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 ϳ5.6% of apoA-I that interacts with cells is degraded and that internalized apoA-I contributes to ϳ1.4% 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.

Apolipoprotein A-I Associated Amyloidoses: The Intriguing Case of a Natively Unfolded Protein Fragment

“…Different results were obtained instead in the case of full-length ApoA-I, as the protein does not appear to be significantly degraded once internalized. However, we observed a strong co-localization of ApoA-I with lysosomes, in agreement with recent reports Denis et al, 2008). Although the question concerning the physiologic role of ApoA-I in lysosomes remains controversial, it might be considered that, even if lysosomes are best known for their role in degradation, they may also fuse with the plasma membrane to release their content in the extracellular medium (Luzio et al, 2007).…”

“…Similar results were obtained for ApoA-I, in agreement with recent reports showing that the majority of cell-associated ApoA-I does not co-localize with ABCA1, although no internalization was observed in cells ABCA1 -/- (Zha et al, 2001). To explain this observation, a model was recently proposed (Denis et al, 2008;Vedhachalam et al, 2007) in which the interaction of a small fraction of lipid-free ApoA-I to ABCA1 is sufficient to activate ABCA1 lipid translocase activity, which in turn promotes the formation of specialized lipid domains, acting as high affinity binding sites for ApoA-I (Vedhachalam et al, 2007). Nevertheless, whether or not ABCA1 has to be considered as an ApoA-I receptor is still ambiguous, as ABCA1 could have a role in inducing modifications of membrane lipid distribution facilitating ApoA-I docking (Vedhachalam et al, 2007).…”

SPTLC1 Binds ABCA1 To Negatively Regulate Trafficking and Cholesterol Efflux Activity of the Transporter

“…Cell surface expression of ABCA1 permits the transporter to transfer cholesterol and phospholipid to apoA-I, which is present in the extracellular environment (23,24). β1-Syntrophin binds ABCA1 through the PDZ motif in the ABCA1 C-terminal domain, and the positive effect that β 1-syntrophin has on the trafficking of ABCA1 likely is mediated by the ability of β 1-syntrophin to recruit utrophin, a cytoskeletal binding protein, to the ABCA1 complex (12,25,26).…”

Ht31, a Protein Kinase A Anchoring Inhibitor, Induces Robust Cholesterol Efflux and Reverses Macrophage Foam Cell Formation through ATP-binding Cassette Transporter A1

“…We have established previously that apoA-I primarily acquires cholesterol from the plasma membrane without the need of endocytosis (15). To test the possibility that st-Ht31 might mimic apoA-I and mediate cholesterol export by lipidating itself at the plasma membrane without entering cells, we incubated cells with Ht31, a nonstearated and thus nonpermeate form.…”