Changes in the lipid and protein components of thymus cell membrane during lipid peroxidation]

Nonenzymatic lipid peroxidation in thymus cell plasma membranes was studied. The composition of lipid and protein components, intensity of fluorescence of the membrane probes (1-anilinonaphthalene-8-sulfonate, 4-dimethylaminochalcon, eosin, pyronin and rhodamine), fluorescence polarization of tryptophan residues of membrane proteins and quenching by acrylamide of intrinsic fluorescence of proteins were determined. Induction of lipid peroxidation by the Fe(2+)-ascorbate system caused changes in the composition and structure of lipids. This was paralleled with changes in the structural-dynamic organization of membrane proteins, transition of some peripheral proteins to the water phase and increased solubilization of integral proteins by Triton X-100.     

X-ray diffraction and electron microscope study of the interactions of myelin components. The structure of a lamellar phase with a 150 to 180 A repeat distance containing basic proteins and acidic lipids

A variety of phases has been studied: those formed by lipids extracted from myelin, the basic myelin proteins A1 (from the central nervous system) and P1 (from the peripheral nervous system) or other basic proteins. A particularly interesting type of phase was observed which consists of one of the basic proteins of myelin, acidic phospholipids and sulphatides; this phase is lamellar and contains two lipid bilayers in its unit cell. The structure of this phase was determined by the pattern recognition technique and by electron microscope observations of OsO₄-flxed and freeze-etched preparations. It is formed by two different lipid bilayers, one containing mainly the phospholipids with the hydrocarbon chains in a liquid-like conformation and the other containing mainly the sulphatides with at least one fraction of the chains stiff and hexagonally packed. Under the effect of high temperature, or if cholesterol is added, this phase is replaced by other phases which lack the large repeat. The segregation of the lipids and their specific associations with the basic proteins are discussed in relation to the structure of myelin.     

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.     

Structural and biochemical insights into lipid transport by VPS13 proteins

VPS13 proteins are proposed to function at contact sites between organelles as bridges for lipids to move directionally and in bulk between organellar membranes. VPS13s are anchored between membranes via interactions with receptors, including both peripheral and integral membrane proteins. Here we present the crystal structure of VPS13s adaptor binding domain (VAB) complexed with a Pro-X-Pro peptide recognition motif present in one such receptor, the integral membrane protein Mcp1p, and show biochemically that other Pro-X-Pro motifs bind the VAB in the same site. We further demonstrate that Mcp1p and another integral membrane protein that interacts directly with human VPS13A, XK, are scramblases. This finding supports an emerging paradigm of a partnership between bulk lipid transport proteins and scramblases. Scramblases can re-equilibrate lipids between membrane leaflets as lipids are removed from or inserted into the cytosolic leaflet of donor and acceptor organelles, respectively, in the course of protein-mediated transport.     

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.     

Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile

Nonbilayer lipids can be defined as cone-shaped lipids with a preference for nonbilayer structures with a negative curvature, such as the hexagonal phase. All membranes contain these lipids in large amounts. Yet, the lipids in biological membranes are organized in a bilayer. This leads to the question: what is the physiological role of nonbilayer lipids? Different models are discussed in this review, with a focus on the lateral pressure profile within the membrane. Based on this lateral pressure model, predictions can be made for the effect of nonbilayer lipids on peripheral and integral membrane proteins. Recent data on the catalytic domain of Leader Peptidase and the potassium channel KcsA are discussed in relation to these predictions and in relation to the different models on the function of nonbilayer lipids. The data suggest a general mechanism for the interaction between nonbilayer lipids and membrane proteins via the membrane lateral pressure.     

Cell membranes: the lipid perspective

Although cell membranes are packed with proteins mingling with lipids, remarkably little is known about how proteins interact with lipids to carry out their function. Novel analytical tools are revealing the astounding diversity of lipids in membranes. The issue is now to understand the cellular functions of this complexity. In this Perspective, we focus on the interface of integral transmembrane proteins and membrane lipids in eukaryotic cells. Clarifying how proteins and lipids interact with each other will be important for unraveling membrane protein structure and function. Progress toward this goal will be promoted by increasing overlap between different fields that have so far operated without much crosstalk.     

Lipid-protein interactions and the function of the Ca2+-ATPase of sarcoplasmic reticulum

Regardless of the nature of the protein constituents of membranes, the molecular arrangement of lipids interacting with them must satisfy hydrophobic, ionic, and steric requirements. Biological membranes have a great diversity of lipid constituents, and this diversity might have functional roles. It has been proposed, for example, that the hydrophobic regions of membrane proteins are stabilized in the membrane through interactions with lipids able to adopt configurations other than the bilayer structure. Progress in understanding at the molecular level how lipid-protein interactions control the properties of membrane proteins has been hindered by the lack of information concerning the structure of the hydrophobic regions of membrane proteins. Nevertheless, there are many examples in the literature describing how changes in the lipid environment affect physical and biochemical properties of membrane proteins. From these studies, discussed in this review, an overall picture of how lipids and proteins interact in membranes is beginning to emerge.     

Endoplasmic Reticulum Structure and Interconnections with Other Organelles

The endoplasmic reticulum (ER) is a large, continuous membrane-bound organelle comprised of functionally and structurally distinct domains including the nuclear envelope, peripheral tubular ER, peripheral cisternae, and numerous membrane contact sites at the plasma membrane, mitochondria, Golgi, endosomes, and peroxisomes. These domains are required for multiple cellular processes, including synthesis of proteins and lipids, calcium level regulation, and exchange of macromolecules with various organelles at ER-membrane contact sites. The ER maintains its unique overall structure regardless of dynamics or transfer at ER-organelle contacts. In this review, we describe the numerous factors that contribute to the structure of the ER.The endoplasmic reticulum (ER) is a dynamic organelle responsible for many cellular functions, including the synthesis of proteins and lipids, and regulation of intracellular calcium levels. This review focuses on the distinct and complex morphology of the ER. The structure of the ER is complex because of the numerous distinct domains that exist within one continuous membrane bilayer. These domains are shaped by interactions with the cytoskeleton, by proteins that stabilize membrane shape, and by a homotypic fusion machinery that allows the ER membrane to maintain its continuity and identity. The ER also contains domains that contact the plasma membrane (PM) and other organelles including the Golgi, endosomes, mitochondria, lipid droplets, and peroxisomes. ER contact sites with other organelles and the PM are both abundant and dispersed throughout the cytoplasm, suggesting that they too could influence the overall architecture of the ER. As we will discuss here, ER shape and distribution are regulated by many intrinsic and extrinsic forces.     

Electron spin resonance in membrane research: protein-lipid interactions

Different electron spin resonance (ESR) methods are described that allow determination of the stoichiometry and selectivity of interaction of spin-labelled lipids with integral transmembrane peptides or proteins, and also with peripheral surface-binding membrane proteins or peptides. In addition, ESR methods for determining the exchange rates of spin-labelled lipids at the protein-lipid interface are described, as well as methods to detect penetration of surface-binding peptides into the hydrophobic membrane core. Instrumental requirements are considered, and also sample handling, spin-labelling techniques and the synthesis of spin-labelled lipids.     

Key phases in the formation of caveolae

Caveolae are abundant plasma membrane pits formed by the coordinated action of peripheral and integral membrane proteins and membrane lipids. Here, we discuss recent studies that are starting to provide a glimpse of how filamentous cavin proteins, membrane-embedded caveolin proteins, and specific plasma membrane lipids are brought together to make the unique caveola surface domain. Protein assembly involves multiple low-affinity interactions that are dependent on ‘fuzzy’ charge-dependent interactions mediated in part by disordered cavin and caveolin domains. We propose that cavins help generate a lipid domain conducive to full insertion of caveolin into the bilayer to promote caveola formation. The synergistic assembly of these dynamic protein complexes supports the formation of a metastable membrane domain that can be readily disassembled both in response to cellular stress and during endocytic trafficking. We present a mechanistic model for generation of caveolae based on these new insights.     

Interaction of membrane-spanning proteins with peripheral and lipid-anchored membrane proteins: perspectives from protein-lipid interactions (Review)

Studies of lipid-protein interactions in double-reconstituted systems involving both integral and peripheral or lipid-anchored proteins are reviewed. Membranes of dimyristoyl phosphatidylglycerol containing either myelin proteolipid protein or cytochrome c oxidase were studied. The partner peripheral proteins bound to these membranes were myelin basic protein or cytochrome c, respectively. In addition, the interactions between the myelin proteolipid protein and avidin that was membrane-anchored by binding to N-biotinyl phosphatidylethanolamine were studied in dimyristoyl phosphatidylcholine membranes. Steric exclusion plays a significant role when sizes of the peripheral protein and transmembrane domain of the integral protein are comparable. Even so, the effects on avidin-linked lipids are different from those induced by myelin basic protein on freely diffusible lipids, both interacting with the myelin proteolipid protein. Both the former and the cytochrome c/cytochrome oxidase couple evidence a propagation of lipid perturbation out from the intramembrane protein interface that could be a basis for formation of microdomains.     

Wrapping it up: the cell biology of myelination

During nervous system development, oligodendroglia in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) synthesise large amounts of specific proteins and lipids to generate myelin, a specialised membrane that spirally ensheathes axons and facilitates fast conduction of the action potential. Myelination is initiated after glial processes have attached to the axon and polarisation of the plasma membrane has been triggered. Myelin assembly is a multi-step process that occurs in spatially distinct regions of the cell. We propose that assembly of myelin proteins and lipids starts during their transport through the biosynthetic pathway and continues at the plasma membrane aided by myelin-basic protein (MBP). These sequential processes create the special lipid and protein composition necessary for myelin to perform its insulating function during nerve conduction.     

Structural and Functional Characterization of Human Peripheral Nervous System Myelin Protein P2

The myelin sheath is a tightly packed multilayered membrane structure insulating selected axons in the central and the peripheral nervous systems. Myelin is a biochemically unique membrane, containing a specific set of proteins. In this study, we expressed and purified recombinant human myelin P2 protein and determined its crystal structure to a resolution of 1.85 Å. A fatty acid molecule, modeled as palmitate based on the electron density, was bound inside the barrel-shaped protein. Solution studies using synchrotron radiation indicate that the crystal structure is similar to the structure of the protein in solution. Docking experiments using the high-resolution crystal structure identified cholesterol, one of the most abundant lipids in myelin, as a possible ligand for P2, a hypothesis that was proven by fluorescence spectroscopy. In addition, electrostatic potential surface calculations supported a structural role for P2 inside the myelin membrane. The potential membrane-binding properties of P2 and a peptide derived from its N terminus were studied. Our results provide an enhanced view into the structure and function of the P2 protein from human myelin, which is able to bind both monomeric lipids inside its cavity and membrane surfaces.     

Protein secretion in the Archaea: multiple paths towards a unique cell surface

Archaea are similar to other prokaryotes in most aspects of cell structure but are unique with respect to the lipid composition of the cytoplasmic membrane and the structure of the cell surface. Membranes of archaea are composed of glycerol-ether lipids instead of glycerol-ester lipids and are based on isoprenoid side chains, whereas the cell walls are formed by surface-layer proteins. The unique cell surface of archaea requires distinct solutions to the problem of how proteins cross this barrier to be either secreted into the medium or assembled as appendages at the cell surface.     

Functions of the tegument of schistosomes: clues from the proteome and lipidome

The tegumental outer-surface of schistosomes is a unique double membrane structure that is of crucial importance for modulation of the host response and parasite survival. Although several tegumental proteins had been identified by classical biochemical approaches, knowledge on the entire molecular composition of the tegument was limited. The Schistosoma mansoni genome project, together with recently developed proteomic and lipidomic techniques, allowed studies on detailed characterisation of the proteins and lipids of the tegumental membranes. These studies identified tegumental proteins and lipids that confirm the function of the tegument in nutrient uptake and immune evasion. However, these studies also demonstrated that compared to the complete worm, the tegument is enriched in lipids that are absent in the host. The tegument is also enriched in proteins that share no sequence similarity to any sequence present in databases of species other than schistosomes. These results suggest that the unique tegumental structures comprise multiple unique components that are likely to fulfil yet unknown functions. The tegumental proteome and lipidome, therefore, imply that many unknown molecular mechanisms are employed by schistosomes to survive within their host.     

The protein-lipid interface: perspectives from magnetic resonance and crystal structures

Lipid-protein interactions in membranes are dynamic, and consequently are well studied by magnetic resonance spectroscopy. More recently, lipids associated with integral membrane proteins have been resolved in crystals by X-ray diffraction, mostly at cryogenic temperatures. The conformation and chain ordering of lipids in crystals of integral proteins are reviewed here and are compared and contrasted with results from magnetic resonance and with the crystal structures of phospholipid bilayers. Various aspects of spin-label magnetic resonance studies on lipid interactions with single integral proteins are also reviewed: specificity for phosphatidylcholine, competition with local anaesthetics, oligomer formation of single transmembrane helices, and protein-linked lipid chains. Finally, the interactions between integral proteins and peripheral or lipid-linked proteins, as reflected by the lipid-protein interactions in double reconstitutions, are considered.     

Progress in understanding the role of lipids in membrane protein folding

Detailed investigations of membrane protein folding present a number of serious technical challenges. Most studies addressing this subject have emphasized aspects of protein amino acid sequence and structure. While it is generally accepted that the interplay between proteins and lipids plays an important role in membrane protein folding, the role(s) played by membrane lipids in this process have only recently been explored in any detail. This review is intended to summarize recent studies in which particular lipids or membrane physical properties have been shown to play a role in the folding of intact, functionally competent integral membrane proteins. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Stoichiometry of lipid interactions with transmembrane proteins--Deduced from the 3D structures

The stoichiometry of the first shell of lipids interacting with a transmembrane protein is defined operationally by the population of spin-labeled lipid chains whose motion is restricted directly by the protein. Interaction stoichiometries have been determined experimentally for a wide range of alpha-helical integral membrane proteins by using spin-label ESR spectroscopy. Here, we determine the spatially defined number of first-shell lipids at the hydrophobic perimeter of integral membrane proteins whose 3D structure has been determined by X-ray crystallography and lipid-protein interactions characterized by spin-labeling. Molecular modeling is used to build a single shell of lipids surrounding transmembrane structures derived from the PDB. Constrained energy optimization of the protein-lipid assemblies is performed by molecular mechanics. For relatively small proteins (up to 7-12 transmembrane helices), the geometrical first shell corresponds to that defined experimentally by perturbation of the lipid-chain dynamics. For larger, multi-subunit alpha-helical proteins, the lipids perturbed directly by the protein may either exceed or be less in number than those that can be accommodated at the intramembranous perimeter. In these latter cases, the motionally restricted spin-labeled lipids can be augmented by intercalation, or can correspond to a specific subpopulation at the protein interface, respectively. For monomeric beta-barrel proteins, the geometrical lipid stoichiometry corresponds to that determined from lipid mobility for a 22-stranded barrel, but fewer lipids are motionally restricted than can be accommodated around an eight-stranded barrel. Deviations from the geometrical first shell, in the beta-barrel case, are for the smaller protein with a highly curved barrel.     

Nucleotide sugar transporters of the Golgi apparatus

Glycosylation, sulfation and phosphorylation of proteins, proteoglycans and lipids occur in the lumen of the Golgi apparatus. The nucleotide substrates of these reactions must be first transported from the cytosol into the Golgi lumen by specific transporters. The topology and structure of these hydrophobic, multi-transmembrane-spanning proteins are beginning to be understood.