Study of polytopic membrane protein topological organization as a function of membrane lipid composition.

A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM). SCAM is adapted to follow changes in membrane protein topology as a function of changes in membrane lipid composition. The strategy described can be adapted to any membrane system.

Lipid-dependent topogenesis and function of membrane proteins

Membranes are essential for the integrity and function of the cell. The collective property of the lipid bilayer is critical in providing an optimal functioning environment for membrane proteins. The simple yet well-characterized bacterium Escherichia coli serves an ideal model system to study the function of specific lipids since its lipid content can be easily manipulated. The most abundant lipid in E. coli membrane is phosphatidylethanolamine (PE, 70-80%). A PE-lacking E. coli mutant displays a complex mixture of deficient phenotypes, suggesting a profound role for PE in different aspects of cell function. A novel role of PE as a topological and functional determinant for membrane proteins has been established using lactose permease (LacY) as a model protein. PE is found to be required for energy-dependent uphill transport process of LacY. In PE-lacking membranes, LacY undergoes a dramatic conformational change, and the first half of the protein adopts an inverted topology with respect to the bilayer plane. ^ The work reported here was initiated to understand the molecular properties of lipids that enable their function as topological and functional determinants for membrane proteins. A glycolipid, monoglucosyldiacylglycerol (MGlcDAG) which shares physicochemical similarities with PE, was introduced to PE-lacking E. coli membranes. The introduction of MGlcDAG suppresses many of the PE-deficient phenotypes, and in particular supports the function and native topology of LacY. ^ The lipid-sensitive topogenic signals encoded in the amino acid sequence of LacY were also identified. Native LacY adopts an inverted topology when synthesized without PE, but mutation of specific acidic residues in the cytoplasmic extra-membrane domains can prevent this inversion and supports a native topological organization of LacY in PE-lacking membranes. These results suggest that it is the interplay between the collective charge properties of the lipid bilayer and extra-membrane loops of protein that determines the final orientation of transmembrane domains. By comparing the similarities as well as differences between these two lipids, we established how specific physical and chemical properties of lipids influence various cell functions and elucidated the molecular basis for the novel role of lipids in determining membrane protein topology. ^

Self-assembly of fibronectin into fibrillar networks underneath dipalmitoyl phosphatidylcholine monolayers: Role of lipid matrix and tensile forces

The cell-mediated assembly of fibronectin (Fn) into fibrillar matrices is a complex multistep process that is incompletely understood because of the chemical complexity of the extracellular matrix and a lack of experimental control over molecular interactions and dynamic events. We have identified conditions under which Fn assembles into extended fibrillar networks after adsorption to a dipalmitoyl phosphatidylcholine (DPPC) monolayer in contact with physiological buffer. We propose a sequential model for the Fn assembly pathway, which involves the orientation of Fn underneath the lipid monolayer by insertion into the liquid expanded (LE) phase of DPPC. Attractive interactions between these surface-anchored proteins and the liquid condensed (LC) domains leads to Fn enrichment at domain edges. Spontaneous self-assembly into fibrillar networks, however, occurs only after expansion of the DPPC monolayer from the LC phase though the LC/LE phase coexistence. Upon monolayer expansion, the domain boundaries move apart while attractive interactions among Fn molecules and between Fn and domain edges produce a tensile force on the proteins that initiates fibril assembly. The resulting fibrils have been characterized in situ by using fluorescence and light-scattering microscopy. We have found striking similarities between fibrils produced under DPPC monolayers and those found on cellular surfaces, including their assembly pathways.

High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes

The EPR spectra of spin-labeled lipid chains in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine containing 40 mol % of cholesterol have been studied in the liquid-ordered phase at a microwave radiation frequency of 94 GHz. At such high field strengths, the spectra should be optimally sensitive to lateral chain ordering that is expected in the formation of in-plane domains. The high-field EPR spectra from random dispersions of the cholesterol-containing membranes display very little axial averaging of the nitroxide g-tensor anisotropy for lipids spin labeled toward the carboxyl end of the sn-2 chain (down to the 8-C atom). For these positions of labeling, anisotropic 14N-hyperfine splittings are resolved in the gzz and gyy regions of the nonaxial EPR spectra. For positions of labeling further down the lipid chain, toward the terminal methyl group, the axial averaging of the spectral features systematically increases and is complete at the 14-C atom position. Concomitantly, the time-averaged 〈Azz〉 element of the 14N-hyperfine tensor decreases, indicating that the axial rotation at the terminal methyl end of the chains arises from correlated torsional motions about the bonds of the chain backbone, the dynamics of which also give rise to a differential line broadening of the 14N-hyperfine manifolds in the gzz region of the spectrum. These results provide an indication of the way in which lateral ordering of lipid chains in membranes is induced by cholesterol.

Equilibration rates in lipid monolayers

A theoretical analysis is given for the rate of change of domain sizes in lipid monolayers at the air–water interface. The calculation is applicable to liquid domains formed from binary mixtures of lipids that form two coexisting liquid phases. Under conditions where the two lipid molecules have approximately equal areas, the equilibration rate does not involve macroscopic hydrodynamic flow in the subphase but rather depends on the diffusion coefficient of the lipid molecules. The calculation shows that the equilibration rate in binary mixtures of cholesterol and phosphatidylcholine is remarkably slow, the radius of a typical 20-μm diameter domain changing by as little as a part in a million per second. Under these circumstances, equilibration times of the order of days or weeks are expected. Even with such long times, the final state reached by the monolayer will in general be a state of metastable equilibrium, rather than true equilibrium.

VIP17/MAL, a lipid raft-associated protein, is involved in apical transport in MDCK cells

Apical proteins are sorted and delivered from the trans-Golgi network to the plasma membrane by a mechanism involving sphingolipid–cholesterol rafts. In this paper, we report the effects of changing the levels of VIP17/MAL, a tetraspan membrane protein localized to post-Golgi transport containers and the apical cell surface in MDCK cells. Overexpression of VIP17/MAL disturbed the morphology of the MDCK cell layers by increasing apical delivery and seemingly expanding the apical cell surface domains. On the other hand, expression of antisense RNA directed against VIP17/MAL caused accumulation in the Golgi and/or impaired apical transport of different apical protein markers, i.e., influenza virus hemagglutinin, the secretory protein clusterin (gp80), the transmembrane protein gp114, and a glycosylphosphatidylinositol-anchored protein. However, antisense RNA expression did not affect the distribution of E-cadherin to the basolateral surface. Because VIP17/MAL associates with sphingolipid–cholesterol rafts, these data provide functional evidence that this protein is involved in apical transport and might be a component of the machinery clustering lipid rafts with apical cargo to form apical transport carriers.

Core lipid structure is a major determinant of the oxidative resistance of low density lipoprotein.

The influence of thermally induced changes in the lipid core structure on the oxidative resistance of discrete, homogeneous low density lipoprotein (LDL) subspecies (d, 1.0297-1.0327 and 1.0327-1.0358 g/ml) has been evaluated. The thermotropic transition of the LDL lipid core at temperatures between 15 degrees C and 37 degrees C, determined by differential scanning calorimetry, exerted significant effects on the kinetics of copper-mediated LDL oxidation expressed in terms of intrinsic antioxidant efficiency (lag time) and diene production rate. Thus, the temperature coefficients of oxidative resistance and maximum oxidation rate showed break points at the core transition temperature. Temperature-induced changes in copper binding were excluded as the molecular basis of such effects, as the saturation of LDL with copper was identical below and above the core transition. At temperatures below the transition, the elevation in lag time indicated a greater resistance to oxidation, reflecting a higher degree of antioxidant protection. This effect can be explained by higher motional constraints and local antioxidant concentrations, the latter resulting from the freezing out of antioxidants from crystalline domains of cholesteryl esters and triglycerides. Below the transition temperature, the conjugated diene production rate was decreased, a finding that correlated positively with the average size of the cooperative units of neutral lipids estimated from the calorimetric transition width. The reduced accessibility and structural hindrance in the cluster organization of the core lipids therefore inhibits peroxidation. Our findings provide evidence for a distinct effect of the dynamic state of the core lipids on the oxidative susceptibility of LDL and are therefore relevant to the atherogenicity of these cholesterol-rich particles.

Association of stomatin with lipid bodies

The oligomeric lipid raft-associated integral protein stomatin normally localizes to the plasma membrane and the late endosomal compartment. Similar to the caveolins, it is targeted to lipid bodies (LBs) on overexpression. Endogenous stomatin also associates with LBs to a small extent. Green fluorescent protein-tagged stomatin (StomGFP) and the dominant-negative caveolin-3 mutant DGV(cav3)(HA) occupy distinct domains on LB surfaces but eventually intermix. Studies of StomGFP deletion mutants reveal that the region for membrane association but not oligomerization and raft association is essential for LB targeting. Blocking protein synthesis leads to the redistribution of StomGFP from LBs to LysoTracker-positive vesicles indicating a connection with the late endosomal/ lysosomal pathway. Live microscopy of StomGFP reveals multiple interactions between LBs and microtubule-associated vesicles possibly representing signaling events and/or the exchange of cargo. Proteomic analysis of isolated LBs identifies adipophilin and TIP47, various lipid-specific enzymes, cytoskeletal components, chaperones, Ras-related proteins, protein kinase D2, and other regulatory proteins. The association of the Rab proteins 1, 6, 7, 10, and 18 with LBs indicates various connections to other compartments. Our data suggest that LBs are not only involved in the storage of lipids but also participate actively in the cellular signaling network and the homeostasis of lipids.

Domains of the TGN: Coats, tethers and G proteins

The trans-Golgi network is the major sorting compartment of the secretory pathway for protein, lipid and membrane traffic. There is a constant flow of membrane and cargo to and from this compartment. Evidence is emerging that the trans-Golgi network has multiple biochemically and functionally distinct subdomains, each of which contributes to the combined sorting and transport requirements of this dynamic compartment. The recruitment of distinct arrays of protein complexes to trans-Golgi network membranes is likely to produce the diversity of structure and biochemistry observed amongst subdomains that serve to generate different carriers or maintain resident trans-Golgi network components. This review discusses how these subdomains may be formed and examines the molecular players involved, including G proteins, clathrin adaptors and golgin tethers. Diversity within these protein families is highlighted and shown to be critical for the functionality of the trans-Golgi network, as a mediator of protein sorting and membrane transport, and for the maintenance of Golgi structure.

ROR alpha regulates the expression of genes involved in lipid homeostasis in skeletal muscle cells - Caveolin-3 and CPT-1 are direct targets of ROR

The staggerer mice carry a deletion in the RORalpha gene and have a prolonged humoral response, overproduce inflammatory cytokines, and are immunodeficient. Furthermore, the staggerer mice display lowered plasma apoA-I/-II, decreased plasma high density lipoprotein cholesterol and triglycerides, and develop hypo-alpha-lipoproteinemia and atherosclerosis. However, relatively little is known about RORalpha in the context of target tissues, target genes, and lipid homeostasis. For example, RORalpha is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for similar to40% of total body weight and 50% of energy expenditure. This lean tissue is a primary site of glucose disposal and fatty acid oxidation. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. In particular, the role of RORalpha in skeletal muscle metabolism has not been investigated, and the contribution of skeletal muscle to the ROR-/- phenotype has not been resolved. We utilize ectopic dominant negative RORalpha expression in skeletal muscle cells to understand the regulatory role of RORs in this major mass peripheral tissue. Exogenous dominant negative RORalpha expression in skeletal muscle cells represses the endogenous levels of RORalpha and -gamma mRNAs and ROR-dependent gene expression. Moreover, we observed attenuated expression of many genes involved in lipid homeostasis. Furthermore, we show that the muscle carnitine palmitoyltransferase-1 and caveolin-3 promoters are directly regulated by ROR and coactivated by p300 and PGC-1. This study implicates RORs in the control of lipid homeostasis in skeletal muscle. In conclusion, we speculate that ROR agonists would increase fatty acid catabolism in muscle and suggest selective activators of ROR may have therapeutic utility in the treatment of obesity and atherosclerosis.

Lipid rafts and plasma membrane microorganization: insights from Ras

The spatial organization of plasma membrane components in discrete microdomains is thought to be a key factor in the generation of distinct signal outputs. A detailed characterization of plasma membrane microdomains, including descriptions of their size, dynamics and abundance, has proved to be a taxing problem for cell biologists and biophysicists. The use of novel techniques is providing exciting new insights into the challenging problem of plasma membrane microstructure and has allowed the visualization of domains with the characteristics expected of lipid rafts - microdomains of the plasma membrane enriched in cholesterol and sphingolipids. This review focuses on some of these recent advances and uses Ras signaling as a paradigm for understanding inner plasma membrane organization and the role of lipid rafts in cellular function.

Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane

The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.

Regulated localization of rab18 to lipid droplets - Effects of lipolytic stimulation and inhibition of lipid droplet catabolism

Rab GTPases are crucial regulators of membrane traffic. Here we have examined a possible association of Rab proteins with lipid droplets (LDs), neutral lipid-containing organelles surrounded by a phospholipid monolayer, also known as lipid bodies, which have been traditionally considered relatively inert storage organelles. Although we found close apposition between LDs and endosomal compartments labeled by expressed Rab5, Rab7, or Rab11 constructs, there was no detectable labeling of the LD surface itself by these Rab proteins. In contrast, GFP-Rab18 localized to LDs and immunoelectron microscopy showed direct association with the monolayer surface. Green fluorescent protein (GFP)-Rab18-labeled LDs underwent oscillatory movements in a localized area as well as sporadic, rapid, saltatory movements both in the periphery of the cell and toward the perinuclear region. In both adipocytes and non-adipocyte cell lines Rab18 localized to a subset of LDs. To gain insights into this specific localization, Rab18 was co-expressed with Cav3(DGV), a truncation mutant of caveolin-3 shown to inhibit the catabolism and motility of lipid droplets. GFP-Rab18 and mRFP-Cav3(DGV) labeled mutually exclusive subpopulations of LDs. Moreover, in 3T3-L1 adipocytes, stimulation of lipolysis increased the localization of Rab18 to LDs, an effect reversed by beta-adrenergic antagonists. These results show that a Rab protein localizes directly to the monolayer surface of LDs. In addition, association with the LD surface was increased following stimulation of lipolysis and inhibited by a caveolin mutant suggesting that recruitment of Rab18 is regulated by the metabolic state of individual LDs.

Lipid droplets: a unified view of a dynamic organelle

Lipid droplets form the main lipid store in eukaryotic cells. Although all cells seem to be able to generate lipid droplets, their biogenesis, regulatory mechanisms and interactions with other organelles remain largely elusive. In this article, we outline some of the recent developments in lipid droplet cell biology. We show the mobile and dynamic nature of this organelle, and advocate the adoption of a unified nomenclature to consolidate terminology in this emerging field.

Structure and lipid interactions of membrane-associated glycosyltransferases : Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine.