Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.06.327825v1?rss=1 Authors: Xavier, B. M., Zein, A. A., Venes, A., Wang, J., Lee, J.-Y. 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 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 ATP catalysis by using three loss-of-function missense variants, two sitosterolemia mutations (E146Q and R543S; within polar relay) and one sterol-binding mutation (A540F; 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 polar relay network by all three mutants. Our data herein provide a biochemical evidence underlying the importance of the polar relay in regulating the catalytic activity of ABCG5/G8 sterol transporter. Copy rights belong to original authors. Visit the link for more info