Apolipoprotein-mediated plasma membrane microsolubilization - Role of lipid affinity and membrane penetration in the efflux of cellular cholesterol and phospholipid

Lipid-free apolipoprotein (apo) A-I contributes to the reverse transport of cholesterol from the periphery to the liver by solubilizing plasma membrane phospholipid and cholesterol. The features of the apolipoprotein required for this process are not understood and are addressed in the current study, Membrane nnicrosolubilization of human fibroblasts is not specific for apo A-I; unlipidated apos A-II, C, and E incubated with the fibroblast monolayers at a saturating concentration of 50 mu g/ml are all able to release cholesterol and phospholipid similarly. To determine the properties of the apolipoprotein that drive the process, apo A-I peptides spanning the entire sequence of the protein were utilized; the peptides correspond to the 11- and 22-residue amphipathic cu-helical segments, as well as adjacent combinations of the helices. Of the 20 helical peptides examined, only peptides representing the N- and C-terminal portions of the protein had the ability to solubilize phospholipid and cholesterol, Cholesterol efflux to the most effective peptides, 44-65 and 209-241, was approximately 50 and 70%, respectively, of that to intact apo A-I. Deletion mutants of apo E and apo A-I were constructed that have reduced lipid binding affinities as compared with the intact molecule. The proteins, apo A-I (Delta 222-243), apo A-I (Delta 190-243), apo E3 (Delta 192-299) and apo E4 (Delta 192-299) all exhibited a decreased ability to remove cellular cholesterol and phospholipid. These decreases correlated with the reduced ability of these proteins to penetrate into a phospholipid monomolecular film, Overall, the results indicate that insertion of amphipathic cu-helices between the plasma membrane phospholipid molecules is a required step in the mechanism of apolipoprotein-mediated cellular lipid afflux. Therefore the lipid binding ability of the apolipoprotein is critical for efficient membrane microsolubilization.