Abstract 1315: Evidence for a Highly Cooperative Interaction Between apoA-I and ABCA1/Phospholipid Microdomain Binding Sites System: Implications for Nascent HDL Speciation and Biogenesis
Recent studies on the lipidation of apoA-I have proposed a two-binding site model for nascent HDL formation that involves both ABCA1 and a putative phosphatidylcholine-rich plasma membrane-high-capacity binding site (HCBS). However, the nature and specifics of apoA-I interaction with this phospholipid-associated membrane domain and its relationship to HDL formation remain poorly understood. To explore this issue, the dynamics of apoA-I dissociation from both ABCA1 and the HCBS were investigated in fibroblasts and BHK cells stably overexpressing ABCA1. We developed a rapid and efficient crosslinking assay to monitor apoA-I association with ABCA1. Fibroblasts or BHK cells expressing ABCA1 were incubated with 125I-apoA-I, crosslinked, and the ABCA1-complex was immunoprecipitated. The amount of 125I-apoA-I associated with ABCA1 or with the HCBS was quantitatively determined. We obtained evidence that the rate of dissociation of 125I-apoA-I from ABCA1 at 37°C was extremely rapid (t1/2 ~8 min), whereas the dissociation of apoA-I from the HCBS was >10-fold slower (t1/2~85 min). We next investigated the lipidation state of apoA-I by monitoring the dissociation of apoA-I from the HCBS over an 8-h period at 37°C. During the first hour of dissociation, apoA-I was released as an apoA-I-phospholipid complex without detectable cholesterol that exhibited α-electrophoretic mobility with a particle size of 8 nm. In contrast, particles generated after one hour dissociation were enriched in cholesterol and phospholipids, having particles size ranging from 9.5 to 20 nm. Taken together, these results provide strong evidence that apoA-I initially contacts the cell via ABCA1 and that this association is rapid but transient. Subsequently, ABCA1 mediates the transfer of apoA-I to the HCBS, thereby allowing first phospholipid and then cholesterol extraction and dissociation of the lipidated product. The differences in the particle size and cholesterol content of nascent LpA-I may reflect the heterogeneity in lipid composition of different membrane environments within the HCBS. Overall, the cooperative and productive interaction between apoA-I and the ABCA1/HCBS system is consistent with a tandem two-binding site model for nascent HDL genesis.