Molecular and cellular physiology of apolipoprotein A-I lipidation by the ATP-binding cassette transporter A1 (ABCA1).
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 (125)I-apoA-I to ABCA1 at 37 degrees C were determined: K(d) = 0.65 microg/ml, B(max) = 0.10 ng/microg cell protein. Lipid-free apoA-I inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than pre-beta(1)-LpA-I, reconstituted HDL particles r(LpA-I), or HDL(3) (IC(50) = 0.35 +/- 1.14, apoA-I; 1.69 +/- 1.07, pre-beta(1)-LpA-I; 17.91 +/- 1.39, r(LpA-I); and 48.15 +/- 1.72 microg/ml, HDL(3)). Treatment of intact cells with either phosphatidylcholine-specific phospholipase C or sphingomyelinase affected neither (125)I-apoA-I binding nor (125)I-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 (125)I-apoA-I from normal cells at 37 degrees C was rapid (t((1/2)) = 1.4 +/- 0.66 h; n = 3) but almost completely inhibited at either 15 or 4 degrees C. A time course analysis of apoA-I-containing particles released during the dissociation period showed nascent apoA-I-phospholipid complexes that exhibited alpha-electrophoretic mobility with a particle size ranging from 9 to 20 nm (designated alpha-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 alpha-LpA-I-like particles. This process plays in vivo a key role in HDL biogenesis.
Denis
Maxime
M
Cardiovascular Genetics Laboratory, Cardiology Division, McGill University Health Center, Royal Victoria Hospital, Montréal, Québec H3A 1A1, Canada.
Haidar
Bassam
B
Marcil
Michel
M
Bouvier
Michel
M
Krimbou
Larbi
L
Genest
Jacques
J
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000077556:Alitretinoin; D016632:Apolipoprotein A-I / Q000378:metabolism* / Q000494:pharmacology; D001667:Binding, Competitive; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D005347:Fibroblasts; D053442:High-Density Lipoproteins, Pre-beta; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology; D007457:Iodine Radioisotopes; D050356:Lipid Metabolism; D008075:Lipoproteins, HDL / Q000378:metabolism; D053440:Lipoproteins, HDL3; D010316:Particle Size; D010713:Phosphatidylcholines / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology; D013109:Sphingomyelins / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology; D014316:Tritium; D010738:Type C Phospholipases / Q000494:pharmacology
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D053442:High-Density Lipoproteins, Pre-beta; D006888:Hydroxycholesterols; D007457:Iodine Radioisotopes; D008075:Lipoproteins, HDL; D053440:Lipoproteins, HDL3; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D014316:Tritium; C003585:22-hydroxycholesterol; D000077556:Alitretinoin; D014212:Tretinoin; D002784:Cholesterol; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase; C105598:phosphatidylcholine-specific phospholipase C