The ATP-binding Cassette Transporter A1 (ABCA1) is required for the biogenesis of HDL through the lipidation of lipid-poor apolipoprotein A-I (apoA-I). ApoA-I has been suggested to internalize with ABCA1, presumably to acquire lipids from the endosomal compartments, an important hub for cholesterol trafficking. The aim of our work is to determine how apoA-I get endocytosed and whether this internalization contributes to the biogenesis of HDL. When examined by confocal microscopy, we found that Cy3.5-apoA-I endocytosed rather slowly (t
1/2
= 15 min, in baby hamster kidney cells expressing ABCA1) and poorly co-localized with transferrin, consistent with a pathway independent of clathrin-coated pits. ApoA-I was instead found perfectly co-localized with FITC-dextran, a bulk phase uptake marker and, at later time points, with LysoTracker. This strongly indicates that majority of internalized apoA-I was delivered to the lysosomes. ABCA1 was not found to co-localize with endocytosed apoA-I. Similar observations were obtained in mouse macrophages and normal human fibroblasts. Next, in order to determine the functional significance of endocytosis to cholesterol efflux, we used sucrose or latrunculin A to inhibit apoA-I endocytosis. We show that apoA-I, transferrin and dextran uptake were abolished, but cholesterol efflux was not decreased. To specifically determine whether internalized apoA-I contributes to the biogenesis of HDL, we developed a method to strip off apoA-I from the cell surface. Our results show that internalized apoA-I do not significantly contribute to the biogenesis of HDL (< 20%). Together, our results suggest that the plasma membrane is the main platform where ABCA1-mediated lipidation of apoA-I occurs. Internalized apoA-I is mostly targeted for lysosomal degradation and therefore does not significantly contribute to the biogenesis of HDL.
Cellular distribution of high and low affinity concanavalin A binding sites on the plasma membrane of normal human fibroblasts.