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 membran e 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 ap o A-I; unlipidated apos A-II, C, and E incubated with the fibroblast monola yers at a saturating concentration of 50 mu g/ml are all able to release ch olesterol and phospholipid similarly. To determine the properties of the ap olipoprotein that drive the process, apo A-I peptides spanning the entire s equence of the protein were utilized; the peptides correspond to the 11- an d 22-residue amphipathic cu-helical segments, as well as adjacent combinati ons of the helices. Of the 20 helical peptides examined, only peptides repr esenting the N- and C-terminal portions of the protein had the ability to s olubilize phospholipid and cholesterol, Cholesterol efflux to the most effe ctive peptides, 44-65 and 209-241, was approximately 50 and 70%, respective ly, of that to intact apo A-I. Deletion mutants of apo E and apo A-I were c onstructed 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 dec reased ability to remove cellular cholesterol and phospholipid. These decre ases correlated with the reduced ability of these proteins to penetrate int o a phospholipid monomolecular film, Overall, the results indicate that ins ertion of amphipathic cu-helices between the plasma membrane phospholipid m olecules is a required step in the mechanism of apolipoprotein-mediated cel lular lipid afflux. Therefore the lipid binding ability of the apolipoprote in is critical for efficient membrane microsolubilization.