suggests the existence of domains or rafts in cell membranes, which behave as protein and lipid platforms. They have a functional role in important cellular processes, like protein sorting or cell signaling, among others. Theoretical work suggests that the interfacial energy at the domain edge, also known as line tension, is a key parameter determining the distribution of domain sizes, but there is little evidence of how line tension affects membrane organization. We have investigated the effects of the line tension on the formation and stability of liquid ordered domains in model lipid bilayers with raft-like composition by means of time-lapse confocal microscopy coupled to atomic force microscopy. We varied the hydrophobic mismatch between the two phases, and consequently the line tension, by modifying the thickness of the disordered phase with phosphatidylcholines of different acyl chain length. The temperature of domain formation, the dynamics of domain growth, and the distribution of domain sizes depend strongly on the thickness difference between the domains and the surrounding membrane, which is related to line tension. When considering line tension calculated from a theoretical model, our results revealed a linear increase of the temperature of domain formation and domain growth rate with line tension. Domain budding was also shown to depend on height mismatch. Our experiments contribute significantly to our knowledge of the physical-chemical parameters that control membrane organization. Importantly, the general trends observed can be extended to cellular membranes.     

Homeostatic restitution of cell membranes. Nuclear membrane lipid biogenesis and transport of protein from cytosol to intranuclear spaces

Our studies on homeostatic restitution of cellular and subcellular membranes showed that vesicular intracellular transport is engaged in systematic and coordinated replacement of lipids and proteins in the membranes of the secretory, non-dividing epithelial cells (Slomiany et al., J. Physiol. Pharmacol. 2004; 55: 837-860). In this report, we present evidence on the homeostatic restitution of lipids in the biomembranes that constitute nuclear envelopes. We investigated nuclear membranes lipid synthesis by employing purified intact nuclei (IN), the outer nuclear membrane (ONM), the inner nuclear membrane (INM) and the cell cytosol (CC). In contrast to Endoplasmic Reticulum (ER) which in the presence of CC generates new biomembrane that forms ER vesicles transporting ER products to Golgi, the IN, ONM and INM are not producing transport vesicles. Instead, the newly synthesized lipids remain in the nuclear membranes. The membranes (INM, ONM) of IN incubated with CC become enriched with newly synthesized phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA). The incubation of separated ONM and INM with CC also enriched the membranes with IN specific lipids identified above. Moreover, the incubation of IN or its membranes with CC afforded retention of numerous CC proteins on the nuclear membrane. Here, we concentrated on 30kDa CC protein that displayed affinity to nuclear membrane PIP2. The 30kDa CC protein bound to PIP2 of IN, INM, and ONM. With IN, initially the PIP2-30kDa CC protein complex was detected on ONM, after 30-120 min of incubation, was found on INM and in nuclear contents. At the same time when the 30 kDa protein was released from INM and found in nuclear contents, the PIP2 of INM and ONM became undetectable, while the lipid extract from the membrane displaced from IN contained labeled PI only. Since ONM is an uninterrupted continuum of ER and INM, we speculate that the synthesis of the lipids in the ER, in the region adjacent to nucleus, is defining nuclear outer and inner biomembrane composition, is responsible for transport of the cytosolic protein into the nucleus and, replenishment of ER membrane used for vesicular transport.     

Life at the border: Adaptation of proteins to anisotropic membrane environment

This review discusses main features of transmembrane (TM) proteins which distinguish them from water‐soluble proteins and allow their adaptation to the anisotropic membrane environment. We overview the structural limitations on membrane protein architecture, spatial arrangement of proteins in membranes and their intrinsic hydrophobic thickness, co‐translational and post‐translational folding and insertion into lipid bilayers, topogenesis, high propensity to form oligomers, and large‐scale conformational transitions during membrane insertion and transport function. Special attention is paid to the polarity of TM protein surfaces described by profiles of dipolarity/polarizability and hydrogen‐bonding capacity parameters that match polarity of the lipid environment. Analysis of distributions of Trp resides on surfaces of TM proteins from different biological membranes indicates that interfacial membrane regions with preferential accumulation of Trp indole rings correspond to the outer part of the lipid acyl chain region—between double bonds and carbonyl groups of lipids. These “midpolar” regions are not always symmetric in proteins from natural membranes. We also examined the hydrophobic effect that drives insertion of proteins into lipid bilayer and different free energy contributions to TM protein stability, including attractive van der Waals forces and hydrogen bonds, side‐chain conformational entropy, the hydrophobic mismatch, membrane deformations, and specific protein–lipid binding.     

The superlattice model of lateral organization of membranes and its implications on membrane lipid homeostasis

Most biological membranes are extremely complex structures consisting of hundreds of different lipid and protein molecules. According to the famous fluid-mosaic model lipids and many proteins are free to diffuse very rapidly in the plane of the membrane. While such fast diffusion implies that different membrane lipids would be laterally randomly distributed, accumulating evidence indicates that in model and natural membranes the lipid components tend to adopt regular (superlattice-like) distributions. The superlattice model, put forward based on such evidence, is intriguing because it predicts that 1) there is a limited number of allowed compositions representing local minima in membrane free energy and 2) those energy minima could provide set-points for enzymes regulating membrane lipid compositions. Furthermore, the existence of a discrete number of allowed compositions could help to maintain organelle identity in the face of rapid inter-organelle membrane traffic.     

The Gbetagamma dimer drives the interaction of heterotrimeric Gi proteins with nonlamellar membrane structures

Heterotrimeric G proteins are peripheral membrane proteins that propagate signals from membrane receptors to regulatory proteins localized in distinct cellular compartments. To facilitate signal amplification, G proteins are in molar excess with respect to G protein-coupled receptors. Because G proteins are capable of translocating from membrane to cytosol, protein-lipid interactions play a crucial role in signal transduction. Here, we studied the binding of heterotrimeric G proteins (Galphabetagamma) to model membranes (liposomes) and that of the entities formed upon receptor-mediated activation (Galpha and Gbetagamma). The model membranes used were composed of defined membrane lipids capable of organizing into either lamellar or nonlamellar (hexagonal H(II)) membrane structures. We demonstrated that although heterotrimeric G(i) proteins and Gbetagamma dimers can bind to lipid bilayers of phosphatidylcholine, their binding to membranes was markedly and significantly enhanced by the presence of nonlamellar phases of phosphatidylethanolamine. Conversely, activated G protein alpha subunits showed an opposite membrane binding behavior with a marked preference for lamellar membranes. These results have important consequences in cell signaling. First, the binding characteristics of the Gbetagamma dimer account for the lipid binding behavior and the cellular localization of heterotrimeric G proteins. Second, the distinct protein-lipid interactions of heterotrimeric G proteins, Gbetagamma dimers, and Galpha subunits with membrane lipids explain, in part, their different cellular mobilizations during signaling upon receptor activation. Finally, their differential interactions with lipids suggest an active role of the membrane lipid secondary structure in the propagation of signals through G protein-coupled receptors.