Apolipoprotein binding to protruding membrane domains during removal of excess cellular cholesterol.

High-density lipoproteins (HDL) are believed to protect against cardiovascular disease by removing excess cholesterol from cells. Lipid-free HDL apolipoproteins remove cellular cholesterol and phospholipids by an active, Golgi-dependent process that is still poorly understood. Here we characterized the morphology of apolipoprotein binding sites on cultured cells by immunogold electron microscopy. After 6 h incubations with lipid-free apoA-I or apoE, immunogold-labeled apolipoproteins were distributed sparsely along the planar surface of human fibroblasts and THP-1 macrophages. Overloading these cells with cholesterol led to a several-fold increase in the concentration of immunogold-labeled apoA-I and apoE on the cell surface, and over 80% of these gold particles were associated with novel electron-opaque structures protruding from the plasma membrane. Protrusions binding apoE were larger (100-200 nm) than those binding apoA-I (10-60 nm), and similar apoA-I-binding structures appeared when cells were incubated with either purified apoA-I or HDL particles. These structures were formed and enlarged by a time-dependent process inhibited by the Golgi disruptor brefledin A, the energy poison NaF, and low temperature. Moreover, formation of these structures was nearly absent in fibroblasts from a subject with Tangier disease, cells that lack a functioning apolipoprotein-mediated lipid removal pathway. Thus, formation of novel apolipoprotein binding structures protruding from the cell surface is an intermediate step in the cellular pathway by which apolipoproteins remove excess cholesterol.