Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis

Abstract

It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL biogenesis and in the human atheroprotective system. However, the nature and specifics of apoA-I/ABCA1 interactions remain poorly understood. Here, we present evidence for a new cellular apoA-I binding site having a 9-fold higher capacity to bind apoA-I compared with the ABCA1 site in fibroblasts stimulated with 22-(R)-hydroxycholesterol/9-cis-retinoic acid. This new cellular apoA-I binding site was designated "high-capacity binding site" (HCBS). Glyburide drastically reduced 125 I-apoA-I binding to the HCBS, whereas 125 IapoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Furthermore, reconstituted HDL exhibited reduced affinity for the HCBS. Deletion of the C-terminal region of apoA-I (#187-243) drastically reduced the binding of apoA-I to the HCBS. Interestingly, overexpressing various levels of ABCA1 in BHK cells promoted the formation of the HCBS. The majority of the HCBS was localized to the plasma membrane (PM) and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C, but not sphingomyelinase, concomitantly reduced the binding of 125 I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux, and the formation of nascent apoA-Icontaining particles. Together, these data suggest that a functional ABCA1 leads to the formation of a major lipidcontaining site for the binding and the lipidation of apoA-I at the PM. Our results provide a biochemical basis for the HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a two binding site model for ABCA1-mediated nascent HDL

Quantitative Analysis of ABCA1-dependent Compartmentalization and Trafficking of Apolipoprotein A-I

“…This is consistent with our previous findings that disruption of the HCBS inhibited the formation of nascent HDL (31). It is possible, however, that the PM could act as an initial tether point or apoA-I reservoir, allowing apoA-I to be brought into close proximity for interaction with the endocytotic pathway.…”

“…In this study we obtained evidence that deletion of the C-terminal region of apoA-I, residues 187-243, drastically reduced the compartmentalization of apoA-I between the PM and ICCs (Fig. 3B), consistent with our previous report documenting that apoA-I ⌬(187-243) exhibited reduced binding to both ABCA1 and the HCBS (31). This is in agreement with a previous study by Zannis and coworkers (9) showing that direct cross-linking of apoA-I ⌬(185-243) and ⌬(220 -243) to ABCA1 produced 3-fold higher K d values for these mutants compared with WT apoA-I.…”

“…4). Our recent results showing that disruption of the high capacity binding site impaired HDL formation (31) indicate that a complex regulation mechanism exists for the lipidation of apoA-I within different subcellular compartments. It is likely that under conditions of continuous exposure to an excess of apoA-I the accumulation of apoA-I within different compartments reflects the balance of two opposing processes of endocytosis from the PM and re-secretion from ICCs.…”

“…These results indicate that the C-terminal region of apoA-I is required for the formation of a productive complex with ABCA1 that leads to the compartmentalization of apoA-I between the PM and ICCs. Loss of compartmentalization by the C-terminal deletion mutant could be due to an inability of apoA-I ⌬(185-243) to bind directly to specialized phospholipid domains, such as the PCcontaining HCBS within the PM (31)(32)(33) and/or to an impairment of the apoA-I endocytotic pathway.…”

“…Modulation of ApoA-I Dissociation from the PM by Phospholipids-Recently, we and others have described a phosphatidylcholine containing non-ABCA1 PM site with a high binding capacity for apoA-I (31)(32)(33). In light of this, we hypothesized that PM-associated apoA-I could be released by treatment with phosphatidylcholine-specific phospholipase C (PC- Confluent normal fibroblasts (D) and ABCA1 mutant (Q597R) fibroblasts (E) were stimulated with 22OH/9CRA.…”