title: ATP-binding cassette A1-mediated lipidation of apolipoprotein A-I occurs at the plasma membrane and not in the endocytic compartments.
abstract: ATP-binding cassette transporter (ABC) A1 is required for the lipidation of apolipoprotein A-I to generate high density lipoprotein (HDL). This process is proposed to occur through a retro-endocytosis pathway in which apoA-I internalizes with ABCA1 and generates HDL from the endosomal compartments before resecretion. The aim of this study was to determine the route of apoA-I endocytosis and whether endocytosis contributes to HDL biogenesis. Using confocal microscopy, we found that internalized apoA-I only transiently colocalized with transferrin, a retro-endocytosis marker. Instead, apoA-I perfectly colocalized with a bulk phase uptake marker (fluorescein isothiocyanate-dextran) and, at later time points, with LysoTracker in several cell models including macrophages, fibroblasts, and baby hamster kidney cells. ABCA1 colocalized poorly with internalized apoA-I. To determine the contribution of internalized apoA-I to HDL biogenesis, we specifically removed apoA-I from the cell surface and analyzed the fate of internalized apoA-I. We found that 23% of cell-associated apoA-I was internalized at steady state. Of internalized apoA-I, only 20% was converted to HDL, and the rest was degraded, consistent with a lysosomal destination. We also found that apoA-I was released approximately five times faster from the plasma membrane than from the intracellular compartments. From these kinetic parameters, we estimated that approximately 5.6% of apoA-I that interacts with cells is degraded and that internalized apoA-I contributes to approximately 1.4% of total HDL production. We also found that blocking endocytosis with sucrose or cytochalasin D did not decrease cholesterol efflux or HDL biogenesis. We therefore conclude that the plasma membrane is the main platform where ABCA1-mediated lipidation of apoA-I occurs.
authors.affiliation: Ottawa Health Research Institute, and Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario K1Y 4E9, Canada.
authors.forename: Maxime
authors.initials: M
authors.lastname: Denis
authors.forename: Yves D
authors.initials: YD
authors.lastname: Landry
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Zha
mesh_terms: D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D021381:Protein Transport / Q000502:physiology; D014168:Transferrin / Q000235:genetics / Q000378:metabolism
chemical_list: C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D014168:Transferrin