Abstract 824: Apolipoprotein A-I Mediates the Continuous Endocytic Recycling of ABCA1 to the Cell Surface: Implications for Determining the Kinetics of Nascent HDL Formation
It has been suggested that the endocytotic pathway is involved in apoA-I-mediated cellular cholesterol efflux via ABCA1. However, the molecular basis for the dynamic regulation of the ABCA1 endocytic trafficking and its relationship to nascent HDL formation remain poorly understood. Using cell surface biotinylation with a cleavable biotin, we found that a pool of ABCA1 was rapidly endocytosed in the presence of apoA-I in BHK cells stably overexpressing ABCA1 and 22OH/9CRA -stimulated human fibroblasts. Within 20 min, the amount of endocytosed ABCA1 reached a plateau in the presence of apoA-I, whereas in the absence of apoA-I no significant ABCA1 endocytosis was observed. Naturally occurring mutations of ABCA1 associated with Tangier disease impaired apoA-I-mediated ABCA1 endocytosis. On the other hand, the presence of apoA-I did not affect the rate of endocytosis of both transferrin receptor and activin receptor type II. Importantly, inhibition of endocytosis by chlorpromazine or treatment of cells with drugs, known to perturb ABCA1 trafficking, such as prubucol and cyclosporine A, drastically reduced the association of apoA-I with intracellular compartments (ICCs), but not plasma membrane (PM) and blocked the internalization of ABCA1. Concomitantly, cholesterol efflux as well as nascent HDL formation was completely inhibited. To better understand the role of the endocytotic pathway in the dynamics of the lipidation of apoA-I, a pulse chase experiment was performed and the dissociation (re-secretion) of 125I-apoA-I from both PM and ICCs was monitored over a 6-h period in BHK cells stably overexpressing ABCA1. Using a quantitative biotinylation assay, we found that the time required for 50% dissociation of 125I-apoA-I from ICCs was faster than that of PM (t1/2 =20 ± 3 min vs. t1/2=82 ± 7 min, respectively). The findings that apoA-I specifically mediates the continuous endocytic recycling of ABCA1, together with the observation that apoA-I associated with ICCs was rapidly re-secreted, support a model in which under conditions of continuous exposure to an excess apoA-I, the internalized apoA-I/ABCA1 complex must dissociate rapidly to allow replenishment of lost surface ABCA1 and completion of large number of apoA-I lipidation cycles.