Abstract
The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.
Highlights
All living cells depend on the ability to translocate nutrients, metabolites, and other molecules across their membranes
We show that cholesteryl hemisuccinate (CHS) stimulates the ATPase activity of the human ABCG5/G8 sterol transporter to a much higher specific activity, as compared to previously reported data (Tables 1 and 2)
The much increased CHS-coupled ATPase activity indicates that ABCG5/G8 may need such a high ATP catalytic rate to achieve the sterol-transport function across the cellular membranes
Summary
All living cells depend on the ability to translocate nutrients, metabolites, and other molecules across their membranes. Mol. Scit.r2a0n2s0p,o2r1t,e8r.74U7sing ATPase activity as functional readout of ABCG5/G8, we show differentially 3 of 20 inhibition of the CHS-stimulated ATPase activity by three LOF missense mutants, two sitosterolemia mutations, and one sterol-binding mutation, where residues bearing the two disease mutations are sltoucadteiedsaolofntghethsee pmoluatrarneltasyw. Using ATPase activity as functional readout of ABCG5/G8, we show differentially inhibition of the CHS-stimulated ATPase activity by three LOF missense mutants, two sitosterolemia mutations, and one sterol-binding mutation, where residues bearing the two disease mutations are located along the polar relay. The sterol-binding mutant G5-A540F, when compared to WT, showed a ~90% reduction of the specific activity in CHS-coupled ATP hydrolysis (90 ± 10 nmol/min/mg). The catalytic rates of the mutants decreased by 70–90%, except for G5-A540F, whereas both G5-E146Q and G8-R543S displayed significantly larger KM(CHS), up to a twofold increase This suggests a more profound impact of sitosterolemia mutations on the ABCG5/G8 ATPase activity Int. J.
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call