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.

Interleukin 1␤ (IL-1␤), a secretory protein lacking a signal peptide, does not follow the classical endoplasmic reticulum-to-Golgi pathway of secretion. Here we provide the evidence for a "leaderless" secretory route that uses regulated exocytosis of preterminal endocytic vesicles to transport cytosolic IL-1␤ out of the cell. Indeed, although most of the IL-1␤ precursor (proIL-1␤) localizes in the cytosol of activated human monocytes, a fraction is contained within vesicles that cofractionate with late endosomes and early lysosomes on Percoll density gradients and display ultrastructural features and markers typical of these organelles. The observation of organelles positive for both IL-1␤ and the endolysosomal hydrolase cathepsin D or for both IL-1␤ and the lysosomal marker Lamp-1 further suggests that they belong to the preterminal endocytic compartment. In addition, similarly to lysosomal hydrolases, secretion of IL-1␤ is induced by acidotropic drugs. Treatment of monocytes with the sulfonylurea glibenclamide inhibits both IL-1␤ secretion and vesicular accumulation, suggesting that this drug prevents the translocation of proIL-1␤ from the cytosol into the vesicles. A high concentration of extracellular ATP and hypotonic medium increase secretion of IL-1␤ but deplete the vesicular proIL-1␤ content, indicating that exocytosis of proIL-1␤-containing vesicles is regulated by ATP and osmotic conditions. INTRODUCTIONInterleukin 1 (IL-1) 1 is a multifunctional cytokine and a major soluble mediator of inflammation (Dinarello, 1991). Although its biological activity is extracellular, this protein lacks a secretory signal sequence (Rubartelli and Sitia, 1997), raising the question of how it can be transported out of the cell. Two forms of IL-1 exist, ␣ and ␤; however, studies on IL-1 secretion mostly focused on IL-1␤, which is the major extracellular form in humans (Dinarello, 1991). IL-1␤ is synthesized by monocytes upon activation as a 35-kDa precursor, which accumulates in the cytosol (Singer et al., 1988) and is proteolytically processed to the mature form of 17 kDa by the caspase IL-1␤-converting enzyme (ICE) (Cerretti et al., 1992;Thornberry et al., 1992). ICE is present in the cell cytosol (Ayala et al., 1994;Singer et al., 1995) as a p45 inactive polypeptide. Where and how maturation of ICE to the active (p10/p20) form and the processing of IL-1␤ take place are unclear; however, the mature form of IL-1␤ seems to be either ¶ Corresponding author. E-mail address: annarub@hp380.ist. et al., 1990). Although the levels of basal release of IL-1␤ are quite low (Rubartelli et al., 1990), secretion is dramatically induced by extracellular ATP (Hogquist et al., 1991;Rubartelli et al., 1993;Perregaux and Gabel, 1994), which can be autocrinally produced by activated monocytes (Ferrari et al., 1997) and interact with P2Z purinoreceptors expressed on their membrane (Hickman et al., 1994;Di Virgilio, 1995). The cellular pathway underlying this ATP-driven secretion is, however, unknown. We have previously shown that IL-1␤ is ...

No abstract

No abstract

Mutations in the gene encoding ATP-binding cassette transporter 1 ( ABC1) have been reported in Tangier disease (TD), an autosomal recessive disorder that is characterized by almost complete absence of plasma high-density lipoprotein (HDL), deposition of cholesteryl esters in the reticulo-endothelial system (RES) and aberrant cellular lipid trafficking. We demonstrate here that mice with a targeted inactivation of Abc1 display morphologic abnormalities and perturbations in their lipoprotein metabolism concordant with TD. ABC1 is expressed on the plasma membrane and the Golgi complex, mediates apo-AI associated export of cholesterol and phospholipids from the cell, and is regulated by cholesterol flux. Structural and functional abnormalities in caveolar processing and the trans-Golgi secretory pathway of cells lacking functional ABC1 indicate that lipid export processes involving vesicular budding between the Golgi and the plasma membrane are severely disturbed.

Phagocytosis is the uptake of large particles by cells by a mechanism that is based on local rearrangement of the actin microfilament cytoskeleton. In higher organisms, phagocytic cells are essential for host defence against invading pathogens, and phagocytosis contributes to inflammation and the immune response. In addition, engulfment, defined as the phagocytic clearance of cell corpses generated by programmed cell death or apoptosis, has an essential role in tissue homeostasis. Although morphologically distinct phagocytic events can be observed depending on the type of surface receptor engaged, work over the past two years has revealed the essential underlying role of Rho family proteins and their downstream effectors in controlling actin dynamics during phagocytosis.

No abstract

Smooth muscle and endothelial cells in vivo are quiescent yet exposed to high levels of lipoprotein lipids. Phospholipid (PL) and free cholesterol (FC) efflux maintain homeostasis. Smooth muscle cells (SMC) expressed high levels of ABC-1 transporter mRNA, and glyburide-dependent PL and FC efflux to apolipoprotein A-1 (apo A-1), the major protein of high-density lipoprotein. FC efflux was inhibited by vanadate and okadaic acid, while PL efflux was not. Phosphatidylcholine was the major PL transferred by both cell types. Stimulation of phosphatidylserine efflux, redistributed within the membrane by this transporter, was only minimally increased. Umbilical vein and aortic endothelial cells expressed little ABC-1 mRNA, nor did these cells promote either PL or FC efflux in response to the presence of apo A-1. To investigate the mechanism of ABC-1-dependent lipid efflux from these cells, apo A-1 was preincubated in the presence of unlabeled SMC or fibroblasts, and the conditioned medium was then transferred to endothelial cells. This medium catalyzed the efflux of FC but not of PL from endothelial cells. Such FC efflux was resistant to glyburide but inhibited by okadaic acid and vanadate. The data suggest that ABC-1-dependent PL efflux precedes FC efflux to apo A-1 and that the complex of apo A-1 and PL is a much better acceptor of FC than apo A-1 itself. Inhibition of FC but not PL efflux by vanadate and okadaic acid suggests these transfers involve different mechanisms.