Lipids in biological membrane fusion

The results reviewed suggest that membrane fusion in diverse biological fusion reactions involves formation of some specific intermediates: stalks and pores. Energy of these intermediates and, consequently, the rate and extent of fusion depend on the propensity of the corresponding monolayers of membranes to bend in the required directions.Proteins and peptides can control the bending energy of membrane monolayers in a number of ways. Monolayer lipid composition may be altered by different phospholipases [50, 85, 90], flipases and translocases [4, 50]. Proteins and peptides can change monolayer spontaneous curvature or hydrophobic void energy by direct interaction with membrane lipids [20, 32, 111]. Proteins may also provide some barriers for lipid diffusion in the plane of the monolayer [83, 141]. If diffusion of lipids at some specific membrane sites (e.g., in the vicinity of fusion protein) is somehow hindered, the energy of the bent fusion intermediates would reflect the elastic properties of these particular sites rather than the spontaneous curvature of the whole monolayers. Proteins may deform membranes while bringing them locally into close contact. The alteration of the geometric (external) curvature will certainly change the elastic energy of the initial state and, thus affect the energetic barriers of the formation of the intermediates [143]. In addition, the area and the energy of the stalk can be reduced by preliminary bending of the contacting membranes [111]. The possible effects of proteins and polymers on local elastic properties and local shapes of the membranes have been recently analyzed [22, 39, 45, 63]. These studies may provide a good basis for future development of theoretical models of protein-mediated fusion.     

Lipid traffic: the ABC of transbilayer movement

Membrane lipids do not spontaneously exchange between the two leaflets of lipid bilayers because the polar headgroups cannot cross the hydrophobic membrane interior. Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other. In addition, cellular membranes contain proteins that facilitate a passive equilibration of lipids between the two membrane halves. In recent years, a growing number of proteins have been put forward as lipid translocators or facilitators. Unexpectedly, some of these appear to be required for efficient translocation of lipids lacking bulky headgroups, like cholesterol and fatty acids. The candidate lipid translocators identified so far belong to large protein families whose other members include pumps for amphiphilic molecules like bile salts and drugs.     

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.     

Membrane bending by protein-protein crowding

Curved membranes are an essential feature of dynamic cellular structures, including endocytic pits, filopodia protrusions and most organelles. It has been proposed that specialized proteins induce curvature by binding to membranes through two primary mechanisms: membrane scaffolding by curved proteins or complexes; and insertion of wedge-like amphipathic helices into the membrane. Recent computational studies have raised questions about the efficiency of the helix-insertion mechanism, predicting that proteins must cover nearly 100% of the membrane surface to generate high curvature, an improbable physiological situation. Thus, at present, we lack a sufficient physical explanation of how protein attachment bends membranes efficiently. On the basis of studies of epsin1 and AP180, proteins involved in clathrin-mediated endocytosis, we propose a third general mechanism for bending fluid cellular membranes: protein-protein crowding. By correlating membrane tubulation with measurements of protein densities on membrane surfaces, we demonstrate that lateral pressure generated by collisions between bound proteins drives bending. Whether proteins attach by inserting a helix or by binding lipid heads with an engineered tag, protein coverage above ~20% is sufficient to bend membranes. Consistent with this crowding mechanism, we find that even proteins unrelated to membrane curvature, such as green fluorescent protein (GFP), can bend membranes when sufficiently concentrated. These findings demonstrate a highly efficient mechanism by which the crowded protein environment on the surface of cellular membranes can contribute to membrane shape change.     

Role of bound water in biological membrane structure: Fluorescence and infrared studies

Bound water is a major component of biological membranes and is required for the structural stability of the lipid bilayer. It has also been postulated that it is involved in water transport, membrane fusion, and mobility of membrane proteins and lipids. We have measured the fluorescence emission of membrane-bound 1-anilino-8-naphthalenesulfonate (ANS) and the infrared spectra of membranes, both as a function of hydration. ANS fluorescence is sensitive to polarity and fluidity of the membrane-aqueous interface, while infrared absorption is sensitive to the hydrogen bonding and vibrational motion of water and membrane proteins and lipids. The fluorescence results provide evidence of increasing rigidity and/or decreasing polarity of the membrane-aqueous interface with removal of water. The membrane infrared spectra show prominent hydration-dependent changes in a number of bands with possible assignments to cholesterol (vinyl CH bend, OH stretch), protein (amide A, II, V), and bound water (OH stretch). Further characterization of the bound water should allow its incorporation into current models of membrane structure and give insight into the role of membrane hydration in cell surface function.     

The hydrophobic insertion mechanism of membrane curvature generation by proteins

A wide spectrum of intracellular processes is dependent on the ability of cells to dynamically regulate membrane shape. Membrane bending by proteins is necessary for the generation of intracellular transport carriers and for the maintenance of otherwise intrinsically unstable regions of high membrane curvature in cell organelles. Understanding the mechanisms by which proteins curve membranes is therefore of primary importance. Here we suggest, for the first time to our knowledge, a quantitative mechanism of lipid membrane bending by hydrophobic or amphipathic rodlike inclusions which simulate amphipathic α-helices—structures shown to sculpt membranes. Considering the lipid monolayer matrix as an anisotropic elastic material, we compute the intramembrane stresses and strains generated by the embedded inclusions, determine the resulting membrane shapes, and the accumulated elastic energy. We characterize the ability of an inclusion to bend membranes by an effective spontaneous curvature, and show that shallow rodlike inclusions are more effective in membrane shaping than are lipids having a high propensity for curvature. Our computations provide experimentally testable predictions on the protein amounts needed to generate intracellular membrane shapes for various insertion depths and membrane thicknesses. We also predict that the ability of N-BAR domains to produce membrane tubules in vivo can be ascribed solely to insertion of their amphipathic helices.     

Flippases and vesicle-mediated protein transport

The best-understood mechanisms for generating transport vesicles in the secretory and endocytic pathways involve the localized assembly of cytosolic coat proteins such as clathrin, coat protein complex (COP)I and COPII onto membranes. These coat proteins can deform membranes by themselves, but accessory proteins might help to generate the tight curvature needed to form a vesicle. Enzymes that pump phospholipid from one leaflet of the bilayer to the other (flippases) can deform membranes by creating an imbalance in the phospholipid number between the two leaflets. Recent studies describe a requirement for the yeast Drs2p family of P-type ATPases in both phospholipid translocation and protein transport in the secretory and endocytic pathways. This indicates that flippases work with coat proteins to form vesicles.     

The membrane transporter lactose permease increases lipid bilayer bending rigidity

Cellular life relies on membranes, which provide a resilient and adaptive cell boundary. Many essential processes depend upon the ease with which the membrane is able to deform and bend, features that can be characterized by the bending rigidity. Quantitative investigations of such mechanical properties of biological membranes have primarily been undertaken in solely lipid bilayers and frequently in the absence of buffers. In contrast, much less is known about the influence of integral membrane proteins on bending rigidity under physiological conditions. We focus on an exemplar member of the ubiquitous major facilitator superfamily of transporters and assess the influence of lactose permease on the bending rigidity of lipid bilayers. Fluctuation analysis of giant unilamellar vesicles (GUVs) is a useful means to measure bending rigidity. We find that using a hydrogel substrate produces GUVs that are well suited to fluctuation analysis. Moreover, the hydrogel method is amenable to both physiological salt concentrations and anionic lipids, which are important to mimic key aspects of the native lactose permease membrane. Varying the fraction of the anionic lipid in the lipid mixture DOPC/DOPE/DOPG allows us to assess the dependence of membrane bending rigidity on the topology and concentration of an integral membrane protein in the lipid bilayer of GUVs. The bending rigidity gradually increases with the incorporation of lactose permease, but there is no further increase with greater amounts of the protein in the membrane.     

Lipid-binding proteins as stabilizers of membrane microdomains--possible physiological significance

Below the melting point temperature of lipids, artificial lipid membranes usually exist in the ordered gel phase. Above these temperatures lipid acyl chains become fluid and disordered (liquid-crystalline phase). Depending on the chemical composition of artificial membranes, phase separation may occur, leading to the formation of transient or stable membrane domains. A similar phase separation of lipids into ordered and disordered domains has been observed in natural membranes at physiological temperature range. Moreover, it has been reported that certain proteins prefer certain organization of lipids, as for example glycosylphosphatidylinositol-anchored proteins or Src family of tyrosine kinases. The aim of present review is to discuss the possibility that some lipid microdomains are induced or stabilized by lipid-binding proteins that under certain conditions, for example due to a rise of cytosolic Ca2+ or pH changes, may attach to the membrane surface, inducing clustering of lipid molecules and creation of ordered lipid microdomains. These domains may than attract other cytosolic proteins, either enzymes or regulatory proteins. It is, therefore, postulated that lipid microdomains play important roles within a cell, in signal transduction and enzymatic catalysis, and also in various pathological states, as Alzheimer's disease, anti-phosphatidylserine syndrome, or development of multidrug resistance of cancer cells.     

Reconstitution of liver endoplasmic reticulum membranes from solubilized proteins and lipids by removal of detergent]

A method for membrane reconstitution from cholate-solubilized microsomal proteins and lipids by a removal of the detergent on a column with charcoal has been developed. A comparative study showed that the membranes reconstituted by a dialysis or absorption do not differ from each other in terms of membrane proteins incorporation into lipid vesicles and cytochrome P-450 reconversion into cytochrome P-450. A possibility of biomembrane reconstitution from membrane proteins and lipids solubilized by a non-ionic detergent Triton X-100 was shown. A removal of the detergent results in a formation of membranes, which are chemically close to the original ones but ultrastructurally very different from the latter. On the other hand, absorption or dialysis of cholate-solubilized proteins and lipids results in reconstituted membranes with asymmetrically arranged intramembrane particles located on the hydrophobic surfaces of the membrane halves. The number and size of these particles are similar to those of the original microsomal membranes.     

From mosaic to patchwork: matching lipids and proteins in membrane organization

Biological membranes encompass and compartmentalize cells and organelles and are a prerequisite to life as we know it. One defining feature of membranes is an astonishing diversity of building blocks. The mechanisms and principles organizing the thousands of proteins and lipids that make up membrane bilayers in cells are still under debate. Many terms and mechanisms have been introduced over the years to account for certain phenomena and aspects of membrane organization and function. Recently, the different viewpoints - focusing on lipids vs. proteins or physical vs. molecular driving forces for membrane organization - are increasingly converging. Here we review the basic properties of biological membranes and the most common theories for lateral segregation of membrane components before discussing an emerging model of a self-organized, multi-domain membrane or 'patchwork membrane'.     

Lipid-Mediated Regulation of Extrinsic Membrane Protein Activities

Abstract

It is increasingly apparent that lipids function not only in the membranous compartmentalization of cell components, but also in the regulation of activities of soluble proteins involved in key cellular events. There are several mechanisms by which membranes and their component lipids affect protein function. Certain proteins are activated by a conformational change that occurs upon association with lipid bilayers. Others appear to be influenced by being recruited to membranes so that they can interact with regulatory factors, or by being sequestered at membranes and thus incapable of interacting with soluble proteins or factors necessary for their function. Finally, membranes regulate many proteins by mechanisms yet to be elucidated. In addition to the lipids in membrane bilayers, products of glycerophospholipid and sphingolipid metabolism, functioning as second messengers, influence certain cytosolic proteins involved in cellular signal signaling pathways. This form of regulation, while important, is not the focus of this review and will only briefly be discussed.     

From mosaic to patchwork: Matching lipids and proteins in membrane organization

Abstract

Biological membranes encompass and compartmentalize cells and organelles and are a prerequisite to life as we know it. One defining feature of membranes is an astonishing diversity of building blocks. The mechanisms and principles organizing the thousands of proteins and lipids that make up membrane bilayers in cells are still under debate. Many terms and mechanisms have been introduced over the years to account for certain phenomena and aspects of membrane organization and function. Recently, the different viewpoints – focusing on lipids vs. proteins or physical vs. molecular driving forces for membrane organization – are increasingly converging. Here we review the basic properties of biological membranes and the most common theories for lateral segregation of membrane components before discussing an emerging model of a self-organized, multi-domain membrane or ‘patchwork membrane'.     

Transbilayer (flip-flop) lipid motion and lipid scrambling in membranes

This paper reviews the current knowledge on the various mechanisms for transbilayer, or flip-flop, lipid motion in model and cell membranes, enzyme-assisted lipid transfer by flippases, floppases and scramblases is briefly discussed, while non-catalyzed lipid flip-flop is reviewed in more detail. Transbilayer lipid motion may occur as a result of the insertion of foreign molecules (detergents, lipids, or even proteins) in one of the membrane leaflets. It may also be the result of the enzymatic generation of lipids, e.g. diacylglycerol or ceramide, at one side of the membrane. Transbilayer motion rates decrease in the order diacylglycerol ? ceramide ? phospholipids. Ceramide, but not diacylglycerol, can induce transbilayer motion of other lipids, and bilayer scrambling. Transbilayer lipid diffusion and bilayer scrambling are defined as two conceptually and mechanistically different processes. The mechanism of scrambling appears to be related to local instabilities caused by the non-lamellar ceramide molecule, or by other molecules that exhibit a relatively slow flip-flop rate, when asymmetrically inserted or generated in one of the monolayers in a cell or model membrane.     

SHIP164 is a chorein motif lipid transfer protein that controls endosome–Golgi membrane traffic

Cellular membranes differ in protein and lipid composition as well as in the protein–lipid ratio. Thus, progression of membranous organelles along traffic routes requires mechanisms to control bilayer lipid chemistry and their abundance relative to proteins. The recent structural and functional characterization of VPS13-family proteins has suggested a mechanism through which lipids can be transferred in bulk from one membrane to another at membrane contact sites, and thus independently of vesicular traffic. Here, we show that SHIP164 (UHRF1BP1L) shares structural and lipid transfer properties with these proteins and is localized on a subpopulation of vesicle clusters in the early endocytic pathway whose membrane cargo includes the cation-independent mannose-6-phosphate receptor (MPR). Loss of SHIP164 disrupts retrograde traffic of these organelles to the Golgi complex. Our findings raise the possibility that bulk transfer of lipids to endocytic membranes may play a role in their traffic.     

How proteins produce cellular membrane curvature

Biological membranes exhibit various function-related shapes, and the mechanism by which these shapes are created is largely unclear. Here, we classify possible curvature-generating mechanisms that are provided by lipids that constitute the membrane bilayer and by proteins that interact with, or are embedded in, the membrane. We describe membrane elastic properties in order to formulate the structural and energetic requirements of proteins and lipids that would enable them to work together to generate the membrane shapes seen during intracellular trafficking.     

Mechanisms of membrane deformation

Membrane traffic requires the generation of high-curvature lipid-bound transport carriers represented by tubules and vesicles. The mechanisms through which membranes are deformed has gained much recent attention. A major advance has been the demonstration that direct interactions between cytosolic proteins and lipid bilayers are important in the acquisition of membrane curvature. Rather than being driven only by the formation of membrane-associated structural scaffolds, membrane deformation requires physical perturbation of the lipid bilayer. A variety of proteins have been identified that directly bind and deform membranes. An emerging theme in this process is the importance of amphipathic peptides that partially penetrate the lipid bilayer.     

Trafficking of malarial proteins to the host cell cytoplasm and erythrocyte surface membrane involves multiple pathways

During the asexual stage of malaria infection, the intracellular parasite exports membranes into the erythrocyte cytoplasm and lipids and proteins to the host cell membrane, essentially "transforming" the erythrocyte. To investigate lipid and protein trafficking pathways within Plasmodium falciparum-infected erythrocytes, synchronous cultures are temporally analyzed by confocal fluorescence imaging microscopy for the production, location and morphology of exported membranes (vesicles) and parasite proteins. Highly mobile vesicles are observed as early as 4 h postinvasion in the erythrocyte cytoplasm of infected erythrocytes incubated in vitro with C6-NBD-labeled phospholipids. These vesicles are most prevalent in the trophozoite stage. An immunofluorescence technique is developed to simultaneously determine the morphology and distribution of the fluorescent membranes and a number of parasite proteins within a single parasitized erythrocyte. Parasite proteins are visualized with FITC- or Texas red-labeled monoclonal antibodies. Double-label immunofluorescence reveals that of the five parasite antigens examined, only one was predominantly associated with membranes in the erythrocyte cytoplasm. Two other parasite antigens localized only in part to these vesicles, with the majority of the exported antigens present in lipid-free aggregates in the host cell cytoplasm. Another parasite antigen transported into the erythrocyte cytoplasm is localized exclusively in lipid-free aggregates. A parasite plasma membrane (PPM) and/or parasitophorous vacuolar membrane (PVM) antigen which is not exported always colocalizes with fluorescent lipids in the PPM/PVM. Visualization of two parasite proteins simultaneously using FITC- and Texas red-labeled 2 degrees antibodies reveals that some parasite proteins are constitutively transported in the same vesicles, whereas other are segregated before export. Of the four exported antigens, only one appears to cross the barriers of the PPM and PVM through membrane-mediated events, whereas the others are exported across the PPM/PVM to the host cell cytoplasm and surface membrane through lipid (vesicle)-independent pathways.     

Cardiolipin binding in bacterial respiratory complexes: Structural and functional implications

The structural and functional integrity of biological membranes is vital to life. The interplay of lipids and membrane proteins is crucial for numerous fundamental processes ranging from respiration, photosynthesis, signal transduction, solute transport to motility. Evidence is accumulating that specific lipids play important roles in membrane proteins, but how specific lipids interact with and enable membrane proteins to achieve their full functionality remains unclear. X-ray structures of membrane proteins have revealed tight and specific binding of lipids. For instance, cardiolipin, an anionic phospholipid, has been found to be associated to a number of eukaryotic and prokaryotic respiratory complexes. Moreover, polar and septal accumulation of cardiolipin in a number of prokaryotes may ensure proper spatial segregation and/or activity of proteins. In this review, we describe current knowledge of the functions associated with cardiolipin binding to respiratory complexes in prokaryotes as a frame to discuss how specific lipid binding may tune their reactivity towards quinone and participate to supercomplex formation of both aerobic and anaerobic respiratory chains. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Molecular Simulation of Mechanical Properties and Membrane Activities of the ESCRT-III Complexes

The endosomal sorting complex required for transport (ESCRT) machinery carries out the membrane scission reactions that are required for many biological processes throughout cells. How ESCRTs bind and deform cellular membranes and ultimately produce vesicles has been a matter of active research in recent years. In this study, we use fully atomistic molecular dynamics simulations to scrutinize the structural details of a filament composed of Vps32 protomers, a major component of ESCRT-III complexes. The simulations show that both hydrophobic and electrostatic interactions between monomers help maintain the structural stability of the filament, which exhibits an intrinsic bend and twist. Our findings suggest that the accumulation of bending and twisting stresses as the filament elongates on the membrane surface likely contributes to the driving force for membrane invagination. The filament exposes a large cationic surface that senses the negatively charged lipids in the membrane, and the N-terminal amphipathic helix of the monomers not only acts as a membrane anchor but also generates significant positive membrane curvature. Taking all results together, we discuss a plausible mechanism for membrane invagination driven by ESCRT-III.