Abstract
Recent studies have identified an ABCA1-dependent, phosphatidylcholine-rich microdomain, called the "high-capacity binding site" (HCBS), that binds apoA-I and plays a pivotal role in apoA-I lipidation. Here, using sucrose gradient fractionation, we obtained evidence that both ABCA1 and [¹²⁵I]apoA-I associated with the HCBS were found localized to nonraft microdomains. Interestingly, phosphatidylcholine (PtdCho) was selectively removed from nonraft domains by apoA-I, whereas sphingomyelin and cholesterol were desorbed from both detergent-resistant membranes and nonraft domains. The modulatory role of cholesterol on apoA-I binding to ABCA1/HCBS was also examined. Loading cells with cholesterol resulted in a drastic reduction in apoA-I binding. Conversely, depletion of membrane cholesterol by methyl-β-cyclodextrin treatment resulted in a significant increase in apoA-I binding. Finally, we obtained evidence that apoA-I interaction with ABCA1 promoted the activation and gene expression of key enzymes in the PtdCho biosynthesis pathway. Taken together, these results provide strong evidence that the partitioning of ABCA1/HCBS to nonraft domains plays a pivotal role in the selective desorption of PtdCho molecules by apoA-I, allowing an optimal environment for cholesterol release and regeneration of the PtdCho-containing HCBS. This process may have important implications in preventing and treating atherosclerotic cardiovascular disease.
Highlights
Recent studies have identified an ABCA1-dependent, phosphatidylcholine-rich microdomain, called the “high-capacity binding site” (HCBS), that binds apoA-I and plays a pivotal role in apoA-I lipidation
We examined the distribution of ABCA1 and the HCBS between membrane microdomains, explored the modulatory role of the lipid environment on ABCA1 function, and examined the potential role of apoA-I in the regulation of the phosphatidylcholine (PtdCho) biosynthesis pathway
Using detergent extraction with 0.2% Triton X-100 followed by sucrose equilibrium density gradient, we examined the localization of ABCA1, ABCA1-associated apoA-I, and HCBS-associated apoA-I to detergent-resistant membranes (DRM) and/or nonraft fractions
Summary
Recent studies have identified an ABCA1-dependent, phosphatidylcholine-rich microdomain, called the “high-capacity binding site” (HCBS), that binds apoA-I and plays a pivotal role in apoA-I lipidation. The finding that the majority of apoA-I associated with the cell was found localized to the HCBS within nonraft domains (Fig. 1) suggests that apoA-I may efficiently mediate PtdCho desorption from these domains.
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