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ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL).The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that ABCA1-GFP was mainly localized to the plasma membrane (PM) but also substantially in intracellular compartments. All three mutant ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Photoaffinity labeling of W590S with 8-azido-[␣-32 P]ATP was stimulated by adding ortho-vanadate in the presence of Mn 2؉ as much as in the presence of wildtype ABCA1. These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis.Cholesterol is not catabolized in the peripheral cells and therefore mostly released and transported to the liver for conversion to bile acids to maintain cholesterol homeostasis. The same pathway may also remove cholesterol that has pathologically accumulated in the cells such as an initial stage of atherosclerosis. Assembly of high density lipoprotein (HDL) 1 particles by helical apolipoproteins with cellular lipid has been recognized as one of the major mechanisms for cellular cholesterol release (1, 2). The importance of this active cholesterolreleasing pathway in regulating cholesterol homeostasis became apparent by the finding that it is impaired in the cells from patients with Tangier disease, a genetic deficiency of circulating HDL (3, 4). Mutations were identified in ATP-binding cassette transporter A1 (ABCA1) of the Tangier disease (TD) patients (5-7), but the molecular mechanism of ABCA1 in the apolipoprotein-mediated HDL assembly remains unclear. Although direct interaction between ABCA1 and apoA-I at the cell surface has been suggested on the basis of chemical crosslinking experiments (8, 9), an indirect role of ABCA1 in the apoA-I binding to the cell was also proposed by a model that ABCA1 induces phosphatidylserine exofacial flopping to generate the microenvironment required for the docking of apoA-I at the cell surface (10). The predominant substrates of the ABCA1-mediated lipid release reaction are still to be determined for the HDL assembly reaction (11, 12). More than 30 mutations have been mapped in the ABCA1 gene in patients with familial hypoalphalipoproteinemia (FHA) and TD (5-7, 13-15). Many mutations have been ...

ABCA7 is highly homologous to ABCA1 and mediates cellular cholesterol and phospholipid release by apolipoproteins when transfected in vitro. However, expression of ABCA7 was downregulated by increased cellular cholesterol while ABCA1 was upregulated, and the results were consistent by forced expression or downregulation of sterol-responsive/ regulatory element (SRE) binding proteins (SREBPs). We analyzed the promoter of the ABCA7 gene and identified the new exon encoding 96 bp (mouse) and 95 bp (human) of the 59 untranslated region and the transcription start site at 1,122 bp (mouse) and 1,260 bp (human) upstream of the initiation methionine codon. At 59 upstream of this exon is the ABCA7 proximal promoter containing multiple binding sites of transcription factors for hematopoiesis and SRE of 9 bp at 212 bp (mouse) and 179 bp (human) upstream of the new exon. The apolipoprotein A-I-mediated lipid release was not influenced by suppression of the endogenous ABCA7 with small interfering RNA in mouse fibroblasts or by its increase in ABCA1-deficient mouse cells. In contrast, phagocytic activity was altered in parallel to the ABCA7 expression in these cells. When phagocytosis was induced, the messages increased for SREBP2, ABCA7, and other SREBP2-regulated proteins. The ABCA1 message decreased in this condition. We conclude that the ABCA7 gene is regulated by sterol in the opposite direction to ABCA1 through SRE/SREBP2 and that expression of ABCA7 by this regulation is associated with phagocytic activity.-Iwamoto, N., S. Abe-Dohmae, R. Sato, and S. Yokoyama. ABCA7 expression is regulated by cellular cholesterol through the SREBP2 pathway and associated with phagocytosis.

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