Changes In Lipid Environment Research Articles

Lipid packing in biological membranes governs protein localization and membrane permeability

Plasma membrane (PM) heterogeneity has long been implicated in various cellular functions. However, mechanistic principles governing functional regulations of lipid environment are not well understood due to the inherent complexities associated with the relevant length and timescales that limit both direct experimental measurements and their interpretation. In this context, computer simulations hold immense potential to investigate molecular-level interactions and mechanisms that lead to PM heterogeneity and its functions. Herein, we investigate spatial and dynamic heterogeneity in model membranes with coexisting liquid ordered and liquid disordered phases and characterize the membrane order in terms of the local topological changes in lipid environment using the nonaffine deformation framework. Furthermore, we probe the packing defects in these membranes, which can be considered as the conjugate of membrane order assessed in terms of the nonaffine parameter. In doing so, we formalize the connection between membrane packing and local membrane order and use that to explore the mechanistic principles behind their functions. Our observations suggest that heterogeneity in mixed phase membranes is a consequence of local lipid topology and its temporal evolution, which give rise to disparate lipid packing in ordered and disordered domains. This in turn governs the distinct nature of packing defects in these domains that can play a crucial role in preferential localization of proteins in mixed phase membranes. Furthermore, we observe that lipid packing also leads to contrasting distribution of free volume in the membrane core region in ordered and disordered membranes, which can lead to distinctive membrane permeability of small molecules. Our results, thus, indicate that heterogeneity in mixed phase membranes closely governs the membrane functions that may emerge from packing-related basic design principles.

Subtle Changes in Lipid Environment Have Profound Effects on Membrane Oxidation Chemistry.

Nature carefully designs the components of amphiphile-composed monolayer and bilayer membranes to deliver specific functions. The compositions of these interfacial layered structures are so delicate that minute modifications can result in huge changes in function. Great effort has been expended to understand membrane physical properties, with only minimum attention given to associated chemical properties. Here we report the first examples of the delicate chemistry associated with membrane amphiphilic components by studying OH-mediated oxidation of six different unsaturated lipids/surfactants and their mixtures at the air-water interface using field-induced droplet ionization mass spectrometry (FIDI-MS). When the packing is loose or perturbed to be loose by other components or prior chemical modification, the double bond is oxidized without cleavage by adding oxygen functionality. In contrast, compact packing results in double bond cleavage through a Criegee intermediate mechanism. We postulate that constrained environments imposed by lipid packing limit the conformations of the reaction intermediates, controlling reaction pathways.

Dynamic membrane protein topological switching upon changes in phospholipid environment

A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. Establishing the principles governing membrane protein folding is central to understanding the molecular basis for membrane proteins that display multiple topologies, the intrinsic dynamic organization of membrane proteins, and membrane protein conformational disorders resulting in disease. We previously established that lactose permease of Escherichia coli displays a mixture of topological conformations and undergoes postassembly bidirectional changes in orientation within the lipid bilayer triggered by a change in membrane phosphatidylethanolamine content, both in vivo and in vitro. However, the physiological implications and mechanism of dynamic structural reorganization of membrane proteins due to changes in lipid environment are limited by the lack of approaches addressing the kinetic parameters of transmembrane protein flipping. In this study, real-time fluorescence spectroscopy was used to determine the rates of protein flipping in the lipid bilayer in both directions and transbilayer flipping of lipids triggered by a change in proteoliposome lipid composition. Our results provide, for the first time to our knowledge, a dynamic picture of these events and demonstrate that membrane protein topological rearrangements in response to lipid modulations occur rapidly following a threshold change in proteoliposome lipid composition. Protein flipping was not accompanied by extensive lipid-dependent unfolding of transmembrane domains. Establishment of lipid bilayer asymmetry was not required but may accelerate the rate of protein flipping. Membrane protein flipping was found to accelerate the rate of transbilayer flipping of lipids.

Local changes in lipid environment of T cell receptor microclusters regulate membrane binding by the CD3epsilon cytoplasmic domain (P1103)

Abstract The CD3epsilon and zeta cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane, and a previous NMR structure showed that both tyrosines of the CD3epsilon Immuno-tyrosine based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3epsilon residues were previously shown to be essential for CD3epsilon and zeta membrane binding. Phosphatidylserine is the most abundant negatively charged lipid on the inner leaflet of the plasma membrane and makes a major contribution to membrane binding by the CD3epsilon cytoplasmic domain. Here we show that T cell receptor triggering by peptide-MHC complexes induces dissociation of the CD3epsilon cytoplasmic domain from the plasma membrane. Release of the CD3epsilon cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and phosphatidylserine density in T cell receptor microclusters. The localized reduction in phosphatidylserine density is explained by reduced diffusion of this lipid into synapses. These changes in the lipid composition of T cell receptor microclusters even occur when receptor signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of the microclusters thus render the CD3epsilon cytoplasmic domain accessible during early stages of T cell activation.

Local changes in lipid environment of TCR microclusters regulate membrane binding by the CD3ε cytoplasmic domain.

The CD3ε and ζ cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane (PM), and a previous nuclear magnetic resonance structure showed that both tyrosines of the CD3ε immunoreceptor tyrosine-based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3ε residues were previously shown to be essential for CD3ε and ζ membrane binding. Phosphatidylserine (PS) is the most abundant negatively charged lipid on the inner leaflet of the PM and makes a major contribution to membrane binding by the CD3ε cytoplasmic domain. Here, we show that TCR triggering by peptide–MHC complexes induces dissociation of the CD3ε cytoplasmic domain from the plasma membrane. Release of the CD3ε cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and available PS in TCR microclusters. These changes in the lipid composition of TCR microclusters even occur when TCR signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of TCR microclusters thus render the CD3ε cytoplasmic domain accessible during early stages of T cell activation.

Lipid Composition Regulates the Orientation of Transmembrane Helices in HorA, an ABC Multidrug Transporter

ATP-binding cassette (ABC) transporters constitute a large class of molecular pumps whose central role in chemotherapy resistance has highlighted their clinical relevance. We investigated whether the lipid composition of the membrane affects the function and structure of HorA, a bacterial ABC multidrug transporter. When the transporter was reconstituted in a bilayer where phosphatidylethanolamine (PE), the main lipid of the bacterial membrane, was replaced with phosphatidylcholine (PC), ATP hydrolysis and substrate transport became uncoupled. Although ATPase activity was maintained, HorA lost its ability to extrude the prototypical substrate Hoechst33342. Attenuated Total Reflection-Fourier Transform Infrared spectroscopy (ATR-FTIR) revealed that, although the secondary structure of the protein was unaffected, the orientation of the transmembrane helices (TM) was modified by the change in lipid composition. The orientation of the backbone carbonyls indicated that the helices opened wider in PE versus PC-containing liposomes, with 10 degrees difference. This was supported by hydrogen/deuterium exchange studies showing increased protection of the backbone from the solvent in PC-containing liposomes. Electron Paramagnetic Resonance was used to further probe the structural change. In the PC-containing liposomes we observed increased mobility of the spin label in TM4, along with increased exposure to molecular oxygen, used as a hydrophobic quencher. This indicates that the lipid change induced modification of the orientation of TM4, exposing Cys-180 to the lipid phase. The lipid composition of the bilayer thus modulates the structure of HorA, and in turn its ability to extrude its substrates.

Effect of cellular inositol content on ethanol tolerance of Saccharomyces cerevisiae in sake brewing

The effect of cellular inositol content on the ethanol tolerance of sake yeast was investigated. In a static culture of strain K901 in a synthetic medium, when cells were grown in the presence of inositol in limited amount (L-cells), the inositol content of cells decreased by one-third that of cells grown in the presence of inositol in sufficient amount (H-cells). L-cells exhibited a higher death rate constant than H-cells in the presence of 12–20% ethanol, while no difference in specific ethanol production rate in the presence of 0–18% ethanol between the two cell types was observed. L-cells leaked more intracellular components, such as nucleotides, phosphate and potassium, in the presence of ethanol than H-cells. L-cells exhibited a lower intracellular pH value than H-cells, which represented the lowering of cell vitality by the decrease in H + extrusion activity. Furthermore, the plasma membrane H +-ATPase activity of L-cells was approximately one-half of that of H-cells. Therefore, it was considered that the decrease in viability in the presence of ethanol due to inositol limitation results from the lowering of H +-ATPase activity, which maintains the permeability barrier of the yeast membrane, ensuring the homeostasis of ions in the cytoplasm of yeast cells. It is assumed that the lowering of H +-ATPase activity due to inositol limitation is caused by the change in lipid environment of the enzyme, which is affected by inositol-containing glycerophospholipids such as phosphatidylinositol (PI), because in the PI-saturated mixed micellar assay system, the difference in H +-ATPase activity between L- and H-cells disappeared. In the early stage of sake mash, inositol limitation lowers the ethanol tolerance due to the decrease in H +-ATPase activity as in static culture. In the final stage of sake mash, the disruption of the ino1 gene responsible for inositol synthesis, resulted in a decrease in cell density. Furthermore, the ino1 disruptant, which was not capable of increasing the cellular inositol level in the final stage, exhibited a significantly higher methylene blue-staining ratio than the parental strain. It was suggested that the yeast cellular inositol level is one of the important factors which contribute to the high ethanol tolerance implied by the increased cell viability in the presence of ethanol.

Effect of Cellular Inositol Content on Ethanol Tolerance of Saccharomyces cerevisiae in Sake Brewing

The effect of cellular inositol content on the ethanol tolerance of sake yeast was investigated. In a static culture of strain K901 in a synthetic medium, when cells were grown in the presence of inositol in limited amount (L-cells), the inositol content of cells decreased by one-third that of cells grown in the presence of inositol in sufficient amount (H-cells). L-cells exhibited a higher death rate constant than H-cells in the presence of 12–20% ethanol, while no difference in specific ethanol production rate in the presence of 0–18% ethanol between the two cell types was observed. L-cells leaked more intracellular components, such as nucleotides, phosphate and potassium, in the presence of ethanol than H-cells. L-cells exhibited a lower intracellular pH value than H-cells, which represented the lowering of cell vitality by the decrease in H+ extrusion activity. Furthermore, the plasma membrane H+-ATPase activity of L-cells was approximately one-half of that of H-cells. Therefore, it was considered that the decrease in viability in the presence of ethanol due to inositol limitation results from the lowering of H+-ATPase activity, which maintains the permeability barrier of the yeast membrane, ensuring the homeostasis of ions in the cytoplasm of yeast cells. It is assumed that the lowering of H+-ATPase activity due to inositol limitation is caused by the change in lipid environment of the enzyme, which is affected by inositol-containing glycerophospholipids such as phosphatidylinositol (PI), because in the PI-saturated mixed micellar assay system, the difference in H+-ATPase activity between L- and H-cells disappeared. In the early stage of sake mash, inositol limitation lowers the ethanol tolerance due to the decrease in H+-ATPase activity as in static culture. In the final stage of sake mash, the disruption of the ino1 gene responsible for inositol synthesis, resulted in a decrease in cell density. Furthermore, the ino1 disruptant, which was not capable of increasing the cellular inositol level in the final stage, exhibited a significantly higher methylene blue-staining ratio than the parental strain. It was suggested that the yeast cellular inositol level is one of the important factors which contribute to the high ethanol tolerance implied by the increased cell viability in the presence of ethanol.

Effects of membrane lipids on ion channel structure and function.

Biologic membranes are not simply inert physical barriers, but complex and dynamic environments that affect membrane protein structure and function. Residing within these environments, ion channels control the flux of ions across the membrane through conformational changes that allow transient ion flux through a central pore. These conformational changes may be modulated by changes in transmembrane electrochemical potential, the binding of small ligands or other proteins, or changes in the local lipid environment. Ion channels play fundamental roles in cellular function and, in higher eukaryotes, are the primary means of intercellular signaling, especially between excitable cells such as neurons. The focus of this review is to examine how the composition of the bilayer affects ion channel structure and function. This is an important consideration because the bilayer composition varies greatly in different cell types and in different organellar membranes. Even within a membrane, the lipid composition differs between the inner and outer leaflets, and the composition within a given leaflet is both heterogeneous and highly dynamic. Differential packing of lipids (and proteins) leads to the formation of microdomains, and lateral diffusion of these microdomains or "lipid rafts" serve as mobile platforms for the clustering and organization of bilayer constituents including ion channels. The structure and function of these channels are sensitive to specific chemical interactions with neighboring components of the membrane and also to the biophysical properties of their membrane microenvironment (e.g., fluidity, lateral pressure profile, and bilayer thickness). As specific examples, we have focused on the K+ ion channels and the ligand-gated nicotinicoid receptors, two classes of ion channels that have been well-characterized structurally and functionally. The responsiveness of these ion channels to changes in the lipid environment illustrate how ion channels, and more generally, any membrane protein, may be regulated via cellular control of membrane composition.

Effect of photoperiod and gibberellic acid on the plasma membrane H<sup>+</sup>ATPase activity of spinach leaves

Plasma membrane H+ATPase extracted from leaves of spinach plants induced to flower by gibberellic acid treatments or by a transfer to a photoperiod of 24 h had a lower Kmapp than that from vegetative plants grown in short days. The Kmapp obtained after inhibition by vanadate was decreased in vegetative plants and increased in induced ones showing a differential effect of this inhibitor on the kinetic properties of the enzyme between vegetative and induced plants. The phospholipid fatty acid analysis of the purified plasma membrane showed an increase of C18:1/C18:2 fatty acid ratio upon induction by light or by gibberellic acid treatments, whereas the saturated to unsaturated fatty acid ratio was kept constant. The decrease in the Kmapp observed after induction may be thus interpreted in terms of the observed changes in lipid environment.