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.