ATP-binding-cassette transporter 1 (ABC1) has been implicated in processes related to membrane-lipid turnover. Here, using in vivo loss-of-function and in vitro gain-of-function models, we show that ABC1 promotes Ca2+-induced exposure of phosphatidylserine at the membrane, as determined by a prothrombinase assay, membrane microvesiculation and measurement of transbilayer redistribution of spin-labelled phospholipids. That ABC1 promotes engulfment of dead cells is shown by the impaired ability of ABC1-deficient macrophages to engulf apoptotic preys and by the acquisition of phagocytic behaviour by ABC1 transfectants. Release of membrane phospholipids and cholesterol to apo-AI, the protein core of the cholesterol-shuttling high-density lipoprotein (HDL) particle, is also ABC1-dependent. We propose that both the efficiency of apoptotic-cell engulfment and the efflux of cellular lipids depend on ABC1-induced perturbation of membrane phosphatidylserine turnover. Transient local exposure of anionic phospholipids in the outer membrane leaflet may be sufficient to alter the general properties of the membrane and thus influence discrete physiological functions.

Membrane rafts are thought to be sphingolipid- and cholesterol-dependent lateral assemblies involved in diverse cellular functions. Their biological roles and even their existence, however, remain controversial. Using an original fluorescence correlation spectroscopy strategy that recently enabled us to identify nanoscale membrane organizations in live cells, we report here that highly dynamic nanodomains exist in both the outer and inner leaflets of the plasma membrane. Through specific inhibition of biosynthesis, we show that sphingolipids and cholesterol are essential and act in concert for formation of nanodomains, thus corroborating their raft nature. Moreover, we find that nanodomains play a crucial role in triggering the phosphatidylinositol-3 kinase/Akt signaling pathway, by facilitating Akt recruitment and activation upon phosphatidylinositol-3,4,5-triphosphate accumulation in the plasma membrane. Thus, through direct monitoring and controlled alterations of rafts in living cells, we demonstrate that rafts are critically involved in the activation of a signaling axis that is essential for cell physiology.

The identification of defects in ABCA1 as the molecular basis of Tangier disease has highlighted its crucial role in the loading with phospholipids and cholesterol of nascent apolipoprotein particles. Indeed the expression of ABCA1 affects apolipoprotein A-I (apoA-I)-mediated removal of lipids from cell membranes, and the possible role of ABCA1 as an apoA-I surface receptor has been recently suggested. In the present study, we have investigated the role of the ABCA1 transporter as an apoA-I receptor with the analysis of a panel of transfectants expressing functional or mutant forms of ABCA1. We provide experimental evidence that the forced expression of a functional ABCA1 transporter confers surface competence for apoA-I binding. This, however, appears to be dependent on ABCA1 function. Structurally intact but ATPase-deficient forms of the transporter fail to elicit a specific cell association of the ligand. In addition the diffusion parameters of membrane-associated apoA-I indicate an interaction with membrane lipids rather than proteins. These results do not support a direct molecular interaction between ABCA1 and apoA-I, but rather suggest that the ABCA1-induced modification of the lipid distribution in the membrane, evidenced by the phosphatidylserine exofacial flopping, generates a biophysical microenvironment required for the docking of apoA-I at the cell surface.The removal of cellular lipids is promoted by high density lipoproteins (HDL), 1 the plasma shuttle mediating reverse cholesterol transport from peripheral tissues to the liver for further uptake and metabolism (1). However, whether the interaction of the lipid-poor apoA-I particle, protein core of the nascent HDL, with cell membranes is mediated by a specific receptor and how its loading with phospholipids and cholesterol occurs is still a matter of debate (2). The recent discovery that a defective ABCA1 transporter leads to Tangier disease (3-9) has directly implicated this transmembrane protein in the active release of cellular lipids and prompted an investigation into its role as a candidate apoA-I receptor (10, 11). Indeed a correlation between the cAMP-induced cell surface apoA-I binding and the expression of ABCA1 in macrophage-like cell lines has been reported (12). Very recently, in addition, a direct molecular interaction between ABCA1 and apoA-I at the cell surface has been proposed on the basis of chemical cross-linking experiments (10, 11). To gain further insight into this issue, we developed an apoA-I binding assay based on the use of a fluorochrome-conjugated ligand. The analysis of apoA-I binding to a panel of transfectants expressing either functionally intact or defective ABCA1 proteins (13) led us to exclude that the transporter behaves as a bona fide receptor for apoA-I. Indeed, whereas surface binding increases with the expression of a functional ABCA1, the expression of structurally intact but functionally impaired ABCA1 proteins fails to elicit specific binding. Considering that, as previously demonstrated, ABCA1 promotes t...

The transport of specific molecules across lipid membranes is an essential function of all living organisms and a large number of specific transporters have evolved to carry out this function. The largest transporter gene family is the ATP-binding cassette (ABC) transporter superfamily. These proteins translocate a wide variety of substrates including sugars, amino acids, metal ions, peptides, and proteins, and a large number of hydrophobic compounds and metabolites across extra-and intracellular membranes. ABC genes are essential for many processes in the cell, and mutations in these genes cause or contribute to several human genetic disorders including cystic fibrosis, neurological disease, retinal degeneration, cholesterol and bile transport defects, anemia, and drug response. Characterization of eukaryotic genomes has allowed the complete identification of all the ABC genes in the yeast Saccharomyces cerevisiae , Drosophila , and C. elegans genomes. To date, there are 48 characterized human ABC genes. The genes can be divided into seven distinct subfamilies, based on organization of domains and amino acid homology. Many ABC genes play a role in the maintenance of the lipid bilayer and in the transport of fatty acids and sterols within the body. Here, we review the current knowledge of the human ABC genes, their role in inherited disease, and understanding of the topology of these genes within the membrane. -Dean, M., Y. Hamon, and G. Chimini. The human ATP-binding cassette (ABC) transporter superfamily.

Abstract. High-frequency, long-term and multisolute measurements are required to assess the impact of human pressures on water quality due to (i) the high temporal and spatial variability of climate and human activity and (ii) the fact that chemical solutes combine short-and long-term dynamics. Such data series are scarce. This study, based on an original and unpublished time series from the Kervidy-Naizin headwater catchment (Brittany, France), aims to determine solute transfer processes and dynamics that characterise this strongly human-impacted catchment.The Kervidy-Naizin catchment is a temperate, intensive agricultural catchment, hydrologically controlled by shallow groundwater. Over 10 yr, five solutes (nitrate, sulphate, chloride, and dissolved organic and inorganic carbon) were monitored daily at the catchment outlet and roughly every four months in the shallow groundwater.The concentrations of all five solutes showed seasonal variations but the patterns of the variations differed from one solute to another. Nitrate and chloride exhibit rather smooth variations. In contrast, sulphate as well as organic and inorganic carbon is dominated by flood flushes. The observed nitrate and chloride patterns are typical of an intensive agricultural catchment hydrologically controlled by shallow groundwater. Nitrate and chloride originating mainly from organic fertilisers accumulated over several years in the shallow groundwater. They are seasonally exported when upland groundwater connects with the stream during the wet season. Conversely, sulphate as well as organic and inorganic carbon patterns are not specific to agricultural catchments. These solutes do not come from fertilisers and do not accumulate in soil or shallow groundwater; instead, they are biogeochemically produced in the catchment. The results allowed development of a generic classification system based on the specific temporal patterns and source locations of each solute. It also considers the stocking period and the dominant process that limits transport to the stream, i.e. the connectivity of the stocking compartment. This mechanistic classification can be applied to any chemical solute to help assess its origin, storage or production location and transfer mechanism in similar catchments.

Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-γ receptor (IFN-γR) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-γR2 subunit, this mutation blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-γ could not occur. Removing IFN-γR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo. VIDEO ABSTRACT.

The ATP binding cassette transporter ABC1 is a 220-kDa glycoprotein expressed by macrophages and required for engulfment of cells undergoing programmed cell death. Since members of this family of proteins such as P-glycoprotein and cystic fibrosis transmembrane conductance regulator share the ability to transport anions, we have investigated the transport capability of ABC1 expressed in Xenopus oocytes using iodide efflux and voltage-clamp techniques. We report here that ABC1 generates an anion flux sensitive to glibenclamide, sulfobromophthalein, and blockers of anion transporters. The anion flux generated by ABC1 is upregulated by orthovanadate, cAMP, protein kinase A, and okadaic acid. In other ABC transporters, mutating the conserved lysine in the nucleotide binding folds was found to severely reduce or abolish hydrolysis of ATP, which in turn altered the activity of the transporter. In ABC1, replacement of the conserved lysine 1892 in the Walker A motif of the second nucleotide binding fold increased the basal ionic flux, did not alter the pharmacological inhibitory profile, but abolished the response to orthovanadate and cAMP agonists. Therefore, we conclude that ABC1 is a cAMP-dependent and sulfonylureasensitive anion transporter.

The production of interleukin-1β (IL-1β), a powerful mediator of inflammation, is tightly regulated at several levels. However, in some pathologic conditions, a pharmacologic treatment is required to control the toxicity of excessive extracellular IL-1β. Because of the heavy side effects of most therapies used in IL-1β–mediated pathologies, a goal of pharmacologic research is the development of selective anti–IL-1β drugs. We show here that the sulfonylurea glyburide, currently used in the oral therapy of noninsulin dependent diabetes, is an inhibitor of IL-1β secretion from human monocytes and mouse macrophages. Glyburide reduces dramatically the recovery of extracellular 17-kD IL-1β in the absence of toxic effects on the cells and without affecting the synthesis or processing of the IL-1β precursor. IL-1β belongs to the family of leaderless secretory proteins released from the cell by a nonclassical secretory route. In bacteria and yeast Atp binding cassette (ABC) transporters are involved in the secretion of leaderless secretory proteins. Interestingly, glyburide blocks the anion exchanger function of ABC1, a mammalian member of the family of ABC transporters. We thus investigated the involvement of ABC1 in IL-1β secretion, through the analysis of the effects of drugs known to inhibit IL-1β secretion, on the activity of ABC1 and in turn the ability of known inhibitors of ABC1 of blocking IL-1β secretion. Our data show that IL-1β secretion and the function of ABC1 as an anion exchanger are sensitive to the same drugs, therefore suggesting an involvement of the ABC1 transporter in the secretion of leaderless proteins in mammals.