Molecular and Cellular Physiology of Apolipoprotein A-I Lipidation by the ATP-binding Cassette Transporter A1 (ABCA1)

• Published in: The Journal of biological chemistry 2004-02, Vol.279 (9), p.7384-7394
• Main Authors: Denis, Maxime, Haidar, Bassam, Marcil, Michel, Bouvier, Michel, Krimbou, Larbi, Genest, Jacques
• Format: Article
• Language: English
• Subjects: Alitretinoin; Apolipoprotein A-I - metabolism; Apolipoprotein A-I - pharmacology; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters - metabolism; Binding, Competitive; Cells, Cultured; Cholesterol - metabolism; Fibroblasts; High-Density Lipoproteins, Pre-beta; Humans; Hydroxycholesterols - pharmacology; Iodine Radioisotopes; Lipid Metabolism; Lipoproteins, HDL - metabolism; Lipoproteins, HDL3; Particle Size; Phosphatidylcholines - metabolism; Protein Binding - drug effects; Sphingomyelin Phosphodiesterase - pharmacology; Sphingomyelins - metabolism; Tretinoin - pharmacology; Tritium; Type C Phospholipases - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M306963200

The dynamics of ABCA1-mediated apoA-I lipidation were investigated in intact human fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Specific binding parameters of 125I-apoA-I to ABCA1 at 37 °C were determined: Kd = 0.65 μg/ml, Bmax = 0.10 ng/μg cell protein. Lipid-free apoA-I inhibited the binding of 125I-apoA-I to ABCA1 more efficiently than pre-β1-LpA-I, reconstituted HDL particles r(LpA-I), or HDL3 (IC50 = 0.35 ± 1.14, apoA-I; 1.69 ± 1.07, pre-β1-LpA-I; 17.91 ± 1.39, r(LpA-I); and 48.15 ± 1.72 μg/ml, HDL3). Treatment of intact cells with either phosphatidylcholine-specific phospholipase C or sphingomyelinase affected neither 125I-apoA-I binding nor 125I-apoA-I/ABCA1 cross-linking. We next investigated the dynamics of apoA-I lipidation by monitoring the kinetic of apoA-I dissociation from ABCA1. The dissociation of 125I-apoA-I from normal cells at 37 °C was rapid (t½ = 1.4 ± 0.66 h; n = 3) but almost completely inhibited at either 15 or 4 °C. A time course analysis of apoA-I-containing particles released during the dissociation period showed nascent apoA-I-phospholipid complexes that exhibited α-electrophoretic mobility with a particle size ranging from 9 to 20 nm (designated α-LpA-I-like particles), whereas lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles. These results demonstrate that: 1) the physical interaction of apoA-I with ABCA1 does not depend on membrane phosphatidylcholine or sphingomyelin; 2) the association of apoA-I with lipids reduces its ability to interact with ABCA1; and 3) the lipid translocase activity of ABCA1 generates α-LpA-I-like particles. This process plays in vivo a key role in HDL biogenesis.