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.
Lin
G
G
Division of Metabolism, Endocrinology and Nutrition, Department of Medicine, University of Washington, Box 356426, Seattle, WA 98195-6426, USA.
Oram
J F
JF
D001054:Apolipoproteins A / Q000378:metabolism* / Q000494:pharmacology; D001057:Apolipoproteins E / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure*; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism*; D008264:Macrophages / Q000000981:diagnostic imaging / Q000187:drug effects / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D012680:Sensitivity and Specificity; D013631:Tangier Disease / Q000378:metabolism; D014463:Ultrasonography
D001054:Apolipoproteins A; D001057:Apolipoproteins E; D008075:Lipoproteins, HDL