Abstract
ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high-density lipoprotein (HDL) metabolism. Although it is predicted that apolipoprotein A-I (apoA-I) directly binds to ABCA1, the physiological importance of this interaction is still controversial and the conformation required for apoA-I binding is unclear. In this study, the role of the two nucleotide-binding domains (NBD) of ABCA1 in apoA-I binding was determined by inserting a TEV protease recognition sequence in the linker region of ABCA1. Analyses of ATP binding and occlusion to wild-type ABCA1 and various NBD mutants revealed that ATP binds equally to both NBDs and is hydrolyzed at both NBDs. The interaction with apoA-I and the apoA-I-dependent cholesterol efflux required not only ATP binding but also hydrolysis in both NBDs. NBD mutations and cellular ATP depletion decreased the accessibility of antibodies to a hemagglutinin (HA) epitope that was inserted at position 443 in the extracellular domain (ECD), suggesting that the conformation of ECDs is altered by ATP hydrolysis at both NBDs. These results suggest that ATP hydrolysis at both NBDs induces conformational changes in the ECDs, which are associated with apoA-I binding and cholesterol efflux.
Highlights
These results suggest that the two intact nucleotide-binding domains (NBD) are required for apolipoprotein A-I (apoA-I) binding and apoA-I-dependent cholesterol efflux by ATP-binding cassette protein A1 (ABCA1)
ABCA1 is induced by the nuclear liver X receptor (LXR) that is activated by oxysterols, which are metabolites of excess cholesterol
Because ABCA1 is a member of the ABC protein family, many of which mediate xenobiotic efflux and lipid transport in an ATP-dependent manner, it has been proposed that membrane phospholipid translocation via ABCA1 generates specific membrane domains that are bound by apoA-I [8, 14,15,16]
Summary
Materials The mouse anti-ABCA1 monoclonal antibody KM3110 was generated against the 20 C-terminal amino acids of ABCA1 [23]. Membranes were incubated on ice with 20 M [␣32P]8N3ATP, 3 mM MgCl2, 2 mM ouabain, 0.1 mM EGTA, and 40 mM Tris-Cl (pH 7.5) in a total volume of 10 l for 10 min and irradiated for 5 min (254 nm, 8.2 mW/cm2) on ice. Unbound ATP was removed by adding 400 l ice-cold 40 mM Tris-Cl buffer containing 0.1 mM EGTA and 1 mM MgCl2 and centrifuging (15,000 rpm, 5 min, 2°C) the samples. Unbound ATP was removed by adding 400 l ice-cold 40 mM Tris-Cl buffer containing 0.1 mM EGTA and 1 mM MgCl2 and centrifuging (15,000 rpm, 5 min, 2°C) the samples The pellets were resuspended in 8 l of TE buffer containing 1 mM MgCl2 and irradiated for 5 min (254 nm, 8.2 mW/cm2) on ice. The samples were electrophoresed on 7% SDS-polyacrylamide gels and analyzed by autoradiography. A P value < 0.05 was considered statistically significant
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