ABCA1 dimer–monomer interconversion during HDL generation revealed by single-molecule imaging

Abstract

The generation of high-density lipoprotein (HDL), one of the most critical events for preventing atherosclerosis, is mediated by ATPbinding cassette protein A1 (ABCA1). ABCA1 is known to transfer cellular cholesterol and phospholipids to apolipoprotein A-I (apoA-I) for generating discoidal HDL (dHDL) particles, composed of 100-200 lipid molecules surrounded by two apoA-I molecules; however, the regulatory mechanisms are still poorly understood. Here we observed ABCA1-GFP and apoA-I at the level of single molecules on the plasma membrane via a total internal reflection fluorescence microscope. We found that about 70% of total ABCA1-GFP spots are immobilized on the plasma membrane and estimated that about 89% of immobile ABCA1 molecules are in dimers. Furthermore, an ATPase-deficient ABCA1 mutant failed to be immobilized or form a dimer. We found that the lipid acceptor apoA-I interacts with the ABCA1 dimer to generate dHDL and is followed by ABCA1 dimermonomer interconversion. This indicates that the formation of the ABCA1 dimer is the key for apoA-I binding and nascent HDL generation. Our findings suggest the physiological significance of conversion of the ABCA1 monomer to a dimer: The dimer serves as a receptor for two apoA-I molecules for dHDL particle generation.membrane protein | transporter P lasma high-density lipoprotein (HDL) is critical for preventing coronary artery disease (1). A member of the ATPdependent transporter family of ABC proteins, ATP-binding cassette protein A1 (ABCA1) initiates the generation of discoidal HDL (dHDL), a bilayer fragment consisting of 100-200 lipids wrapped by two molecules of apolipoprotein A-I (apoA-I) (2-4), by exporting cholesterol and phospholipids to lipid-free apoA-I in serum (5). More than 70 mutations have been identified in the ABCA1 gene. Indeed, mutations in ABCA1 lead to Tangier disease, which is characterized by plasma HDL deficiency (6-10). ABCA1 has two large extracellular domains (ECDs), and the two intramolecular disulfide bonds between them are necessary for apoA-I binding and HDL formation (11-13) (Fig. 1A). Two pieces of evidence suggest that apoA-I interacts with a specific conformation of the ECDs in an ATP-dependent manner: Chemical cross-linkers can cross-link apoA-I with ABCA1, and ATPasedeficient ABCA1 mutants fail to mediate apoA-I binding and crosslinking. However, the importance of direct binding of ABCA1-apoA-I in HDL formation is still controversial and, furthermore, how a dHDL particle containing two molecules of apoA-I is formed from lipid-free apoA-I monomers and membrane lipids is unknown.To address these issues, we performed single-molecule fluorescence imaging (14, 15) of ABCA1 and apoA-I on the plasma membrane (PM) via a total internal reflection fluorescence (TIRF) microscope in living cells. We examined the dynamic behaviors of ABCA1 as well as the interaction of ABCA1 with apoA-I, and found that ABCA1 forms an immobile dimer on the PM; the ABCA1 dimer then dissociates into diffusing monomers upon interaction with apoA-I, finally gen...

ATP-Binding Cassette Transporters, Atherosclerosis, and Inflammation

“…15,35 The direct interaction of apolipoproteins with ABCA1 is of key importance to ABCA1-mediated cholesterol efflux; however, the detailed molecular mechanisms are still elusive and several models have been proposed. 37,38 Using a novel single-molecule fluorescence tracking technique, Nagata et al 38 have shown that lipid efflux to apoA-I involves the ATPase-dependent conversion of mobile ABCA1 monomers into immobile homodimers in the plasma membrane: the model proposes that ABCA1 monomers translocate lipids at the plasma membrane and form dimers; only the dimers can bind apoA-I, with one molecule of apoA-I binding to each ABCA1 molecule in the dimer. ApoA-I is subsequently lipidated, and a discoidal HDL particle containing 2 apoA-I molecules is formed.…”

ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis

“…3 ) Real-time tracking of single ABCA1 molecules showed that ABCA1 forms immobile homodimers in the absence of apoA-I. Upon apoA-I addition, ABCA1 rapidly became mobile ( 61 ). A GPI-anchored protein, a typical raft-associated molecule, also forms homodimers and becomes immobile, which results in the formation of homodimer rafts in a cholesterol-dependent manner ( 62 ).…”

Regulation of High-Density Lipoprotein Metabolism

“…8 Additional lipidation of apoA-I takes place in the golgi and at the plasma membrane in processes that are dependent on a dimeric form of ABCA1. 11,12 ApoA-I is also secreted from the liver into the extracellular space in a lipid-free or lipid-poor form. After secretion from the liver, the C-terminal domain of lipid-free apoA-I interacts with an extracellular loop of ABCA1 in a process that initiates the biogenesis of discoidal HDLs ( Figure 1B).…”