Bile acids stimulate ATP hydrolysis in the ABCG5/G8 cholesterol transporter

ABCG5 and ABCG8 are half-size ATP-Binding Cassette (ABC) transporter proteins that function together as a heterodimer (ABCG5/G8) in the intestines and liver to limit the accumulation of cholesterol and other dietary sterols in the body by transporting into the intestinal lumen and bile. Mutations in either ABCG5 or ABCG8 can cause sitosterolemia, a rare disease characterized by cholesterol and plant sterol accumulation that can result in premature atherosclerosis. Although sitosterolemia is rare, the discovery of ABCG5 and ABCG8 provided evidence that specific molecular mechanisms regulate cholesterol absorption in the intestines and cholesterol excretion in the liver. Disruption of either ABCG5 or ABCG8 in mice results in increased absorption and decreased excretion of cholesterol and other dietary sterols. Overexpression of the human genes in ABCG5/G8 knock-out mice decreased sterol absorption from the diet and increased plant sterol excretion into the bile. Recent studies confirmed that ABCG5/G8 directly transport cholesterol and sitosterol across membranes in an ATP-dependent manner, but questions remain as to how this transporter pair utilizes ATP to facilitate the movement of cholesterol and other sterols across the membrane into the intestinal lumen and bile. Substrate binding to the transmembrane domains of ABC transporters typically stimulates ATP hydrolysis in their nucleotide binding domains so that energy from hydrolysis can be used to facilitate substrate transport across the membrane. The ABCG5/G8 substrates, cholesterol and plant sterols, do not stimulate purified ABCG5/G8’s ATPase activity, indicating ABCG5/G8 may require a cholesterol acceptor or another substrate to stimulate ATP hydrolysis. ABCG5/G8 is expressed in locations where bile acids are present in high concentrations, and bile acids were shown to increase ABCG5/G8 specific cholesterol efflux in cell models. It is not clear how bile acids stimulated cholesterol efflux in these studies, but they may have served as an acceptor for cholesterol. We hypothesized that bile acids stimulate ATP hydrolysis in ABCG5/G8. To test this hypothesis, we expressed ABCG5/G8 in the yeast Pichia pastoris, purified the proteins, and investigated the effects of bile acids on ATP hydrolysis. We found that bile acids stimulated ABCG5/G8 ATPase activity ~30-fold. The bile acid stimulated ATP hydrolysis in ABCG5/G8 was further characterized in terms of kinetic parameters, inhibitor profiles, and lipid requirements. These studies provide evidence that bile acids stimulate ATP hydrolysis in the sterol transporting ABCG5/G8. This interaction may provide insight into how ABCG5/G8 moves cholesterol out of the membrane and lead to innovative strategies to reduce cholesterol levels in the blood and prevent atherosclerosis.