10.1021/bi0608055.s001 Zhanling Wang Zhanling Wang Lindsay D. Stalcup Lindsay D. Stalcup Brandy J. Harvey Brandy J. Harvey Joachim Weber Joachim Weber Maja Chloupkova Maja Chloupkova Mark E. Dumont Mark E. Dumont Michael Dean Michael Dean Ina L. Urbatsch Ina L. Urbatsch Purification and ATP Hydrolysis of the Putative Cholesterol Transporters ABCG5 and ABCG8<sup>†</sup> American Chemical Society 2006 ABCG 8 ABCG 5 Putative Cholesterol Transporters ABCG 5 gel filtration columns ATPase activity ABCG 8 genes yeast Pichia pastoris transporters ABCG 5 2006-08-15 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Purification_and_ATP_Hydrolysis_of_the_Putative_Cholesterol_Transporters_ABCG5_and_ABCG8_sup_sup_/3065197 Mutations in the ATP-binding cassette (ABC) transporters ABCG5 and ABCG8 lead to sitosterolemia, a disorder characterized by sterol accumulation and premature atherosclerosis. ABCG5 and ABCG8 are both half-size transporters that have been proposed to function as heterodimers in vivo. We have expressed the recombinant human <i>ABCG5</i> and <i>ABCG8</i> genes in the yeast <i>Pichia pastoris</i> and purified the proteins to near homogeneity. Purified ABCG5 and ABCG8 had very low ATPase activities (<5 nmol min<sup>-1</sup> mg<sup>-1</sup>), suggesting that expression of ABCG5 or ABCG8 alone yielded nonfunctional transporters. Coexpression of the two genes in <i>P. pastoris</i> greatly increased the yield of pure proteins, indicating that the two transporters stabilize each other during expression and purification. Copurified ABCG5/G8 displayed low but significant ATPase activity with a <i>V</i><sub>max</sub> of ∼15 nmol min<sup>-1</sup> mg<sup>-1</sup>. The ATPase activity was not stimulated by sterols. The catalytic activity of copurified ABCG5/G8 was characterized in detail, demonstrating low affinity for MgATP, a preference for Mg as a metal cofactor and ATP as a hydrolyzed substrate, and a pH optimum near 8.0. AlFx and BeFx inhibited MgATP hydrolysis by specific trapping of nucleotides in the ABCG5/G8 proteins. Furthermore, ABCG5/G8 eluted as a dimer on gel filtration columns. The data suggest that the hetero-dimer is the catalytically active species, and likely the active species in vivo.