Cellular Localization and Trafficking of the Human ABCG1 Transporter.

We have developed a suitable heterologous cell expression system to study the localization, trafficking, and site(s) of function of the human ABCG1 transporter. Increased plasma membrane (PM) and late endosomal (LE) cholesterol generated by ABCG1 was removed by lipoproteins and liposomes, but not apoA-I. Delivery of ABCG1 to the PM and LE was required for ABCG1-mediated cellular cholesterol efflux. ABCG1 LEs frequently contacted the PM, providing a collisional mechanism for transfer of ABCG1-mobilized cholesterol, similar to ABCG1-mediated PM cholesterol efflux to lipoproteins. ABCG1-mobilized LE cholesterol also trafficked to the PM by a non-vesicular pathway. Transfer of ABCG1-mobilized cholesterol from the cytoplasmic face of LEs to the PM and concomitant removal of cholesterol from the outer leaflet of the PM bilayer by extracellular acceptors suggests that ABCG1 mobilizes cholesterol on both sides of the lipid bilayer for removal by acceptors. ABCG1 increased uptake of HDL into LEs, consistent with a potential ABCG1-mediated cholesterol efflux pathway involving HDL resecretion. Thus, ABCG1 at the PM mobilizes PM cholesterol and ABCG1 in LE/LYS generates mobile pools of cholesterol that can traffic by both vesicular and non-vesicular pathways to the PM where it can also be transferred to extracellular acceptors with a lipid surface.

Structure/function relationships of apolipoprotein a-I mimetic peptides: implications for antiatherogenic activities of high-density lipoprotein.

RATIONALE: Apolipoprotein (apoA)-I mimetic peptides are a promising type of anti-atherosclerosis therapy, but how the structural features of these peptides relate to the multiple antiatherogenic functions of HDL is poorly understood. OBJECTIVE: To establish structure/function relationships of apoA-I mimetic peptides with their antiatherogenic functions. METHODS AND RESULTS: Twenty-two bihelical apoA-I mimetic peptides were investigated in vitro for the capacity and specificity of cholesterol efflux, inhibition of inflammatory response of monocytes and endothelial cells, and inhibition of low-density lipoprotein (LDL) oxidation. It was found that mean hydrophobicity, charge, size of hydrophobic face, and angle of the link between the helices are the major factors determining the efficiency and specificity of cholesterol efflux. The peptide with optimal parameters was more effective and specific toward cholesterol efflux than human apoA-I. Charge and size of hydrophobic face were also the major factors affecting antiinflammatory properties, and the presence of cysteine and histidine residues was the main factor determining antioxidant properties. There was no significant correlation between capacities of the peptides to support individual functions; each function had its own optimal set of features. CONCLUSIONS: None of the peptides was equally effective in all the antiatherogenic functions tested, suggesting that different functions of HDL may have different mechanisms and different structural requirements. The results do suggest, however, that rationalizing the design of apoA-I mimetic peptides may improve their therapeutic value and may lead to a better understanding of mechanisms of various antiatherogenic functions of HDL.

The ABCA1 transporter functions on the basolateral surface of hepatocytes.

ABCA1 on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was currently assessed by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. These findings suggest an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL.